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Rôle de la régulation stomatique et de la capacité de détoxication

1. GLR25 Control Ozone Face Abaxial Adaxial I T Carpaccio Cima Robusta Carpaccio Robusta GLUR32 Control Ozone Face Abaxial Adaxial Carpaccio Cima Robusta Carpaccio Robusta GORK12 Control Ozone Face Abaxial Adaxial Carpaccio Cima Robusta Carpaccio Robusta KAT12 Control Ozone Face Abaxial ir Adaxial Carpaccio Robusta Carpaccio Robusta 2 0 i Normalized expression o o a 0 0 o a o Normalized expression o a 0 0 1 2 o o Normalized expression o o o o N 0 0 Normalized expression N o h 1 GLUR3 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta GORK4 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta GORK Control Ozone Carpaccio Cima Robusta Carpaccio Robusta KAT36 Control Ozone Carpaccio Robusta Carpaccio Cima Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial 2 5 EN o i o Normalized expression o in 0 0 o Normalized expression o a 0 0 1 54 1 0 Normalized expression 0 0 3 0 in o a Normalized expression o 0 5 0 0
2. O puns oArjejnd O Tepus ose1ojsuen AqiourAxodp Aq OULIOS 0 oaned foseroysueT AJOUT AXOIPAU 8 10002990 IT S697 T00 INXII W 00LI109710 nod VOCOIA OT J 8d pysoussy jxgnso 78788000 0078 20 0900 1104 6IOIIIX OT 234 pysoussy jxgnso IT O961 700 INXlI W 00900010304 01000 00 0 8 T 90E8TETOO 000 lt lt 09 lt 001 04 lt 802000 pLWHSs 11986006200 00206092001 04 LLODVODVOODVVOLLLLVO VOVDLIOOVDVIODDILVOVV LOOLVODDOLVVIVVILOLOL JDIVVOVIVVVODDDODLV LL DLVVOOVOOVOVOVOLVOLL JIILLVILIJIDIVLLLLIDV VDV IOODIOVDOLVOVOOOLL IL295 LLI00VDDIOOODVLVV L LVIOLOODVOLVVVVOLlOIlDO IOLLLVOOVVIVOJLIOVODOLID 0981052100 1404 0998059000 ULdOd O6LEOSETOO ULdOd 08705000 ULdOd 0806082000 ULdOd TILVDOD 9LVDOD ETEND SAWD vVTVAID L SOUVV C991pOVV ETSSOAV V c LLAVV 6T TLWVV 9SELEOVV 8991FOVV 191 9650154 QOSSOUVV T8SSOAVV TETILNVV 60CLLINVV 5 lt lt 0 6SSSOUVV TSTISAVV TESSOAVV LZZILINVV JO 80 L 8O 0U110 09 0109 9f au OSBJOUIUAS OJ 1e rurs 96 9v VO DEAL V S96004 V8ZETIZI 8007SAVV
3. Control Ozone E Carpaccio Cima Robusta Carpaccio Cima Robusta CAX1 Control Ozone T oe LL Carpaccio Robusta Carpaccio Robusta CAX16 Control Ozone Carpaccio Robusta Carpaccio Robusta CLCa Control Ozone Carpaccio Cima Robusta Carpaccio Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial 3 0 a Normalized expression o a 0 0 2 0 Normalized expression o o in 0 0 o o Normalized expression o f o 0 0 o o Normalized expression o 0 0 CA4 Control Ozone Carpaccio Robusta Carpaccio Robusta CAX3 Control Ozone Carpaccio Robusta Carpaccio Robusta CHL Control Ozone Carpaccio Robusta Carpaccio Robusta CLCa6 Control Ozone Carpaccio Robusta Carpaccio Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial 1 5 1 0 Normalized expression 0 0 M m o in o a Normalized expression o a 0 0 1 2 o o E o o Normalized expression o T 02 0 0 25 m o in o Normalized expression o 0 0
4. euno suem ercehso SZHS9 HH GSHETIGHETIGSHEZT L GHETLIGHECTLIGHETI ajewemn 9 euues KERNO epyns uoiejuisse EI LL GHhETIGHETIGHEZTI euiue v eje AxoAI B ajejo9A16 Sisouju soiq euoiujejn s R sultats There were only few genes that showed very contrasting response to ozone between genotypes Thus the expression of 410 decreased strongly in Carpaccio while it was quite stable in response to ozone in the other two genotypes The expression of G76 and GT16 decreased in Cima but increased in Carpaccio and Robusta The expression of GSH13 was more than 1 8 fold lower in ambient condition in Robusta than in Cima and Carpaccio and was also less increased in Robusta 2 1 fold than in Carpaccio 3 5 fold and Cima 2 3 fold Figure 6A The expression of GSH25 and THA13 were 3 6 fold and more than 1 5 fold higher in ambient condition in Carpaccio than in the other two genotypes Figure 6B C In response to ozone the expression of GSH25 was still 2 4 fold higher in Carpaccio than in the other two genotypes Figure 6B The expression of 7HA13 increased very strongly in Carpaccio in response to ozone 5 3 fold while the increase was much lower in Cima 1 4 fold and Robusta 1 5 fold Figure 6C Effects of ozone on amino acid contents Ozone treatment had a contr
5. _ 50 C D 7 40 ORC A p 30 EH 7 222 7 10 4 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 L Ad tt tt _ 25 LE LF L 2 0 F 4 I Q M T 7 zZ 2 1 0 L 4 Lost 4 tt tp a 1 4 t L L LG T H px kkk 7 amp 1 10 F 1 lt Zo E 5 pas pe a qe 4 ge d 4 Tac E uc 6 12 18 6 12 18 Time d Fig 8 Effect of treatment on NADPH A NADP C D NADH F and NAD G H contents nmol g FW in leaf protein extracts of Carpaccio A C E G and Robusta D F H genotypes n 4 st Significant differences between the Os exposed and control extracts are indicated by P lt 0 05 P lt 0 01 and P lt 0 001 Table 3 NAD P H NAD P ratios and total NAD P H nmolg FW pools in the leaves of control C and 15 day ozone treated O3 Carpaccio and Robusta genotypes C C C O3 R C R O3 NADH NAD O22 0 11 0 26 0 11 NADPH NADP 0 33 049 0 36 0 26 NAD H nmol g FW 8 0 15 8 8 8 13 8 Y NADP H nmol g FW 52 7 41 6 46 2 41 7 7 Total pyridine pool 9 FW 60 7 574 550 55 5 Processed by cytosolic NADP ICDH this reaction also yields NADPH The higher stimulation of NADP ICDH activity in Carpaccio may contribute to maintain higher levels of NADPH in the cytosol of the tolerant genotype Sta
6. 8 71 0 requis T asojaejes T 03 1e rurs L9EE8HV Jo c Jo c ostjonpoi ojox opre 0j 0628 10 ds 7 01 ets 06 55591 9 sisdopiqpay 8 71 0 requis T asojaejes T 03 1e rurs 0079 lt Jo 801040156 ISL I9JSUP IJOUTUIR 8 Tepus 041616000 11 99 86 00 T 619ETETOO 16696516200 06 0XI OT J uid pusous3 1 59 0021800600 1104 6041 OT 034 pysoussy 1xq1so 001 8201001194 9LHOIAX DT Oud pusouosy jxgiso TELOV d 0022 109107004 001S 09010 104 DVOLVOOLVODO LLLIOVDO LL DLLLV IVOOLVVOOLOLODD 0658056000 ULdOd 6HGI D T OVDOLLVODVVDLIVVVVDD0D VOLLDOVVVOOLVODIVOLOD 066051000 ULdOd DOVOVOVVVOOOVVOVVIOV ODLVIVOLVOLLODIOVIOO 016189100 ULdOd VIOVOVOLVOLIODVIODVIO DIOLVVOLVOOLOVOOLVVO 085080100 ULdOd 0119 05897811 V OTISSSSIV Jo D So oq10 09 Z 9 0j Je ruis L6190v VO 680 0V 666800VV 09t0LJVV BLSTEdVV OSLSSVVA LOLTEJVV 155 866800 0L9SOVVE 6 791 66 0d 9IST8EV VO 9S00TVVO 11 30 01 BO OUMO 09 5 u
7. 8 Terus 77 SOAVV 991 ETSSOAV v CETILINVY 61 LLIANVV 9SELEOVV 8991FOVV LLOIPOVY 9STI SAV QOSSOUVV 18650 TETILNVV 60C LLINVV TSSSOAVV 6SSSOUVV TSTISAVV TESSOUVV LZZILINVV JO 8 JO 8O 04110 09 2100 0 au DYO oururejn s OJ 1e rurs c991rOVV ETSSOAV V CETILINVV 6r LLINVV 9SELEOVV 8991 LLOITOVV 9 lt 0154 QOSSOUVV I8SSOUVV IETILNVV 60C LLINVV TSSSOAVV 6SSSOUVV TSTISAVV TESSOAVV LZZILINVV JO 830 8 So oquo 09 02097 2u124150 y40 OSBJOUIUAS oururejn s 1e rurs SCICII OT OTA IOSIMOUOD IXAIS9 S SDLAI4d T 8EzLHI AAS OOTTETOLIO Hd 0600ccI 2 84 pysouasy 1XAISI SOdON4d IT 6L967ETO0 XI 00LP 0DS10 1nod 000IIX DT Oud pysouasy jxgiso 5 76928 16000 WXUOU 006 FOD710 Md VVDLVOOLVODODIOLVVVD VIOVOLOVOLLLVODVVDD DVOVVOOLLOLVIOVVOOOL DLVVOOODLVVDLIOLLLVOD OVOLLLVVVOOV IOLODVOO LLLVOOLOLLOOLLOIOODL OLLCOSLIOO ULdOd 010508100 ULdOd 060 082100 ULdOd 1150 SISO cISD OTEET8TIV 0850 813 JO c JO 1 02 51 ose1ojsuejJourue 8 7 ICIS 09968 pav Jo Jo So oqo oo z ose1ojsuemourure 8 O IUS Jos1nooud IEUS Z osedojsuedjourue eje Axo AT 8 9590 45 oAnejnd
8. GSH11 AMBIENT nil T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA OZONE sampling dates day 2 4 11 15 17 gt GS8 AMBIENT OZONE sampling dates day o 15 17 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Normalized expression o a 0812 AMBIENT OZONE sampling dates 1 5 day 2 74 11 15 17 0 0 CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o Normalized expression o a GS17 AMBIENT sampling dates day 12 la 11 15 0 5 17 CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA OZONE o L Normalized expression GSH213 AMBIENT OZONE 1 2 o sampling dates day iy 2 r 4 11 15 17 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o 1 o Normalized expression o P o 1 GT3 AMBIENT OZONE sampling dates day 2 4 11 15 17 T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day 2 E 4 11 15 17 ROBUSTA Normalized expression o o o o N gt o 0 0 GT7 AMBIENT OZONE T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA o 1 Normalized expression o a GT10 AMBIENT OZONE sampling dates day GT6 OZONE 1 0 0 8 sampling dates day 0 6 Lp 4 11 04 15 1
9. 02 0 au DYO oururejn s OJ 1e rurs 991 ETSSOAV V cLLNVV 6T LLNVV 9SELEOVV 8991 POVV LLOTFOVV 9SZTISAVV 90 lt lt 0 I8SSOUVV TECILWNV V 60c LLINVV ZSSSOUVV 6SSSOUVV TSTISAVV TESSOUVV LZZILINVV JO 8 JO 8O 04L10 09 vvo au DNO OSBJOUIUAS oururejn s OJ TP TLUIS SOAVV 9917 ETSSOAVV c LLINVV E6IILINYYV OSELEOVV 8991rOVY LLOIPOVV 9SZISAVV OOSSOUVV I8SSOUVV CTECLLINVV 60cLLINVY ZSSSOUVV 6SSSOUVV TSTISAWV TESSOAVV LCCLLINV V JO 830 9 So oquo 09 02097 eur AO OSBJOUIUAS oururejn s TP ILIIS DT jxgiso L SDdON Id 06 000 0016c0DOI0 tod OGLTIIIA DT 0 84 puseuesy 8 SDdON d T L69T1ET00 NXIOM 0010075800 tod 6 0 OT 0 84 pysoucsy jxgqso 0L6L0SLOO0 ALdOd SDdON d U O 901 C00 009690000 tod LLODIVDOIOVOVVVVOOLOD LLVODIDOIODIOOOVDVVVIL IOIVVOLLOLOJIVODOOVOO IOLOLOLLOVVVDDOLVODO VOLLOLVIVODVDODVODVOV IOLLVOLLLVODLLODOIVOL 010 lt 080100 ULdOd 0940758000 ULdOd 0961051000 ULdOd 0150 850 150 e96 9VWO S9600d V8ZETIZI 8007SAVV T6HEOAV V e6L0cdV V 6cLT0OVV TILTOS 082804 6LEETAVW 4660019 530 02 snjoaspyd DYyO AOUPTY 2050350 O eurues 7 9 esesi
10. gt adAjoUas x su su su su su su su su su su su su su su 8800 0 su 1200 c 0 0 su 9000 689000 su su su su su su su su su su su su su su su su su su 1000 gt su su su su GELEO O F 8L L TSI SFSZ ELSI 2581 190 F 1261 0T 6l S F ZZ S 0t 488 0688 LL O F SCVI EST LF 8E 6 6 F 92801 FV TIL FGE VI T 90 669 F PIPL 9 OV v Iv 0 85 8TLE PLOFLVVL LE FG8 OF FG8 9 9 L6C F 2912 1 c 1 2 su su su su su su su su LYTOO O 9002 F VLC9 68EZ 2189 9821 1809 ttv L9V 907 LL9 601 F VELI T 5 2 su su su su su su su su su 1991 LZES 2502 0 99 6267 084 ISIC 021 60EZ 2 pue p y adAjouas pue jusurjea1 Joye uone muisse _S _ uorerreA Uoneqrwuisse 5 joum paads Suson ejeurojs 5 A SON ejeurojs 10 uoxej 5 1 S 329M 10 T 193je JUILUJLII 0 1JU09 JOU 10 EQ jo our 021 o3 5 eysnqoy pue eun oroedie sadAjouas 1e dod 2211 JO 45 F eq 80 AdA P 03 sesuodsa1 38UPUIXS ses rureuAq 9 t9 oroed1e ejsnqoy o ejsnqoy euir EQ euirr oroedae j
11. 11 2000 7 Quantit Carpaccio Robusta Carpaccio Cima Robusta Galactose AMBIANT OZONE 200004 15000 4 Temps jour 2 2 4 100004 0000 11 17 5000 d Carpaccio Cima Robusta Carpaccio Robusta Mannose AMBIANT OZONE 6000 7 Temps 4000 lour 2 2 t 4 5 1 8 1 17 E gt Carpaccio Cima Robusta Carpaccio Cima Robusta Sucrose AMBIANT OZONE 1000000 4 750000 4 d Temps jour 2 2 500000 id 2 11 o 17 250000 4 o 7 r B Carpaccio Cima Robusta Carpaccio Cima Robusta Figure 52 Concentration en A fructose B glucose C raffinose D galactinol E galactose F mannose et G sucrose dans les feuilles de trois g notypes de peuplier Carpaccio Cima et Robusta apr s 2 4 11 15 et 17 jours de traitement ozone ou ambiant R sultats entraine une hausse des besoins en glucides et une baisse de l assimilation en carbone Des changements physiologiques et biochimiques se mettent alors en place pour permettre de soutenir les besoins prioritaires de la respiration et des processus m taboliques n cessaires pour faire face au stress comme dans le cas de l ozone les processus de d toxication et de r paration Les sucres solubles agissent comme mol cules de signal r gulant l exp
12. 39UPJINPUOD jo ueds 6 ur 58y syoam 10 Z 1 Joye aueureaa 01JU09 JOU 10 EQ Jo our 021 pejjruqns eysnqoy pue oed re sed joueS rejdod 22113 Jo AS sueeur 5 Jouni QE 030 1480 e sesuodso1 528 orureu AG IALL su su su su su su su 97700 su su su su su su su su su su su su su su su su su su su 811000 su su su su su su su su 10007 10007 10007 0 00 su su su su su su 872000 S 600 0 su su su su su su su su su 1000 gt 100 0 gt su su 61 0 0 su 100707 96000 su su su su su su su su su su su su su su su 96200 su su su su su su 791100 5609100 su F 6L I 9COTcC IVOTSUI ZCOTLVC 9 97F LI TFII 6c9 1589 500 F 621 ELO F 6TT TO F POT FLO FCI TOO ICT 6 L 9 F 8L 1 91 96 1 69vS WOFILC LVOTVvYVC 691 SCO TSCC 100 791 Ict CL 9 9011 6 0 680 6GEOFEEI 90 61 CVOFSEI 920 691 90059 1 III peF6L VIT 801 2991 9 0 1671 60 0 F 900 870 670 861 YTO FESI F VOI ve F LIL ST 06 6 18 615 66EL LVOFSUI 6LOFLGI 200 221 6GLOFLEI SVO TVVI VEO TFOI9CI 67671 S FELI 9F 191 06 OPOL Z 1 c I t IV su su su su su su J Dumont et al Environmen
13. NHX1 _ Control Ozone Carpaccio Robusta Carpaccio Cima Robusta NHX113 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta OST2 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta PHOT4 Control Ozone Carpaccio Robusta Carpaccio Cima Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial 3 0 in o a L Normalized expression o 0 5 0 0 1 2 o o Normalized expression o o gt o o 0 0 o in o Normalized expression o a 0 0 2 0 o in Normalized expression o in 0 0 Control NHX110 Ozone mM Carpaccio Robusta Carpaccio Robusta NHX114 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta OST2 18 Control Ozone Carpaccio Robusta Carpaccio Robusta PHOT2 Control Ozone Carpaccio Robusta Carpaccio Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial Control Ozone 3 c 4 e 5 2 o o N E o 21 0 Carpaccio Cim
14. Unicolore Turanga p non Crevass e grandeur supporte la Afrique pruniosa collant Dentel e 111 RN ilm Nord cili e d jet salinit Eurasie glauca Large Houppier Est et heterophylla Brun peu Bicolore 5 5 ample Ornemental Leucoides ps XE Circulaire non Crevass e Am rique lasiocarpa collant Cr nel e im Feuillage en Europe cili e du Nord brillant alba guzmanantlensis Eurasie monticola Afrique simaroa Bicolore Lisse et Leuce or du Nord adenopoda ay Cr nel e Circulaire Large claire Grand Peupliers Populus et gamblei t lob e ou Cili e Crevass e d veloppement forestiers Am rique grandidentata lobul e au pied du Nord sieboldii tremula tremuloides Uni ou Eurasieet deltoides Rouge ou Circulaire Large Rugueuse oe Ligniculture Aigeiros Am rique fremontii Brun S ouunpeu non Sillonn e put Prairie rhombo dale P Si troit du Nord nigra collant 22545 anguleux cili e longitudinalement Po Hors for t Cr nel e verticill augustifolia balsamifera cathayana ciliata ue Eurasie koreana A Ligniculture Est et laurifolia Rouge Oblongue Large Lisse Pyramidale Prairies et Tacamahaca oe ES Brun anguleux puis l g rement port large for ts dans Am rique maximowiczli ovale M a di Nord simonii collant cili e fissur e verticill son aire suaveolens d origine szechuanica trichocarpa yunnanensis For t Abaso Mexique mexicana tropicale Orneme
15. 5 iu 110 4 all all 421 3 51 2 3C AIR 2 441 A 4 1 451 5019 MR 1 2 14R 58 Zo GRO MB SOR 22 140 131 13C IR 9 5 2 Ape 3a 421 451 126 58 E 2R A A4R 45 A2R WC 2 X a3R A4R 3C E JC cate SAT N 43 i3R 14R 2 3 N a3R 4 4 14R J 5 9 8 7 6 5 4 3 2 JU 0 1 2 3 4 5 6 7 8 9 PCI 52 SIMCA P 12 0 1 2013 02 04 16 06 30 UTC 1 Figure 47 Analyse en composantes principales bas e sur les concentrations des carot noides des chlorophylles et des terp nols En rouge le traitement control en bleu le traitement ozone Les chantillons sont repr sent s par un chiffre correspondant aux pr l vements 1 2 3 4 5 pour 2 4 11 15 et 17 jours de traitement respectivement et une lettre pour le g notype C I R pour Carpaccio Cima et Robusta respectivement R sultats 2 EFFETS DE L OZONE SUR LES CONCENTRATIONS EN COMPOSES PHENOLIQUES ET EN CAROTENO DES L ascorbate et le glutathion sont deux acteurs majeurs du processus de d toxication mais d autres m tabolites sont capables de d toxiquer les ROS produits par l ozone Parmi ces m tabolites on peut citer les compos s ph noliques et les carot no des En effet les compos s ph noliques sont connus comme repr sentant des compos s de d fense importants contre les dommages dus l ozone a t montr chez diff
16. 9948 191880200 T L8ESEETOO WXIIoW 1 08801ET00 T09S60 000 ETES LINHS 0076019700 mod l O 86 T IMS 6LNHS 0007179100 tod 800088 pysoussy jxgnso CYINHSAd 9LINHS 0065091101504 Tr60X DT Oud pysouasy jxgiso TINHSJd LLINHS 00276 0010 04 TMS T INHS 1d CLINHS 0067009800 1104 DLVOVIVIODIDODLIOLVOV LLOLLVDODIOLIOIODVVODL DVILVVVVIVVLOLIDIIVI DLLVOIODILLODVOVIOJIVO ODVVVDOVODLLOVOLVOLV I9O9I15DLIVVDIlVODD LLOLL ODVVVDOLOVVVDVIOVODD ILVVODVVVDDVD IODI5DVOO LLLOIVIOVODOOLOLOOLL VVOVVODLIOVVOIVOLIOOOL 0660192000 ULdOd OS6ITSI000 ULdOd 0098051100 ULdOd O T9CS0100 ULdOd 050088000 ULdOd TVAIO 61VAID LIVAID OTVAIO 8V IO r061 Pd V 09 8 JO c Jo c 09 pays o8pa1paW OO oseyuds 8 juopuodop HAVN 01 epus 0 1 0993 OTTIPSTIV p99cedV V 820901 Jo T JO 1 BOTOUNO 09 v42314n228 y 40 oseyuds 8 juopuodop urxopo119 1e rurs lorsszssiv JO c o 1 80100 00 022 26610 SNPIYAIA 20 woy gug osejourido 20805 erus uro301d juspuodop GYN lorsszssiv JO c Jo T 3o ou10 09 0 CL 66 1D snjj1214424 2 1 woy OUI osejourido 20805 ejus uro301d 9VL990LT 1D
17. S cher les surnageants sous atmosph re azot e Dissoudre les surnageants d ss ch s dans 600 uL de dichlorom thane Ajouter 1 4mL de m lange ac tonitrile m thanol 6 1 les proportions finales de ACN MeOH DCM seront 60 10 30 139 Annexes Pr lever deux aliquots de 400 uL dans des bouteilles opaques Lancer l analyse d un des deux sous HPLC S cher l autre sous atmosph re azot e et le stocker 20 C HPLC MS Volume d injection 10 uL colonne Kinetex 2 6 u C18 150 x 4 6 mm flux 1 6 mL min temp rature de colonne 25 C temp rature de plateau 4 C d tection UV 240 700 nm ACN MeOH 75 25 v v B ACN MeOH dichlorom thane 75 10 15 v v v avec un gradient de 0 B 50 B en 4 min isocratique 50 B pendant 6 min et 9 min d quilibration avec 096 B avant l chantillon suivant APCI en mode positif balayage total des ions de 300 1000 m z PROTOCOLE D ANALYSE DES SUCRES COMPOSES PHENOLIQUES ET ACIDES AMINES 1 EXTRACTION Transf rer 30mg de mat riel v g tal dans un tube Eppendorf de 2mL et le plonger dans de l azote liquide Broyer les chantillons avec une spatule refroidie au pr alable dans de l azote liquide Ajouter une bille m tallique refroidie au pr alable Broyer les chantillons dans les tubes Eppendorf ins r s dans des blocs froids dans un TissueLyser 3 fois pendant 15 s avec une vitesse de 20 fois s la premi re fois puis 24 fois s En
18. l int rieur de la chambre de mesure de l appareil Li 6400 SOMMAIRE SOMMAIRE SYNTHESE 1 Ts OZONE 2 1 G n ralit s Sur zx tee eee tee bE e tee edis dd 2 2 Cycle de ozone A e stet ok Kod SRL ans DR IA ed 2 3 Evolution des concentrations en ozone troposph rique eese 3 4 Impact de l ozone sur la v g tation seen 4 Sites d action de l oZon6 cn Sete epe ob e e HR ne oe Deed og Pe e im p bert rise 4 Effets visibles sur les e e PRU Ir eg te dd Map i eb lide der A tot 6 Effets sur la biomasse et la croissance sn 7 Effets Sur les changes gazeux foliaies ui Deer agate ede dep d e ete eod aie 7 Effets biochimiIques dee t E 8 Il R LE DES STOMATES acere O aane eaten teta ne ve etate dite 8 1 G n ralit s aii EAT REG e TRU ETE 8 2 Mod les de conductance stomatique sense 9 3 R gulation des mouvements stomatiques sense 10 Effet du VPD Vapor Pressure Deficit et de la temp rature 3a CO teta edid uU ideis auus EINER Eftet di C mai educit eei e nie ed oda eqs 12 Bffets deT0z0one a oe bebe tee deemed ped dee
19. 4 C puis le lendemain 3 lavages de 5 min Postfixation avec le tetroxide d osmium 2 tamponn avec le tampon phosphate 0 1 M pH 7 2 pendant une heure 4 C Lavage dans le tampon phosphate 3 fois 5 min puis dans l eau distill e D shydratation dans des bains croissants d ac tone 10 15 min 20 15 min 40 15 min 60 15 min ou la nuit 80 15 min ou la nuit 95 20min X2 100 20 min X3 Impr gnation dans la r sine Si les chantillons restent la surface un l ger passage sous vide sera n cessaire les temps sont indicatifs peuvent tre diminu s ou augment s suivant les chantillons Pour les temps longs bien fermer les tubes et mettre 4 C 1 3 r sine 2 3 ac tone 24 heures 1 2 r sine 1 2 ac tone une journ e 54 r sine 1 4 ac tone une journ e R sine pure une nuit Le lendemain la r sine est remplac e par de la r sine fraiche laisser au moins 1 2 journ e puis inclure dans les capsules ou des moules viter les bulles d air les retirer si il y en a 154 Annexes Mettre polym riser 60 C dans une tuve pendant 48 heures Attendre que la r sine polym ris e revienne temp rature ambiante avant de d mouler les chantillons 155 R F RENCES 156 R f rences Aasamaa K S ber A Responses of stomatal conductance to simultaneous changes in two environmental factors Tree physiology 2011 31 855 8
20. 6 0 e6L0cdV V 67LIOOVV TILTOS 8804 6LEETAVV Le600rJo S JO T 2 s1408jna snjoaspyd DYyO Kouprpy oi oso34o O merus T 1 E 9 esesi 8 1e murs 0 71 0801 02019 80 p99ced vv 8cc901 Jo T JO c 09 v42314n228 y 40 oseyuds 8 juopuodop urxopod19 1e rurs r06 1r V O9PESSSIV Jo e Jo 1 09 pays o3r21pojy tyq4O oseyuds 8 juopuodop HQ VN 01 1e murs U 6rc90 c00 JWXII W U 6r8 0 200 INXII W T L8STTETOO XI IT OOPITETOO IWXII W STEEA OT IOSIMOUOD IXAIS9 007 60D 00 1104 9970AI OT Oud pusouos jxqiso vV SOLAOJd 0078007001204 OLTOIAX OT 234 jxquso 0069009101204 CLIOAX DT 234 pysouasy 1 59 C LLTOAd 00SLTODST0 04 LLILDVODVODVOVOVOLLDD JIIVVVDIDJDILVDLVVILLI VVOLOODLIODLLOVOLIOVVD LLOLOOOODVVOVVOLOOVV LLOIVOJIODOLLOIODIVOD VVOLVOVOVIOJIOOVVOOOO VIODVVVDLLODIODVVODV VOLLVOLVVODLLOVODODL 019608000 ULdOd SSD O8E80SP000 ULdOd 50 0 lt 9 lt 059100 1404 91LVDOD 056108100 ULdOd SLLVDOD 991 ETSSOAV V 6T LLNVV 9SELEOVV 8991 POVV LLOTFOVV 9STISAVV QOSSOUVV I8SSOUVV IETILNVV 60C LLINVV 5 lt 50 6SSSOUVV TSTISAVV TESSOAVV LZZILINVV JO 8 JO
21. V rifier qu il reste suffisamment de place sur le disque dur Vider la bouteille poubelle Nettoyer la surface dans le bouclier ionique avec 50 50 MeOH H20 Purger les canaux Rincer les t tes de pompe avec de l eau Millipore pur lentement l aide d une seringue au moins deux fois Purger avec de l eau Millipore pendant au moins 2 min Laver avec 98 de MeOH 0 4 mL min pendant au moins 30 min jusqu ce que la colonne semble propre en allumant de spectrom tre de masse Laver le spectrom tre de masse avec 50 50 MeOH H20 0 2 mL min pendant 10 20 min Le tube de transfert d ions doit tre lav tous les jours Laisser le spectrom tre de masse allum pendant 5 min suppl mentaire avec un flux nul Purger les canaux utilis s C et D Laisser la phase soluble se stabiliser avant de lancer une nouvelle analyse environ 10 min La pression de d part doit toujours tre la m me d pend de la colonne Les appareils peuvent tre teints apr s chaque analyse Ne pas oublier de lester les pompes binaires une fois par semaine Vider la bouteille poubelle de nouveau apr s avoir nettoy le syst me quand il n y a pas d analyse lancer le m me jour 146 Annexes Apr s avoir fini toutes les analyses nettoyer les analyses dans un bain ultrasons avec de l eau Millipore puis 5 min dans du MeOH Purger les canaux utilis s C et D en utilisant la bouteille d eau pure Millipore pendant 10 30
22. and phytotoxic ozone dose over a threshold of 1 nmol m s Significant stomatal closure was observed after treatment for the three genotypes Fig It was linked with a significant decrease in net assimilation Fig 4D F Robusta differed significantly once again with higher stomatal conductance values and thus higher net assimilation Ci values were stable throughout the experiment data not shown Stomatal conduc tance differences led to genotype differences in the amplitude variations of POD Fig 6 The effect of O3 on chlorophyll content in relation to POD was modelled as a linear adjustment A polynomial 5 10 R 0 9862 Relative of diameter POD mmol m 72 5 1 in Carpaccio Two adjustment models were used a linear regression lin solid line and a polynomial regression of order 2 pol dotted line with y 1 3327x 2 2598 and y 0 1407x 0 0596x 0 4639 as equations respectively Table 2 Evolution of the numbers of leaves of fallen leaves and of biomass means SE of the three poplar genotypes Carpaccio Cima and Robusta before TO and after being submitted to 120 nmol mol of or not control treatment for 18 days T18 Treatment and genotype effect are considered as significant when p 0 05 using the contrast method Stem biomass g Root biomass g Total biomass g Root shoot ratio Leaf biomass g Number of fallen leaves Number of
23. de limiter les pertes conomiques 132 ANNEXES 133 Annexes I PROTOCOLE DU DOSAGE DE L ASCORBATE ET DU GLUTATHION FORMES OXYDEES ET REDUITES EN MICROPLAQUE La vitamine C et le glutathion ne sont pas stables surtout temp rature ambiante par cons quent essayer de travailler rapidement et laisser les extraits sur la glace FAIRE LES PLAQUES LE MEME JOUR QUE L EXTRACTION et commencer par l ascorbate On peut cependant doser les extraits pour le glutathion pendant 1 semaine s ils sont stock s 80 C 1 roborack 21 blancs 3 extraits de r f rence extrait dont on connait la teneur en ascorbate et glutathion 72 chantillons Pour chaque roborack d extraction faire 2 plaques de dosage r plica technique Attention pour chaque produit pipet par le robot compter 15mL suppl mentaire de volume mort 5 EXTRACTION Peser dans des tubes micronics 1 mL 20 mg d chantillon Attention ne pas laisser l chantillon d congeler Plonger le tube dans l azote liquide vider l azote et faire la tare Mettre 20 mg de poudre et attendre la stabilisation replonger aussit t dans l azote Une fois les roboracks pr ts les stocker 80 C Mettre un petit film d azote dans une cuvette et y disposer le roborack D capsuler les tubes Attention ne pas d congeler les chantillons Ne pas h siter remettre le roborack sur l azote Une fois d capsul s mettre 20 C Mettre la centrifugeuse
24. effet de champ coupl des platines de cryofracture pour viter les mouvements d l ments solubles et mobiles De plus une tude de cin tique journali re compl te jour nuit permettrait de tester un ventuel effet d accumulation de certains ions sous stress ozone Enfin les flux d ions aboutissant des flux d eau l ozone pourrait aussi agir sur les aquaporines qui sont principalement des canaux plasmiques et tonoplastiques PIP TIP facilitant les mouvements d eau L expression des g nes de cette large famille de prot ines semble r gul e sous l effet de diff rentes contraintes abiotiques Cohen et al 2013 Elle pourrait donc l tre aussi par l ozone au sein des cellules de garde et ainsi modifier les vitesses d ouverture et de fermeture des stomates Pour am liorer les indicateurs de seuils de risque il faut aussi prendre en compte les processus de d toxication qui limitent les effets biologiques dus l ozone Pour tenir compte de la part d ozone d toxiqu e les indicateurs incluent un seuil de concentration en ozone dans leur calcul N anmoins les capacit s de d toxication variant d une esp ce une autre d un g notype un autre le seuil ne peut tre fixe Il faut donc int grer diff remment les processus de d toxication dans le calcul des indicateurs de seuils de risque L ascorbate apoplastique est le premier acteur de la d toxication agir sur les ROS produits par l ozone Certains mod les se
25. 4 C et pr parer le robot remplir une cuve avec de l acide m taphosphorique 5 Lancer le programme et apporter les roboracks quand n cessaire Le robot va ajouter chaque tube 400 uL d acide m taphosphorique 5 gard au pr alable dans la glace Il va ensuite agiter V rifier que l agitation tait suffisante Sinon capsuler les tubes de nouveaux pour les agiter manuellement 134 Annexes Centrifuger 10 min 4 C 4000 g puissance max Une fois centrifug s remettre dans la glace Solution pr parer Acide m taphosphorique 5 5 g dans 100 mL d eau se conserve 1 mois 4 C 6 DOSAGE ASCORBATE Placer 4 fois plus de plaques num rot es que de nombre de roboracks d extraits sur le robot Pr parer une cuvette de NEM 0 5 une cuvette de DTT 5 mM et une cuvette de tampon phosphate 0 4 M pH7 4 Remplacer 6 blancs par une gamme talon Ascorbate nmol dans le puit 0 5 10 15 20 30 Ascorbate 5 mM uL 0 25 50 75 100 150 Acide m taphosphorique 5 uL 500 475 450 425 400 350 Le robot va d poser 20 uL d extrait dans chaque plaque Il va ajouter 20 uL de tampon phosphate dans la moiti des plaques et 20 uL de DTT dans l autre moiti Commence par une plaque Tampon puis une DTT et ainsi de suite Mettre les plaques incuber 20 min 37 C Pendant ce temps pr parer le mix A B Remplacer la cuve de tampon par une c
26. 8 AT AVPD ASE 1 exp k 86 38 AT AVPD M Vs M Vp genae Fai Be gemm A Pe FUP 1 09 expo St 02 9 if VPD gt exp bo m 8 73 Soum if VPD exp h E m In VPD b eise n ea T T TET Taia fom r exp 222 Gm mee GFAC P ETP Bour gt m m X E Ese zd if Yi 1 MPa Samui A gi a 1100 67 6 8 ges gena 1k exp 6 BL famm frema min l ks DOY 8 8i a exp ABA B exp 4 ABA anse 2 1 58 J b Re A Eie 356 Re expl B1A8AD m LEE 5 MM VED F Kan VA Y 8 architecture description fers l exp s E P Y 1 X cg ee CELA KL Kam if Pen lt M Y Y 4 Kus Kaag re if Mon gt P gt w if Pin gt U CH T Poor R x P P g z Ak AW ay E k dan B exp 5 egi 8 4 gt 0 g 3 mP g VPD KAYV Y amp AT P Synth se bibliographique 2 MODELES DE CONDUCTANCE STOMATIQUE Comme la r gulation de la conductance stomatique joue un r le cl dans l adaptation aux variations des conditions environnementales notamment en situation de stress il apparait d une importance capitale d am liorer notre compr hension de ces m canismes afin de pouvoir mod
27. Les ADNc ainsi obtenus ont ensuite t purifi s avec le kit DNA Micro Qiagen et quantifi s par Nanodrop Toutes les matrices ont t dilu es pour atteindre une concentration de 5 ng uL 36 Mat riel et M thodes DESSIN DES AMORCES Le dessin des amorces s est fait de la m me mani re que d crit pr c demment mais pour des cibles diff rentes Ont t choisis comme cibles des g nes codant pour les canaux plasmiques ou vacuolaires des stomates connus pour jouer un r le dans les mouvements stomatiques ainsi que les r cepteurs de la lumi re bleue phytotropines et du CO les anhydrases carboniques La sp cificit des couples d amorces a t test e par qPCR sur de l ADNc de chacun des 3 g notypes tudi s ainsi que par s quengage de l amplicon par l entreprise Beckman coulter genomic PCR EN TEMPS REEL QPCR La PCR en temps r el a t r alis e par le syst me Mx3000P QPCR Agilent technologies en suivant les recommandations du fabricant pour le programme 10 min 95 C 40 cycles de 5 s 95 C et 20 s 58 ou 60 C en fonction des amorces suivie d une courbe de dissociation pour v rifier la sp cificit de l amplification Le MIX r actionnel contenait 10 uL de Brillant III Ultrafast SYBR green QPCR Master mix 0 3 uL de ROX dilu au 500 Agilent Technologies 250 nM d amorces sp cifiques et 2 2uL de cDNA 5 ng uL pour un volume total de 20 uL par puit Des contr les NTC
28. Measurements began on day 4 after fumigation started and stopped the day after fumigation ended day 18 Sampling procedure Samples were taken on ML along the 17 day long exposure on days 2 4 7 11 and 17 after the beginning of fumigation Two leaves located next to each other were sampled per tree and per chamber Midribs and petioles were cut off from the foliar limb using a razor blade Leaf samples were collected in aluminum paper rapidly frozen in liquid nitrogen and immediately stored at 80 C Protein extraction and quantification A leaf powder extract 300mg and 1096 w w polyvinylpolypyrrolidone were mixed with 3 5ml of extraction buffer containing HEPES KOH pH 7 5 100 mM PVP 25 6 m m PEG 20 7 m m MgCl 5 mM EGTA 5 mM glycerol 10 v v and 5 v v protease inhibitor mixture Sigma Aldrich P9599 The crude extracts were then centrifuged at 36 000 g and at 4 C The supernatants were filtered through Sephadex G25 columns Pharmacia PD 10 Orsay France accord ing to the manufacturer s instructions The elution buffer contained HEPES KOH pH 7 5 glycerol 1096 v v MgCl 5mM DTT 2 mM The eluted volume was homogenized and stored at 80 C for further analysis Total soluble proteins were determined according to Wildhagen et al 2010 a protocol adapted for microplate spectrophotometry Ten microliter duplicates of protein extracts were pipetted into the wells of a 96 well plate and mixe
29. de d fense D toxication et processus de r paration Figure 27 Absorption d ozone et effets biologiques Tausz et al 2007 Objectifs de recherche 2 POD YL max Fetj Y 0 At Fst repr sentant les flux d ozone stomatique nmol m s En pr sence d ozone les plantes disposent de deux moyens de r sistance la r gulation de la conductance stomatique pour limiter l entr e d ozone dans les feuilles et un syst me de d toxication complexe pour liminer l ozone et les ROS form s L valuation des seuils de risque l ozone doit prendre en compte les diff rentes barri res et d fenses des v g taux et ne pas seulement tenir compte des concentrations en ozone ext rieures Karlsson et al 2007 Il reste maintenant prendre en compte les aspects de d toxication des feuilles en plus des flux entrants d ozone afin d expliquer la variabilit inter et intra sp cifique due ces deux facteurs 2 FLUX EFFECTIF D OZONE On peut utiliser la notion de flux effectif d ozone balance entre les flux stomatiques et l intensit de la d toxication cellulaire Musselman et al 2006 Une chaine de 3 ou 4 v nements est d crite suivant les auteurs Dizengremel et 2009 Tausz et al 2007 Figure 27 e concentration du polluant dans l atmosph re conditions environnementales e d p t de l ozone sur les surfaces sols feuilles exposition e degr d intrusion de l ozone dans les feuilles trave
30. 15 15 17 17 2000 50000 0 0 r CARPACCIO ROBUSTA CARPACCIO ROBUSTA CARPACCIO ROBUSTA CARPACCIO ROBUSTA Asparagine ASN Aspartic acid ASP AMBIENT OZONE AMBIENT OZONE 80000 3000 quantity 8 1000 CARPACCIO CIMA Cysteine CYS T T ROBUSTA CARPACCIO CIMA ROBUSTA AMBIENT OZONE 50 quantity E 50 0 T T a T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day 2 4 11 15 17 sampling dates day 2 j 4 11 15 17 quantity 60000 40000 20000 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Glutamic_acid GLU AMBIENT OZONE 80000 60000 40000 20000 0 antity T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day 2 4 11 15 17 sampling dates day 2 E 4 11 15 17 Glutamine GLN AMBIENT OZONE 20000 15000 sampling dates day 2 Lp 4 10000 11 15 17 5000 CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Histidine HIS AMBIENT OZONE 600 sampling dates day 2 400 _ t 4 3 2 11 L il 15 17 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Glycine_GLY AMBIENT OZONE 600 500 400 1 2 3 5 300 200 100 0 CARPACCI
31. 3 5 10004 11 o n o 15 15 50000 500 o 4 7 Carpaccio Robusta Carpaccio Robusta Carpaccio Robusta Carpaccio Robusta Malate AMBIANT OZONE AMBIANT OZONE 3000 4 150000 4 2000 gt Temps Temps jour lour 2 2 2 100000 4 4 5 5 11 n 15 15 17 17 1000 4 50000 4 4 0 Carpaccio Robusta Carpaccio Cima Robusta Carpaccio Cima Robusta Carpaccio Cima Robusta Figure 51 Concentration en A citrate B succinate C fumarate D malate dans les feuilles de trois g notypes de peupliers Carpaccio Cima et Robusta apr s 2 4 11 15 et 17 jours de traitement ozone ou ambiant Fructose AMBIANT 120000 4 OZONE 90000 4 Temps jour 2 60000 4 11 17 30000 Carpaccio Cima Robusta Carpaccio Cima Robusta Quantit Glucose AMBIANT OZONE 05 Temps jour 2 L 4 054 3 4 5 11 17 2 05 han 0e 00 i B a B Carpaccio Cima Robusta Carpaccio Robusta Raffinose mu AMBIANT OZONE 200004 150004 Temps 2 2 t 4 S 10000 4 3 10000 11 17 5000 4 1 Carpaccio Cima Robusta Carpaccio Robusta Galactinol AMBIANT OZONE 6000 rh 4000 Temps
32. Boccalandro HE Giordano CV Ploschuk EL Piccoli PN Bottini R Casal JJ Phototropins but not cryptochromes mediate the blue light specific promotion of stomatal conductance while both enhance photosynthesis and transpiration under full sunlight Plant physiology 2012 158 1475 1484 157 R f rences Bienert GP Moller ALB Kristiansen KA Schulz A Moller IM Schjoerring JK Jahn TP Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes Journal of Biological Chemistry 2007 282 1183 1192 Brunner AM Busov VB Strauss SH Poplar genome sequence functional genomics in an ecologically dominant plant species Trends in Plant Science 2004 9 49 56 Bunce JA Effects of humidity on short term responses of stomatal conductance to an increase in carbon dioxide concentration Plant Cell amp Environment 1998 21 115 120 Caban M Pireaux J C Leger E Weber E Dizengremel P Pollet B Lapierre C Condensed lignins are synthesized in poplar leaves exposed to ozone Plant physiology 2004 134 586 594 Cape JN Surface ozone concentrations and ecosystem health Past trends and a guide to future projections Science of the Total Environment 2008 400 257 269 Castagna A Ranieri A Detoxification and repair process of ozone injury From O 3 uptake to gene expression adjustment Environmental Pollution 2009 157 1461 1469 Cohen D Bogeat Triboulot M B Vialet Chabrand S Merret R
33. Ishida H Nishimori Y Sugisawa M Makino A Mae T 1997 The large subunit of ribulose 1 5 bisphosphate carboxylase oxygenase is fragmented into 37 kDa and 16 kDa polypeptides by active oxygen in the lysates of chloroplasts from primary leaves of wheat Plant Cell Physiol 38 471 479 Junqua M Biolley JP Pie S Kanoun M Durand R Goulas P 2000 In vivo occurrence of carbonyl residues in Phaseolus vulgaris proteins as a direct consequence of a chronic ozone stress Plant Physiol Biochem 38 853 861 Karnosky DF Gagnon ZE Dickson RE Coleman MD Lee EH Isebrands JG 1996 Changes in growth leaf abscission and biomass associated with seasonal tropospheric ozone exposures of Populus tremuloides clones and seedlings Can J Forest Res 26 23 37 Karnosky DF Pregitzer KS Zak DR Kubisce ME Hendrey GR Weinstein D Nosal M Percy KE 2005 Scaling ozone responses of forest trees to the ecosystem level in a changing climate Plant Cell Environ 28 965 981 Karnosky D Skelly J Percy K Chappelka A 2007 Perspectives regarding 50 years of research on effects of tropospheric ozone air pollution on US forests Environ Pollut 147 489 506 Landolt W Gunthardt Goerg MS Pfenninger I Einig W Hampp R Maurer S Matyssek R 1997 Effect of fertilization on ozone induced changes in the metabolism of birch Betula pendula leaves New Phytol 137 389 397 Maier A Zell MB Maurino VG 2011 Malate decarboxylases evolution and roles of NAD P
34. Les aliquots de surnageants qui seront utilizes pour les analyses de composes ph noliques et les analyses en GC doivent tre compl tement vapor s sec le jour m me de l extraction Prendre deux aliquots de 150 uL pour les analyses en GC deux aliquots de 150 uL pour les analyses d acides amin s et deux aliquots de 500 uL pour les analyses de compos s ph noliques V rifier la temp rature d vaporation 30 C Il est important qu aucun r sidu d eau ne reste pour les analyses en GC 141 Annexes Les chantillons sont stock s dans des flacons en verre D gazer les chantillons avec un jet d azote avec la fermeture du flacon Les chantillons peuvent tre stock s 70 C jusqu aux analyses mais pas plus d un mois pour les analyses en GC 2 ANALYSE DES SUCRES Apr s avoir sorti les chantillons du cong lateur Laisser temp rature ambiante pendant une heure avec d ouvrir les flacons Ajouter 20 uL de standard de temps de r tention C7 C40 alcanes satur s dans de l hexane Sigma Ass cher vaporation rapide Ajouter 80 uL de MAHC 20 mg mL de methoxyamine hydrochloride dans de la pyridine faire le m lange chaque jour laisser d river 90 min 37 C Ajouter80uL de MSTFA N methyl N trimethylsilyl trifluoro acetamide laisser d river60 min 37 C Transf rer les chantillons dans des inserts et lancer l analyse Notes Les chantillons sont extraits et analys s dans un ordre
35. ME isoforms in species performing C4 and C3 photosynthesis J Exp Bot 62 3061 3069 Matyssek R Innes JL 1999 Ozone a risk factor for trees and forests in Europe Water Air Soil Poll 116 199 226 Matyssek R Wieser G Nunn AJ Kozovits AR Reiter IM Heerdt C Winkler JB Baumgarten M Haberle K H Grams TEE Werner H Fabian P Havranek WM 2004 Comparison between AOT40 and ozone uptake in forest trees of different species age and site conditions Atmos Environ 38 2271 2281 Matyssek R Le Thiec D L w M Dizengremel P Nunn AJ Haberle KH 2006 Interaction between drought stress and stress in forest trees Plant Biol 8 11 17 McLaughlin SB Nosal M Wullschleger SD Sun G 2007 Interactive effects of ozone and climate on tree growth and water use in a southern Appalachian forest in the USA New Phytol 174 109 124 Mhamdi A Mauve C Houda G Saindrenan P Hodges M Noctor G 2010 Cytosolic NADP dependent isocitrate dehydrogenase contributes to redox homeostasis and Physiol Plant 148 2013 the regulation of pathogen responses in Arabidopsis leaves Plant Cell Environ 33 1112 1123 M ller IM Rasmusson AG 1998 The role of NADP in the mitochondrial matrix Trends Plant Sci 3 21 27 Nemoto Y Sasakuma T 2000 Specific expression of glucose 6 phosphate dehydrogenase G6PDH gene by salt stress in wheat Triticum aestivum L Plant Sci 158 53 60 Noctor G 2006 Metabolic signalling in defence and stress the ce
36. OZONE 300 250 0 8 quantity 8 8 T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA M ROBUSTA sampling dates day 2 4 11 15 17 sampling dates day 2 4 11 15 17 sampling dates day 2 4 11 15 17 Proline PRO AMBIENT OZONE 1500 1000 00 quantity sampling dates day 2 El 4 11 15 17 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Serine SER AMBIENT OZONE ex 6000 4000 2000 0 T T CARPACCIO CIMA ROBUSTA quantity Threoni sampling dates day 2 Ej 4 11 15 17 T CARPACCIO CIMA ROBUSTA ne_THR AMBIENT OZONE 4000 3000 antity sampling dates day 2 4 11 15 17 22000 1000 T T CARPACCIO CIMA ROBUSTA T CARPACCIO CIMA ROBUSTA Tryptophan_TRP AMBIENT OZONE 400 quantity 8 K H T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Valine VAL AMBIENT OZONE 3000 2500 2000 quantity 1000 500 T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day sampling dates day 2 4 11 15 17 quantity Tyrosine_TYR AMBIENT OZONE 50 4 CARPACCIO CIMA sampling dates day T T ROBUSTA CARPACCIO CIMA ROBUSTA
37. PROTOCOLE DE DOSAGE DES ARNS EN RIBOGREEN Pr parer le tampon TE 1 mL de TE20X 19 mL d eau Pr parer la solution Ribogreen en la diluant 200 fois dans du tampon TE Pr parer la solution m re d ARN pour la gamme talon 1 aliquot de 2 uL 1 ug uL 998 uL de TE dilution au 500X Si n cessaire diluer les chantillons pour avoir des concentrations comprises entre 10 et 100 ng L Sur une plaque faire 3 r plicas de gamme 0 5 10 20 40 60 80 100 ng 100uL final dans le puit D poser 50 uL d chantillon avec 50 uL de solution Ribogreen M langer et centrifuger quelques secondes 2500 g Lire la plaque le Ribogreen est quivalent au r glage Sybrgreen sur la machine 150 Annexes VIII PROTOCOLE DE RETROTRANSCRIPTION AVEC LE KIT ISCRIPT ET L ALIEN Effectuer les dilutions des chantillons pour avoir 10 uL 70 ng uL D naturer 5 min 70 C et conserver dans la glace Proc der la dilution de la solution m re en suivant les tapes suivantes M langer 1 uL d alien stock avec 59 uL d eau Concentration finale 5 10 copies uL En prendre 10 pL et ajouter 90 uL d eau Recommencer avec la nouvelle dilution 3 fois pour avoir une concentration finale de 5 10 copies uL Faire un mix avec 1 uL de RTase 4 uL de buffer 5X 3 uL d eau et 2 uL d alien dilu Mettre 10 uL de MIX dans chaque chantillon Vortexer et centrifuger quelques secondes 2500 g Proc der un
38. amp Giersch 2004 Lohammer er al 1980 Monteith 1995 Monteith 1995 Delwiche amp Cooke 1977 Dewar 1995 Ball er af 1987 Aphalo amp Jarvis 1993 Leuning 1990 Leuning 1995 Supply function and biochemical model of Farquhar amp Wong 1984 Jarvis amp Davies 1998 Stewart 1988 Misson et al 2004 MacFarlane er al 2004 Ogle amp Reynolds 2002 Tenhunen er 1990 Baldocchi 1997 Sala amp Tenhunen 1996 Misson er al 2004 Wang amp Leuning 1998 Van Wijk er al 2000 Uddling er ai 2005 Tardieu amp Davies 1993 Gutschick amp Simonncau 2002 Oren eral 1999 Tyree amp Sperry 1988 Sperry er al 1998 er al 2003 Jones amp Sutherland 1991 Cochard er ai 1996a Lu er al 1996 Dewar 2002 Gao er ai 2002 Buckley er al 2003 T AVPD FA C AUA amp amp AVPD Bie Bess MiN AQ f VPD 1 8 pm jt Bs VPD g a l b E 1 1 Le f Pe Pust a set of diflential equations integrated P et P water potentials osmotic potentials water vapour resistances and water fluxes B x P P g zl r AW 8 8 Bo Ac B eges L2 VPD k ks T VPD H 8 G T ten UT 8 AG es 3 FES fiam Vos Ri 0 Ha Ti re E Vous Ty Ri R BP s G G sE
39. duite qui est observ e 75 R sultats e l expression des g nes impliqu s dans les voies de biosynth se de l ascorbate est globalement induite par l ozone et la r gulation semble s effectuer gr ce au g ne VTC2 principalement l ozone entra ne une augmentation des concentrations en glutathion et le ratio redox est maintenu Cette augmentation est plus intense chez le g notype le plus r sistant l expression des g nes impliqu s dans les voies de biosynth se du glutathion est globalement induite par l ozone et la r gulation semble s effectuer gr ce aux g nes codant pour les enzymes GSHI et GSH2 principalement Chez le g notype le plus r sistant Carpaccio une tr s forte induction de l expression d un g ne codant pour la thr onine aldolase pourrait permettre la synth se de glycine partir de la thr onine e ozone provoque une remobilisation des acides amin s vers la synth se d antioxydants et de nouvelles sources d nergie Nous observons une hausse de la cyst ine disponible pour la synth se de glutathion une hausse des acides amin s aromatiques utilis s pour la synth se des ph nols des flavonoides et une hausse de la putrescine acteur de la d toxication induisant une augmentation de l activit de certaines enzymes antioxydantes surtout chez le g notype le plus r sistant Ces r sultats ont t soumis dans Plos One 76 R sultats Ozone effects on ascorbate and glutathione biosy
40. en suivant les recommandations du fabricant pour le programme 10 min 95 C 40 cycles de 5 s 95 C et 20 s 58 ou 60 C en fonction des amorces suivie d une courbe de dissociation pour v rifier la sp cificit de l amplification Le MIX r actionnel contenait 10 uL de Brillant III Ultrafast SYBR green QPCR Master mix 0 3 uL de ROX dilu au 500 Agilent Technologies 250 nM d amorces sp cifiques et 2 uL de cDNA dilu au 1 5 pour un volume total de 20 uL par puit Des contr les NTC ont t ajout s chaque plaque Des gammes ont t r alis es en triplica pour chacun des g nes et chacun des g notypes L efficience de la PCR a ensuite t calcul e gr ce elles pour chaque g ne et chaque g notype Les valeurs de Ct ont t caract ris es pour un m me seuil L expression des g nes a ensuite t normalis e gr ce 2 g nes de r f rence alien et UBL et deux g nes d int r t stables GOGAT6 et VTC21 d termin s par le logiciel GeneNorm 3 ANALYSES TRANSCRIPTOMIQUES A PARTIR DE STOMATES MICRODISSEQUES PREPARATION DES MATRICES L extraction des ARNs totaux a t r alis e partir d chantillons de 500 stomates microdiss qu s en suivant les instructions du fabricant avec le kit Qiagen Micro Plus Qiagen Les ARNs ont t par la suite retrotranscrits et les ADNc amplifi s en suivant les recommandations du fabricant avec le kit TransPlex Complete Whole Transcriptome Amplification Sigma
41. explain ozone tolerance among poplar genotypes through a comprehensive study of interactions between gene expression and metabolites 80 R sultats Materials and Methods Ethics statement We declare that cuttings have been bought from National Forest State nursery of Gu men Penfao where poplar genotypes and provenances are conserved France Loire Atlantique 47 37 46 N 1 53 33 W 20 m a s l The master certificate has the number F52 11B004 The plant health passport has the number SPV F PL00736 Plant culture and exposure conditions Three Euramerican hybrid poplar genotypes Populus deltoides x Populus nigra Carpaccio Cima and Robusta differing in ozone sensitivity based on foliar injuries leaf fall and total biomass losses were studied The plant material was grown as described in 30 Briefly rooted cuttings were planted and kept in growth chambers situated at Lorraine University for 5 weeks at 23 C 21 C day night with 14 h light period 250 300 umol m s photon flux at leaf height At the end of this period 40 individuals of each genotype were divided in eight phytotronic chambers with the same environmental conditions The plants were let to acclimate to phytotronic chambers for seven days Four phytotronic chambers were used for each treatment during 17 days 1 ambient treatment with charcoal filtered air concentration lt 4 nmol and 2 ozone treatment 120 nmol mol
42. for 13 h from 09 00 to 22 00 Fumigation started one hour after the beginning of the photoperiod and ran until its end Sampling and storage After 2 4 11 15 and 17 days from the beginning of the fumigation the 10 and 11 leaves from the top were sampled These leaves were mature at the start of the fumigation 81 R sultats Each time different plants were used for sampling The leaves were cut in half by removing the midrib and were immediately flash frozen in liquid nitrogen For RNA extraction ascorbate and glutathione quantification half of each leaf was crushed into powder in liquid and stored at 80 C For metabolomic analyses the other half was freeze dried at 40 C for 40 h at a pressure of 10 Pa in a FreeZone Plus freeze dryer Labconco Kansas City Missouri and then the temperature was turned back progressively to 20 C within 10 h Freeze dried samples were crushed with TissueLyser Qiagen Helsinki Finland 3x15 s in Safe Lock Eppendorf tube and stored at 80 C Leaf fall and biomass measurement At the end of the experiment the number of fallen leaves was count and all the plants were harvested divided into stems leaves and roots and oven dried 60 C until constant weight was reached and then weighed Glutathione and ascorbate extraction and analysis Ascorbate and glutathione were measured at 2 4 11 and 17 days They were extracted from 20 mg of fresh leaf powder in 400 uL of metaph
43. il ne reste que des morceaux d piderme Figure 45 C et D 2 DETERMINATION DE LA DENSITE STOMATIQUE ET DE LA TAILLE DES STOMATES MICROSCOPIE ELECTRONIQUE A BALAYAGE PRINCIPE DE L APPAREIL Le Microscope Electronique Balayage MEB permet d obtenir des images en haute r solution du relief de la surface d un chantillon Un faisceau d lectrons vient balayer la surface de l chantillon qui va alors mettre des particules lectrons secondaires lectrons r trodiffus s rayons X Figure 46 qui sont collect es par des d tecteurs Les informations collect es par les d tecteurs d lectrons permettent alors de reconstruire une image en trois dimensions et ceux collectant les rayons X permettent de d terminer la composition min rale de l chantillon En effet les nergies des rayons X mis sont sp cifiques des l ments min raux constituant l chantillon UTILISATION Trois chantillons de feuilles par arbre pr lev s l emporte pi ce ont t lyophilis s Ils ont ensuite t d coup s en deux et d pos s sur portoirs de mani re avoir un demi disque laissant voir la face sup rieure et l autre la face inf rieure de la feuille Les portoirs ont ensuite t rendus conducteurs aux lectrons par d p t d une couche de carbone pulv risation cathodique Des observations au MEB ont permis de d terminer gr ce l utilisation du d tecteur d lectrons r trodiffus s BSD la densit stoma
44. liser le mieux possible cet l ment cl De par son importance le calcul de la conductance stomatique a t largement tudi au cours des 50 derni res ann es Il existe un nombre important de mod les essentiellement empiriques tr s peu tant m canistes Tableau 1 La majorit est toutefois semi empirique associant des fonctions empiriques des hypoth ses physiologiques Damour et al 2010 Certains mod les g n raux int grent les principaux facteurs environnementaux r gulant la conductance stomatique gr ce des fonctions empiriques param trer en fonction des situations C est le cas du mod le multiplicatif de Jarvis 1976 et du mod le de White et al 1999 qui mettent en relation les diff rentes fonctions du mod le par rapport la conductance maximale puis celui de Noe et Giersch 2004 qui pond re l influence des fonctions environnementales en ne prenant que le minimum de ces limitations D autres mod les calculent la r ponse de la conductance stomatique non pas en fonction des param tres environnementaux directement mais en fonction de facteurs physiologiques comme la photosynth se C est le cas du mod le de Ball Woodrow et Berry 1987 qui a ensuite t am lior par Aphalo et Jarvis 1993 pour prendre en compte de mani re explicite la d pendance de la conductance stomatique l humidit de l air et la temp rature D autres modifications du mod le ont t faites afin d en am liorer les r
45. panneaux contreplaqu s Le sciage voligeage sous toitures palettes literie Les panneaux d riv s fibres agglom r s La p te papier Le bois nergie taillis courte rotation ou tr s courte rotation TCR TTCR e La construction C est aussi une essence de pleine lumi re tr s exigeante en eau et en l ments nutritifs On le retrouve donc fr quemment en ripisylves et en fond de vallon Afin d avoir la meilleure production possible diff rents cultivars vari t s obtenues en culture sont utilis s en fonction des stations sol climat de son comportement face aux maladies et au vent de sa facilit de conduite de ses qualit s de bois La majorit des plantations fran aises sont 22 Tableau 5 Les surfaces en ha de peuplier de production de bois en France et par r gion Peupleraies en plein Peupleraies en plein R gions Source Source SCEES Teruti Lucas 2006 Cadastre 2003 Alsace 2 000 2 176 Aquitaine 17 200 24 974 Auvergne 1 400 3 329 Basse Normandie 4 700 4 586 Bourgogne 11 900 14 442 Bretagne 6 100 8 235 Centre 22 600 22 814 Champagne Ardenne 21 100 26 864 Corse 0 0 Franche Comt 2 900 4 134 Haute Normandie 600 890 Ile de France 2 400 9 576 Languedoc Roussillon 900 1 143 Limousin 900 329 Lorraine 3 600 3 540 Midi Pyr n es 12 500 14 824 Nord Pas de Calais 9 000 12 863 Pays de la Loire 18 900 22 147 Picardie 23 900 32 108 Poitou Charentes 13 000 12
46. tation Soutenance publique le 19 avril 2013 Membres du jury Pr sident M Yves JOLIVET Professeur Universit de Lorraine Nancy Rapporteurs M Daniel LAFFRAY Professeur Universit Paris Est Cr teil Paris Mme Nathalie LEONHARDT Charg e de Recherche CEA Cadarache Examinateurs M Pierre DIZENGREMEL Professeur m rite Universit de Lorraine Nancy M Markku KEINANEN Senior assistant University of Eastern Finland Joensuu M Didier LE THIEC Directeur de Recherche INRA Nancy Directeur de th se Mrs Elina OKSANEN Professor University of Eastern Finland Joensuu UMR1137 INRA UL Ecologie et Ecophysiologie Foresti res Facult des Sciences amp Technologies 54500 Vandceuvre l s Nancy A mes parents qui m ont toujours soutenue et aim e c est gr ce vous que je suis arriv e jusque l soyez fiers de vous A Fabien sans qui cette th se ne serait pas la m me merci pour tout ce que tu m apportes chaque jour A mon oncle et parrain qui m a appris que la vie vaut le coup qu il ne faut jamais abandonner qu il faut se battre pour le bonheur qui nous entoure pour la vie tout simplement Regarde moi je suis heureuse Dans la vie tout n est qu une question de volont REMERCIEMENTS Je remercie l ANR Vulnoz et la R gion Lorraine pour avoir cofinanc cette th se ainsi que l INRA l IFR 110 EFABA le COST Action FP0903 the University of Eastern Finland la plate forme M tabolome Fluxom
47. 0 100 1 0 1200 3 0 Initial 25 0 3 25 0 3 25 0 3 25 0 4 Temp rature C Final 25 0 3 25 0 3 25 0 3 25 0 4 Lumi re Tnitial 0 0 200 30 5 0 0 0 800 30 5 0 rouge bleue pmol m s Final 0 30 1 800 30 5 0 0 0 800 30 5 0 VPD Initial 0 8 0 2 0 8 0 2 08202 0 8 0 2 kPa Final 0 8 0 2 0 8 0 2 3 0 0 3 0 8 0 2 Figure 43 Plateau robotis de ph notypage enzymatique haut d bit Mat riel et M thodes Chaque semaine nous avons mesur la r ponse de la conductance stomatique et de la photosynth se une variation de lumi re bleue de lumi re rouge de concentration en CO et de VPD dans les conditions r sum es dans le Tableau 7 Afin de s affranchir des effets dus la photosynth se la r ponse la lumi re bleue a t mesur e dans l obscurit tout comme la r ponse au VPD afin de permettre une fermeture des stomates plus rapide Les mesures r alis es l obscurit taient faite le matin pour que les arbres ne soient pas mis en pr sence de lumi re avant la mesure et les autres mesures taient faites l apr s midi V ANALYSES METABOLOMIQUES PRINCIPES DES ANALYSES Le COST Action FP0903 a financ une mission scientifique de 3 mois me permettant de partir dans le laboratoire du Pr Elina Oksanen University of Eastern Finland Joensuu afin de proc der des analyses m tabolomiques sur les chantillons de feuilles enti res J ai dos les compos s ph noliques
48. 17 days Mean SE Genotype effect tested by a Student test and considered significant if p lt 0 05 R sultats a 2 fold increase after 11 days of treatment Figure 2A The concentration of DHA increased significantly in Robusta in response to ozone with the strongest response of 1 8 fold at the last sampling date in contrast to Cima that was characterized by a decreased DHA concentration after 11 days of ozone treatment whereas no effect was observed in Carpaccio Figure 2B The concentration of total ascorbate increased significantly in all three genotypes in response to ozone with the strongest response at the last sampling date with a 1 4 fold 1 3 fold and 1 7 fold increase in Carpaccio Cima and Robusta respectively Figure 2C As a result of the lack of increase in the concentration of DHA in Carpaccio and Cima an increase in the ascorbate redox ratio took place in response to ozone with a 1 6 fold and 1 7 fold increase respectively at day 11 Figure 2D For detailed statistical analyses see Supporting Information Table S1 Effects of ozone on glutathione concentrations The constitutive concentration of total glutathione was significantly higher in Carpaccio than in Cima or Robusta Table 2 The concentration of GSH increased progressively and significantly in all three genotypes in response to ozone with the strongest response in Carpaccio with a 2 6 fold increase compared to a 1 6 fold and 2 2 fold increase in Cima
49. 50 5 17 z 0 0 T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GlyA23 AMBIENT OZONE 10 c08 9 sampling dates 2 5 2 Qos o 4 o 11 04 15 5 17 02 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GOGAT12 AMBIENT OZONE sampling dates day o Normalized expression o a 3 oo Coal eli i T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GOGAT16 AMBIENT OZONE 124 T sampling dates day 2 4 11 15 17 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Normalized expression o o o o 1 gt d 1 GS5 AMBIENT OZONE sampling dates day 2 4 11 15 17 Normalized expression o o o o o o N gt o T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o Normalized expression o o Normalized expression o a 0 0 o o Normalized expression o o gt 0 0 o o Normalized expression o o 0 0 o o GS7 AMBIENT OZONE sampling dates day T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GS10 I um m dates T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA OZONE GS15 AMBIENT IF T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day
50. 64 79 82 Pandey S Zhang W Assmann SM Roles of ion channels and transporters in guard cell signal transduction FEBS 2007 581 2325 2336 Paoletti E Ozone slows stomatal response to light and leaf wounding in a Mediterranean evergreen broadleaf Arbutus unedo Environmental Pollution 2005 134 439 445 Paoletti E Grulke NE Ozone exposure and stomatal sluggishness in different plant physiognomic classes Environmental Pollution 2010 158 2664 2671 Pleijel H Danielsson H Vandermeiren K Blum C Colls J K Stomatal conductance and ozone exposure in relation to potato tuber yield Tresults from the European CHIP programme European Journal of Agronomy 2002 17 303 317 Renaut J Bohler S Hausman JF Hoffmann L Sergeant K Ashan N Jolivet Y Dizengremel P The impact of atmospheric composition on plants a case study of ozone and poplar Mass Spectrometry Reviews 2009 28 495 516 165 R f rences Rhoads DM Umbach AL Subbaiah CC Siedow JN Mitochondrial reactive oxygen species Contribution to oxidative stress and interorganellar signaling Plant physiology 2006 141 357 366 Rice Evans CA Miller J Paganga G Antioxidant properties of phenolic compounds Trends in Plant Science 1997 2 152 159 Rosa M Prado C Podazza G Interdonato R Gonzalez JA Hilal M Prado FE Soluble sugars Metabolism sensing and abiotic stress Plant Signaling amp Behavior 2009 4 388 393 S
51. 8 see also Supporting Information Figure S3 for complete dataset Asp which is one of the most abundant amino acids decreased strongly thus there was a strong decrease in total amino acids content Figure 9 Supporting Information Figure S3 Discussion Reduced ascorbate and glutathione availability may be linked to ozone tolerance in poplar Each compartment controls the redox signaling independently by its own antioxidative capacities 39 Ozone generates ROS in apoplastic space before to spread in other cellular compartments Ascorbate is present in various cell compartments including apoplast where it is limited in O5 scavenging because of low levels of its reduced form 37 38 Transport of ascorbate in the cell is necessary for its regeneration thus we focus on the overall response of ascorbate Carpaccio the least sensitive genotype to ozone 23 30 had the highest total leaf ascorbate concentration but not higher constitutive ASA level than Cima and Robusta showing that constitutive ASA level is insufficient for explaining ozone resistance in poplar 40 21 In response to ozone Carpaccio and Cima showed a strong increase in total ascorbate due to a strong increase in ASA In contrast in Robusta the most sensitive to ozone the total ascorbate concentration increased due to an increase of DHA concentration while ASA concentration remained stable Thus Robusta produced de novo ascorbate in response to ozone but
52. A B C D Figure 8 Exemples de sympt mes foliaires sur des feuilles de peupliers soumis un traitement l ozone Photos F Spicher A D coloration feuille de droite par rapport une feuille t moin feuille de gauche B et C n croses plus ou moins importantes entre les nervures de feuilles de peuplier et D marbrures sur des aiguilles de pin d Alep Photo V Calatayud 30 20 10 0 10 20 Total biomass i 406 97 ppb Leaf biomass 0H H 336 95 ppb 1 Leaf arca M5 86ppb Aboveground woody biomass HOH i 279 97 ppb 2 Shoot biomass FCH H 370 100 ppb Root biomass HOH h 353 101 ppb 2 1 s Root to shoot ratio HH 371 101 ppb 2 1 Height HOH 225 95 ppb Diameter HH 82 81 ppb 4 4 30 20 10 0 10 20 Percent change from charcoal filtered control Figure 9 Variation en pourcentage de la biomasse totale biomasse foliaire surface foliaire par unit de surface biomasse ligneuse aerienne biomasse a rienne biomasse racinaire et ratio feuille racine hauteur et diam tre de tous les arbres expos s des concentrations lev es d ozone par rapport aux t moins expos s l air filtr par charbon actif Les symboles sont encadr s par des intervalles de confiance 9596 les degr s de libert et les moyennes des concentrations l ozone sont donn es le long de l axe Y Wittig et al 2009 Synth se bibliographique EFFETS SUR LA
53. B 100 O x T 60 T ns 40 TA 0 3 10 15 20 160 4 C 140 120 100 4 Robusta Height cm Time day Fig 2 Effects of treatment W Ozone Ambient Ozone 13 4 D 1D 5 10 2 go 8 7 4 6 TT T TT T 0 5 10 15 20 g E 5 5 E S a 20 8 amp 5 E S a 20 Time day Ambient on height cm A B and C and stem diameter mm D E and F of Carpaccio A and D Cima B and E and Robusta C and F ns no significant effect and significant differences between treatments T are indicated by p lt 0 05 p lt 0 01 or p lt 0 001 important effect of O3 appeared more or less rapidly in all geno types from 3 days after O3 treatment started in Cima to one week in the other two genotypes Fig 4A C During the experimentation although leaves were fully expanded chlorophyll content continued to increase in ambient conditions and then tended to stabilize Exposure to O3 resulted in a decrease in chlorophyll content with 36 41 and 30 less chlorophyll under exposure than in ambient conditions for Carpaccio Cima and Robusta respectively 105A 2 8 E en 6 3 R2 0 98 ax 4 R 0 9422 5 U 2 5 2 y 0 P 0 5 10 POD mmol m Fig 3 Relationships between height A and diameter B relative to the control treatment
54. Ces niveaux tant r guli rement d pass s Figure 7 un impact sur la v g tation et les cosyst mes ainsi que des baisses de productivit cons quentes sur l agriculture et les activit s foresti res sont pr voir Par exemple en 2002 en Gr ce dans la r gion de Thessalonique on estime la perte conomique totale due l impact de l ozone sur les cultures 43 Vlachokostas et al 2010 Au niveau de la production foresti re on estime la perte conomique 2004 au niveau de la Su de 56 Karlsson ef al 2005 De plus l ozone a un effet indirect sur les capacit s de s questration du CO des cosyst mes agissant ainsi doublement sur le changement climatique Sitch et a 2007 SITES D ACTION DE L OZONE Les flux d ozone dans l atmosph re sont d pendants des r sistances qui s y opposent r sistance a rodynamique r sistance de couche limite r sistance a rodynamique de la canop e r sistance intrins que du sol r sistance de la cuticule En contact avec les feuilles l ozone est confront une surface h t rog ne Les flux d ozone travers la cuticule sont n gligeables Kerstiens and Lendzian 1989 Ils se r alisent essentiellement en passant 4 Synth se bibliographique par les stomates Il convient alors d identifier correctement les flux stomatiques entrants d ozone Le nombre de stomates ainsi que leur ouverture influent sur l entr e d O Castagna and Ranieri 2
55. Courty P E Moretti S Bizet F Guilliot A Hummel I Developmental and environmental regulation of aquaporin gene expression across Populus species Divergence or redundancy PLoS ONE 8 e55506 Damour G Simonneau T Cochard H Urban L An overview of models of stomatal conductance at the leaf level Plant Cell amp Environment 2010 33 1419 1438 Danielsson H Karlsson GP Karlsson PE H kan P Ozone uptake modelling and flux response relationships an assessment of ozone induced yield loss in spring wheat Atmospheric Environment 2003 37 475 485 Day ME Influence of temperature and leaf to air vapor pressure deficit on net photosynthesis and stomatal conductance in red spruce Picea rubens Tree Physiology 2000 20 57 63 Del Rio LA Sandalio LM Corpas FJ Palma JM Barroso JB Reactive oxygen species and reactive nitrogen species in peroxisomes Production scavenging and role in cell signaling Plant physiology 2006 141 330 335 158 R f rences Dghim AA Dumont J Hasenfratz Sauder M P Dizengremel P Le Thiec D Jolivet Y Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity Physiologia Plantarum 2012 doi 10 1111 j 1399 3054 2012 01686 x Dickmann DI Kuzovkina J Poplars and willow of the world with emphasis on silviculturally important species In Isebrands JG Richardson J Chapter 2 eds Poplars and willows in the world meeting the needs of
56. D F Long S P 2009 Quantifying the impact of current and future tropospheric ozone on tree biomass growth physiology and biochemistry a quantitative meta analysis Global Change Biology 15 396 424 R sultats 2 REPONSES DISTINCTES DES STOMATES SUR LA FACE ABAXIALE ET ADAXIALE EN REPONSE A L OZONE CHEZ TROIS GENOTYPES DE PEUPLIER EURAMERICAIN Sous l effet de l ozone nous avons montr que la conductance stomatique des feuilles baisse et que les vitesses d ouverture et de fermeture des stomates aux diff rents facteurs environnementaux sont ralenties Les mouvements stomatiques sont li s des flux d ions impliquant des canaux plasmiques et vacuolaires L impact de l ozone sur les mouvements stomatiques en r ponse aux variations de param tres environnementaux pourrait tre le r sultat de diff rents effets directs sur les cellules de garde comme une modification de l ultrastructure une perturbation du contenu en l ments min raux ou encore une alt ration des flux par les canaux ioniques Nous avons donc cherch comprendre par quels m canismes les mouvements stomatiques taient perturb s par l ozone gr ce la combinaison d approches microscopiques et g nomique De plus le peuplier tant une esp ce amphistomatique nous avons choisi d tudier l effet de l ozone sur la conductance stomatique de chacune des faces de la feuille distinctement afin de voir s il existait des diff rences Cette tude
57. Dghim Jennifer Dumont 5 Marie Paule Hasenfratz Sauder Pierre Dizengremel Didier Le Thiec 5 and Yves Jolivet b e aUMR1137 EEF Universit de Lorraine F 54500 Vandoeuvre l s Nancy Cedex France PUMR1137 EEF INRA F 54280 Champenoux France SIFR110 EFABA F 54500 Vandoeuvre l s Nancy Cedex France Correspondence Corresponding author e mail jolivet scbiol uhp nancy fr Cell capacity for cytosolic NADPH regeneration by NADP dehydrogenases was investigated in the leaves of two hybrid poplar Populus del toides x Populus nigra genotypes in response to ozone O3 treatment 120 ppb for 17 days Two genotypes with differential Os sensitivity were selected based on visual symptoms and fallen leaves Robusta sensitive and Carpaccio tolerant The estimated flux PODy that entered the leaves was similar for the two genotypes throughout the treatment In response to that foliar flux assimilation was inhibited to the same extent for the two genotypes which could be explained by a decrease in Rubisco EC 4 1 1 39 activity Conversely an increase in PEPC EC 4 1 1 31 activity was observed together with the activation of certain cytosolic NADP dehydrogenases above their constitutive level i e NADP G6PDH EC 1 1 1 49 NADP ME malic enzyme EC 1 1 1 40 and NADP ICDH NADP isocitrate dehydrogenase EC1 1 1 42 However the activity of non phosphorylating NADP GAPDH EC 1 2 1 9 remained unchanged F
58. ET CHLOROPHYLLES eee eee e ee e nen e eren 25 1 Mesures de hauteur de diam tre et chutes de feuilles 25 2 Mesures de DIOmasses is is r di R A ad hehe P umida 25 3 Mesures du contenu en chlorophylles 26 Principe del appareil ese i AS EB RUBRO de SEP edicta medie A ARR Uber RE 26 Utilisation cg SESS ERA Ron eiae dig e Ede Si 26 MESURES DES ECHANGES GAZEUX iranin o Po Sp deett nn GSP Puede deeds 26 1 Conductance stomatique la vapeur d eau porom tre 26 Principe de l appareil EE A 26 WHS ALON se ER 27 2 Assimilation nette de CO et conductance stomatique la vapeur d eau 11 6200 27 Principe de l appareil eese ERI RR FEN ERI EQ RT IEEE 27 UtiliSatiotis sette tre tene ER ph ape e eti ttt ee im ee Ae E E ED 28 3 Assimilation nette de et conductance stomatique la vapeur d eau Li 6400 29 Principe de dle n 29 Utilisation 4c 5 ebbe e dee t adeo de ee e e de db a p ded E e eb rode be oet eet 30 ANALYSES METABOLOMIQUES n i ere d avis dei rer eene 31 1 Principes des analyses ig tte repe ee ou 31 2 Ascorbate et glutathion 31 3 Bo ST EE 32 4 Compos s p
59. Gout E Douce R et al 1999 Glycine and serine catabolism in non photosynthetic higher plant cells their role in C1 metabolism Plant J 20 197 205 58 Caban M Pireaux JC Leger E Weber E Dizengremel P et al 2004 Condensed lignins are synthesized in poplar leaves exposed to ozone Plant Physiol 134 586 594 59 Joshi V Laubengayer KM Schauer N Fernie AR Jander G 2006 Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool Plant Cell 18 3564 3575 60 Bick JA Setterdahl AT Knaff DB Chen Y Pitcher LH et al 2001 Regulation of the plant type 5 adenylyl sulfate reductase by oxidative stress Biochemistry 40 9040 9048 102 R sultats 61 Janzik I Preiskowski S Kneifel H 2005 Ozone has dramatic effects on the regulation of the prechorismate pathway in tobacco Nicotiana tabacum L cv Bel W3 Planta 223 20 27 62 Betz GA Gerstner E Stich S Winkler B Welzl G et al 2009 Ozone affects shikimate pathway genes and secondary metabolites in saplings of European beech Fagus sylvatica L grown under greenhouse conditions Trees 23 539 553 63 Yamaji K Julkunen Tiitto R Rousi M Freiwald V Oksanen E 2003 Ozone exposure over two growing seasons alters root to shoot ratio and chemical composition of birch Betula pendula Roth Glob Chang Biol 9 1363 1377 64 Peltonen PA Vapaavuori E Julkunen Tiitto R 2005 Accumulat
60. Le Thiec Didier ABSTRACT Ozone induces stomatal sluggishness impacting photosynthesis and transpiration Here we seek the origin of stomatal movements change on abaxial and adaxial sides of the leaf of three euramerican poplar genotypes differing in ozone sensitivity Effect of ozone on stomatal conductance of each leaf side was studied by gas exchange measurements Ultrastructural modifications of guard cells were studied by TEM Gene expression of ions channels and transporters involved in stomatal movements and the related element contents were quantified by qPCR and X ray microanalyses directly in stomata We report a more important impact of ozone on stomatal conductance of the adaxial side of leaf correlated with higher calcium content in guard cells The expression of H Ca antiports CAX1 and CAX3 homologues and f carbonic anhydrases BCA1 and BCA4 was strongly decreased Decoupling between the rate of stomatal opening and potassium concentrations in guard cells seems to settle earlier in the sensitive genotype Robusta The number of mitochondria in guard cells was increased with ozone Ozone impacts stomatal movements via ion fluxes with a specific calcium signal The increased number of mitochondria could answer to an increasing energy request for detoxification and repair processes and may act as calcium buffer Key words Populus deltoides x nigra ozone guard cell abaxial and adaxial sides gas exchange element content
61. Nettoyer les filtres des bouteilles de solvant dans un bain ultrasons avec de l eau Millipore puis 5 min dans du MeOH Si un filtre avec de l ac tonitrile est mis directement dans une solution contenant des sels il s encrasse Ne pas oublier de lester les pompes binaires une fois par semaine L azote doit tre coup pour le Corona avant de vider les bouteilles poubelles du Corona Le MS doit tre calibr une fois par mois pour cela Pr parer un m lange frais de calibration les solutions de stockage peuvent tre utilis es pendant 4 mois si elles sont gard es 4 C Utiliser la seringue r serv e cette usage R gler la position de la sonde R gler 195 1 et sauvegarde le fichier de r glage Autocalibrer et nettoyer toutes les surfaces et tuyauteries apr s calibration 4 ANALYSE DES ACIDES AMIN S Appliquer exactement le protocol tablit par le producteur du kit EZ faast sauf pour le volume de r actif 1 50 uL au lieu de 100 uL Bri vement Sortir les r actifs du frigo Sortir les chantillons du cong lateur 20 C Pr parer le m lange 3A 3B dans la bouteille bouchon rouge ex 8 chantillons pr parer pour 9 avec le blanc 1200 uL et 800 uL 3B Mettre un flacon allong chantillon 1 et les identifier Mettre 100 uL d chantillons sauf pour le blanc 144 Annexes Ajouter 50 uL dans tous de R actif 1 Mettre un piston dans une seringue de 1 5
62. Protocole d taill en annexe 4 Un traitement la Dnase I Ambion enl ve l ADN r siduel Protocole d taill en annexe 5 La qualit des ARNs ainsi obtenus est v rifi e par le syst me de gel d lectrophor se automatis Experion Bio Rad Protocole d taill en annexe 6 et une quantification pr cise est permise gr ce une mesure au Quant iT riboGreen Invitrogen Protocole d taill en annexe 7 La r trotranscription a t r alis e sur 700 ng d ARNs gr ce au kit iScript cDNA Synthesis Bio Rad en contr lant l efficacit par l ajout du kit Alien QRT PCR Inhibitor Alert Stratagene suivant les recommandations du fabricant Protocole d taill en annexe 8 34 Mat riel et M thodes DESSIN DES AMORCES Toutes les cibles ont t identifi es dans la litt rature sur Arabidopsis thaliana Les homologues chez le peuplier ont ensuite t d termin s gr ce l outil de bioinformatique phytozome V8 0 Goodstein et al 2012 sur la base de blast de s quences prot iques et g nomiques Les g nes les plus proches des cibles chez le peuplier ont t tudi s Des amorces ont t dessin es en se basant sur la version 2 du g nome de Populus trichocarpa et sur les transcrits pr dits par phytozome Certaines r gles ont t suivies pour le dessin des amorces e Taille des amorces gales 20 ou 21 bases b Pourcentage de compris entre 38 et 50 Les amorces doivent avoir le
63. Song C P 2001 K channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells Cell Research 11 195 202 Zhang X Wang H Takemiya A Song C Kinoshita T amp Shimazaki K 2004 Inhibition of blue light dependent H pumping by abscisic acid through hydrogen peroxide induced dephosphorylation of the plasma membrane H ATPase in guard cell protoplasts Plant Physiology 136 4150 4158 74 10 000 OT Ouid pusouosj LLLLVOLOOVVDVDVOVDOD T OPSI ETOO INXHON OSLEISOI00 ULdOd VODVLLVOVVOV LLODDODL 00 LTID010 H10d OI9 LIA TV 009898 T1V 08r S C9 IV OLPLISTIV Jo So ouo OYLIIS8000 ALdOd LVLODOVOWVDLLVDLODDL sisdopiqv4y urojo1d passeidxa oj IIS T OSETIEZOO INXH9A USSIUIIIA VVDOVOVVODLLOLOOVVOV 00981 10800 1304 89LWTV Orr8 TS civ JO Sojoquo DUVIJVYI O 90SST00 ULdOd LLODVLVVOLODVODDOLVL sisdopiqv4y ur woord posso1dxo oj reprums 1T HT98TETOO INXUOI T 9ET T8 TMS IOLOLLOODIOVIODIOVVV 0075005101204 9LWTY OET TIL 00777 HIV 86S ILVVO OLTIOS 000 ULdOd Jo Sojoquo 090 v1914 DYO Guoursey II MS VOOLLODVOOOLOLVVOLVD uvoq ouueuo uinissejod oj 1e rurs VVDVVOVVODDDVVDIDLOD 0088109 00 1304 TLAV EESLL V8 ICC82OVO 90869 2 416 lt lt 90pp9VVO PCCSCOVO S P6TOVO Fly cel TESLLIVE OL9TISSIV rc869d VO Os6LvS eV Lers8g vo Jo TOT 012108100 1404 09 vo1s42d snun4q cyqO s
64. The physiological transcriptional and genetic responses of an ozone sensitive and an ozone tolerant poplar and selected extremes of their F2 progeny Environ Pollut 159 45 54 Szal B Dabrowska Z Malmberg G Gardestr m P Rychter A 2008 Changes in energy status of leaf cells as a consequence of mitochondrial genome arrangement Planta 227 697 706 Turner WL Waller JC Vanderbeld B Snedden WA 2004 Cloning and characterization of two NAD kinases from Arabidopsis identification of a calmodulin binding isoform Plant Physiol 135 1243 1255 UNECE 2004 Mapping Manual UNECE Convention for Long Range Transboundary Air Pollution Manual on methodologies and criteria for modelling and mapping critical loads and levels and air pollution effects risks and trends http www oekodata com icpmapping pub manual 2004 mapman 2004 pdf Edited by P Gardestr m 50 UNECE 2005 Forest Trees Working Group Report Critical Level of Ozone Further applying and developing the fluxed based concept Obergurgl Tyrol Austria pp 15 19 Valderrama R Corpas FJ Carreras A Gomez Rodriguez MV Chaki M Pedrajas JR Fernandez Ocana A Del rio LA Barroso JB 2006 The dehydrogenase mediated recycling of NADPH is a key antioxidant system against salt induced oxidative stress in olive plants Plant Cell Environ 29 1449 1459 Voll LM Zell MB Engelsdorf T Saur A Wheeler MG Drincovich MF Weber APM Maurino VG 2012 Loss of cytosolic NADP mali
65. ULdOd Jo Sojoquo wnayys wnsig DO T ESOTT IMS OLLVODLIOOIVOVIOLOLDOL aseury 605860000 WXPA T TLOHd d VOLDOLODOVVOLLOLLVOOL 000Z7PED 100 10d LLOHd 8 061O VV 66L68VVO 96p588VO 6r 18V VV IP06IOVV LTTUINAX DT I9siMouon jxqiso Jo GOT Sojoquo 09 t ppimouna 00L 0S8100 LLdOd DVIVIVOLOOLDOOLLOLLOO 08r21poj y4O Sed LW H 01 repris UIVHA3d DVVDIODLOVOLVVODIDLV 0009009810 1304 8ICLSO 8 06LO VV 66 6 lt 2 96r68d VO 6r T8VVV 06 SEOOLHT 0 84 yxs OTSPTBTIV Jo GOT 00 t ppmouna 0668789000 LLdOd VVOLLLODDOLOLOOOOLVVIL 8 1 esed LV H NS 1 89L0EET00 NX TIVHA3d LVVVVDODVVDDLODDVVDV 000 79900 1104 92150 L6190v VO S80S0V VH 666800VV O9FOLAVV SLSTEd VV OSLS8SVVA L0LTEJVV TSSEETVV 866800VV OLISOV VA 6cr9LV VH 66 0d 9508 2 95001 2 JO 1130 01 090255000 ULdOd V6VOA DT O TA 8 DLLOLDLODVOODOLDOVIOL sun DO unmbrqn pod reps 1 68990 700 vIdns3d LOVVDDVDDDDVLLODDVDL 0048619500 1304 6Lc9 LIV V 06 80010 00 DOUPUDSDYO DXOOdS unissejod 107 OSSLESTOOO ULdOd IOIOLLIOVOLOVOLLODOLVO Jouueyo SurAgroor pregno snutA nd oj IIS l9 886cc00 INXBos UIFLET IAS VOVVDODVOLVOVVVD
66. Upper 90 1 30 4 0 2 15 8 0 2 R Lower 144 2 33 5 0 2 17 9 0 2 Upper 87 1 30 6 0 3 15 4 0 2 Statistical analyses Genotype lt 0 001 lt 0 001 ns effect Carpaccio lt 0 001 ns ns Side effect Cima lt 0 001 lt 0 001 lt 0 001 Robusta lt 0 001 lt 0 001 lt 0 001 Carpaccio Lower ns ns ns Upper ns ns 0 001 Ozone Cima Lower ns 0 0128 ns effect Upper ns lt 0 001 lt 0 001 Lower ns lt 0 001 ns Robusta Upper ns ns ns R sultats genotypes Table 1 Robusta has the highest stomatal density on both sides with 6 more stomata mm than the other two genotypes on the lower side and 6 and 18 more than Cima and Carpaccio respectively on the upper side of the leaf Stomata are equally large in all the genotypes but a little bit longer in Carpaccio than in Cima or Robusta The size of stomata is the same between the two sides of leaf in Carpaccio but stomata are 1196 larger and around 6 longer on the lower side of the other two genotypes The ultrastructure observation with TEM revealed no difference between side and genotype Fig 2 Ozone has no effect on the stomatal density on both sides of the leaves Table 1 and only minimal effects on the size of stomata In ozone conditions the length of stomata in Cima increases slightly by only 4 on the upper side and 3 on the lower side and in Robusta by 6 only on the lower face Regarding the width of stomata it is slightly decreased only on the upper
67. al Environmental Pollution 173 2013 85 96 R 0 9985 5 R 0 9984 5 0 5 10 15 20 Relative chlorophylls a R 0 9965 5 R 0 9871 Cima Relative chlorophylls R 0 9666 R 0 9525 15 20 Robusta Relative chlorophylls POD mmol m R 0 9756 5 D R 0 9477 10 20 lt x o 8 o LA R 0 9498 824 R 0 8632 20 lt 8 o aA aF R 0 8657 5 R 0 8639 5 0 PT 20 lt 15 x pp 35 45 55 POD mmol m Fig 6 Relationships between chlorophyll content A B and C and CO net assimilation D E and F relative to the control treatment in Carpaccio A and D Cima B and E and Robusta C and F and phytotoxic ozone dose over a threshold of 1 nmol m s y y y y abiotic stress tolerance Lee and Luan 2012 In fact response to pathogens leads to ABA signal which via NADPH oxidase produce H202 Ton et al 2009 Ozone mimics this ROS production which acts as a signal to regulate several ion channels finally causing a stomatal closure Wilkinson and Davies 2010 If O3 can change the functioning of different channels as in the study of Vahisalu et al 2010 it would be relevant to study their gene expression in response to directly in guard cells 4 3 Differences between the three genotypes The three genotypes are characterized by diffe
68. and Robusta at four sampling times 2 4 11 and 17 days Total glutathione relative to Oxidised glutathione relative Reduced glutathione relative Redox ratio relative to ambient to ambient to ambient ambient n E PET ON BO o N 2 4 halaz Robusta 2 l 2 halaz Figure 3 Ozone effects glutathione concentration and redox state in poplar leaf Effects of ozone treatment on glutathione concentrations percentage relative to ambient with reduced glutathione GSH A oxidised glutathione GSSG B total glutathione GSH GSSG C and glutathione redox ratio reduced form total D on three poplar genotypes Carpaccio Cima and Robusta at four sampling times 2 4 11 and 17 days Ascorbate biosynthesis L galactose pathway GDP D Mannose gt 10 123451234512345 LLL SEN more m jsp CE MMM GDP L Galactose 123451234512345 1 0 x fold expression change 7 4162 vTC416 VTC46 0 1 L Galactono 1 4 lactone Salvage pathway 123451234512345 coe L gulose Animal like pathways Ascorbate Figure 4 Ozone effects on the expression of genes encoding enzymes involved in ascorbate biosynthesis Heat maps representing the effects of ozone on the express
69. and the capacity of ROS detoxification processes which limits biological effect of ozone are the two major protections involved in ozone effective flux calculation Dizengremel et al 2008 Castagna and Ranieri 2009 The regulation of stomatal conductance plays a key role in plant adaptation to environmental conditions changes especially under stress It has been shown that ozone impairs the stomatal movements leading to perturbation of the stomatal responses to environmental parameters and sluggish stomatal behaviour Indeed after ozone treatment stomata behave slower to open or close when changing light conditions VPD or CO concentrations Paoletti 2005 Grulke et al 2007 Paoletti amp Grulke 2010 Dumont et al 2013 Stomatal movements are due to ion fluxes which require the participation of many plasma membrane and vacuolar ion channels Pandey et al 2007 Stomatal opening is driven by a membrane hyperpolarisation which leads to an accumulation of K and sugars accompanied by their counter ions and malate leading to an increase of osmotic potential and water uptake in apoplasm Fischer 1968 Outlaw amp Manchester 1979 The closure is the 58 R sultats reverse situation under the control of a Ca signal which induces anion efflux leading to membrane depolarisation and K efflux In the present study we examine whether the direct effect of ozone on stomatal conductance is different on each leaf
70. avoid rehydration and subsequent RNA degradation RNA extraction and cDNA amplification Total RNA was extracted from 500 microdissected stomata using Qiagen Micro Plus Kit Qiagen according to the manufacturer s instructions TransPlex Complete Whole Transcriptome Amplification Sigma was used for RNA reverse transcription and cDNA amplification according to the manufacturer s instructions Amplification was performed on a Mx3000P QPCR System Agilent technologies SYBR Green Sigma Aldrich and ROX Agilent Technologies were added to the reaction mixture to monitor amplification yield Amplified cDNA was purified using the QIA amp DNA Micro Kit Qiagen according to the manufacturer s instructions and quantified on a Nanodrop ND 1000 spectrophotometer Thermo Fischer cDNA concentration was adjusted to 5 ng pL for each sample Quantitative real time polymerase chain reaction qPCR analyses Gene specific primers Supporting Information Table S1 were designed based on the last version of Populus Trichocarpa genome sequence assembly on Phytozome V8 0 Goodstein et al 2012 using Primer 3 software Untergasser et al 2012 Primer specificity was checked by sequencing PCR products Beckman Coulter Genomics Quantitative PCR was performed using an Mx3000P QPCR System Agilent technologies according to the manufacturer s recommended cycling program 10 min 95 C 40 cycles of 5 s at 95 C and 20 s at 58 or 60 C depending on primers A dis
71. avoir clamp la feuille nous avons attendu la stabilisation de la conductance gso puis nous avons fait varier un seul param tre environnemental avant d attendre qu elle se stabilise de nouveau 2 1 Nous avons ensuite calcul la vitesse de r ponse v 10 gr ce au temps n cessaire la stabilisation de la conductance stomatique apr s variation d un param tre t Figure 42 10 V 9s1 Jso t 30 Volume tampon 1 Entr e d air 1 Homog n iser l air afin de minimiser les variations de CO et de temp rature Pi ge CO 2 Bonbonne d eau 3 Sature l air en eau Le syst me de pi ge eau 4 Modifie la temp rature Appareil de mesure d changes gazeux de type Licor 6400 5 pour condenser l eau l aide d l ments Peltier Figure 41 Sch ma du dispositif de mesure avec 3 appareils Li 6400 intercalibr s B g 250 E Ag e Ag E x ef lt gt t gsl gs0 t Temps s Temps s Figure 42 Exemple de r ponses de la conductance stomatique A une augmentation de lumi re bleue et B une augmentation de VPD Tableau 7 Conditions environnementales avant et apr s variation d un des param tres l int rieur de la chambre de mesure de l appareil Li 6400 Param tres Param tre Lumi re Lumi re VPD CO environnementaux test bleue rouge CO Initial 400 2 0 400 3 0 100 1 0 400 3 0 umol mol Final 400 2 0 400 3
72. catalase Dizengremel et al 2008 Moller et al 2007 Figure 22 rendant le maintien d un pool de NADPH par le m tabolisme primaire essentiel la d toxication inductive 2 VOIE D HALLIWELL ASADA Le r le antioxydant de l ascorbate est directement d pendant de la capacit de la cellule le maintenir son tat r duit La voie d Halliwell Asada Figure 23 est alors sollicit e Dizengremel et al 2008 L entr e d ozone entra ne l apparition d H5O dans l apoplasme L ascorbate peroxydase r duit en eau en utilisant le pouvoir r ducteur de l ascorbate ASA vitamine C qui devient du monod hydroascorbate MDHA 17 2 ASA gt 2 H 0 2 MDHA Du d hydroascorbate DHA se forme ensuite spontan ment par dismutation de deux MDHA 18 2 MDHA gt DHA Le DHA passe ensuite dans le cytosol puis dans les chloroplastes ventuellement On y retrouve les m mes r actions 17 et 18 pour neutraliser l H2O r ussissant passer la membrane De plus la monod hydroascorbate r ductase MDHAR pr sente dans ces compartiments permet de r g n rer l ascorbate en utilisant du NAD P H selon la r action suivante 19 2 MDHA NAD P H 2 ASA NAD P 14 cw P Carbon metabolism modified Alteration of protein synthesis decrease in photosynthesis increase in respiration Signal 0 Increase maintenance of NAD P H Asc C GSSG um DHA
73. conditions for stomatal opening are optimal Concerning the effect of O4 on stomatal closure in response to high CO or VPD although there was no impact on Ags the speed of the response was once again slowed down so that more time was required to achieve a steady state under new environmental conditions Trees with slower reactions may take more time to close their stomata when the air gets dry and then may lose more water because of higher transpiration rates This stomatal sluggishness should be taken into account in the models for calculating conductance by adding an function not only linked to phenology but also to the other functions because the responses to variations in the environmental parameters we tested i e blue light red light CO and VPD were altered under O3 expo sure in accordance with other studies Grulke et al 2007 Hayes et al 2012 Hoshika et al 2012 Onandia et al 2011 Paoletti and Grulke 2010 The mechanisms that account for sluggish stomatal responses during O3 exposure are not fully understood yet O3 exposure mediates increases in apoplastic H202 which alters membrane permeability specifically to cation influx Castillo and Heath 1990 Le Thiec et al 1994 McAinsh et al 2002 Pei et al 2000 Torsethaugen et al 1999 Recent advances have demon strated that ABA plays an important role in response to pathogens and that the signalling pathways are very similar to the one of 92 J Dumont et
74. cumulative Figure 12 Ainsworth et al 2012 Suite une exposition chronique l effet de l ozone sur la conductance stomatique a t attribu un effet direct sur la photosynth se li une baisse de l activit de la Rubisco notamment due une diminution des concentrations de l enzyme entra nant une hausse de la concentration interne en CO Ci et une fermeture stomatique Figure 13 Dizengremel 2001 Fiscus et al 2005 N anmoins il existe un effet direct de l ozone sur le fonctionnement des cellules stomatiques induisant des changements de la r ponse dynamique des stomates impliquant un d couplage de la photosynth se et la conductance stomatique Lombardozzi et al 2012 De plus si l ozone r duit les capacit s photosynth tiques la respiration est l inverse stimul e tout comme l activit de la PEPC ce qui permet de contrebalancer en partie la diminution de la photosynth se Dizengremel 2001 Dghim et al 2012 40 30 20 10 0 10 9 aan i 72 92 ppb ymnosperms BAYS A 114 74 ppb ait m Total biomass 160 92 ppb AIH biomass 102 75 ppb r O E i 9 9 10 73 ppb I 1 Lat FO 6008 ppb o EIN oc o 47 101 ppb 2 gt 92 73 ppb I O 34 a 46 100 ppb I A 87 80 ppb Root biomass I 44 103 ppb Root to shoot ratio 101 74 ppb O n 40 30 20 10 0 10 Percent change from charcoal fillered control Fi
75. d eau Attention on en donne jamais a boire a un acide Ajouter 0 3 g de Ferric Chloride 0 6 B 4 2 2 bipyridyl 1 g dans 25 mL 136 Annexes Chaque r actif est stable temp rature ambiante dans le noir pendant 2 3 semaines MAIS PAS LE MELANGE DES DEUX Pr parer le r actif colorant chaque jour au dernier moment en ajoutant 2 2 mL de A 0 8 mL de B 5 mM d acide ascorbique 1 mg mL d acide m taphosphorique stocker 20 C R f rences Gatzek S Wheeler G L and Smirnoff N 2002 Antisense suppression of L galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l galactose synthesis Plant J 30 541 553 Kampfenkel K Van Montagu M and Inze D 1995 Extraction and determination of ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165 167 7 DOSAGE GLUTATHION Placer 4 fois plus de plaques num rot es que de nombre de roboracks d extraits sur le robot Remplacer 6 blancs par une gamme talon GSH Prendre 10 uL GSH 10 mM dans 990 uL d eau 0 1 mM final Mettre 500 uL d acide m taphosphorique dans 6 tubes Ajouter 500 uL de GSH 0 1 mM dans le premier 50 uM final pr lever de ce tube 500 uL et les m langer avec le suivant Proc der cette dilution en cascade pour tous les tubes sauf le 0 pour avoir Concentration uM 50 25 125 6 25 312 0 nmol par puit 0 5 025 01
76. dans les processus de d toxication c est surement leurs concentrations constitutives qui permettent de lutter plus ou moins efficacement face au stress oxydatif induit par l ozone L identification des compos s discriminant les profils de chaque g notype pourrait peut tre r v ler un facteur de tol rance l ozone 106 R sultats 3 CAPACITE DE REGENERATION DU NADPH DANS LES FEUILLES DE DEUX GENOTYPES DE PEUPLIER PRESENTANT DES DIFFERENCES DE SENSIBILITE A L OZONE Si la synth se de novo est importante pour maintenir un pouvoir r ducteur suffisant elle doit tre coupl e avec de bonnes capacit s de r g n ration des mol cules oxyd es La r g n ration repose sur une suite de r actions d oxydo r duction qui se basent principalement sur la disponibilit en NADPH Or la capacit de r g n ration du NADPH est fortement d pendante de r actions enzymatiques li es au m tabolisme carbon L assimilation de carbone est r alis e par l interm diaire des enzymes carboxylantes Rubisco et PEPC En r ponse l ozone la photosynth se est limit e provoquant un d ficit nerg tique et une diminution de l assimilation de carbone Afin de d terminer ce qui provoque la baisse de l assimilation de carbone le contenu en chlorophylle et les activit s des enzymes carboxylantes ont t mesur s De plus cette tude s int resse l effet de l ozone sur le contenu en NADPH ainsi que sur l activit des enzymes
77. des concentrations lev es en ozone par rapport aux t moins expos s l air filtr par charbon actif Les degr s de libert d f et les concentrations ambiantes moyennes en ozone O sont donn s le long de l axe y Wittig et al 2009 Figure 14 A Stomate et B piderme inf rieur de feuille de peuplier observ s au microscope lectronique balayage Synth se bibliographique EFFETS BIOCHIMIQUES Au niveau cellulaire l ozone entraine une hausse des ROS qui va induire toute une cascade d v nements et des modifications biochimiques importantes Outre la r duction de l activit et des concentrations de la Rubisco la r duction du contenu en chlorophylles provoqu e par l ozone va impacter n gativement la disponibilit en carbone Figure 13 Un co t en carbone est ajout pour lutter contre les effets de l ozone sous la forme d une synth se de m tabolites impliqu s dans les processus de d toxication des ROS ascorbate glutathion carot noides flavonoides d une production suppl mentaire des enzymes neutralisant les ROS peroxydase SOD d une stimulation de la voie de synth se de la lignine et d un cycle de r paration intense des prot ines d grad es Caban et al 2004 Ainsworth et al 2012 Les ROS produits suite l entr e d ozone alt reront l expression de nombreuses prot ines Heath 2008 II ROLE DES STOMATES Comme nous l avons mis en avant pr c demment les stomat
78. doit prendre en consid ration ces deux aspects afin de caract riser au mieux les diff rences de sensibilit l ozone intra et inter sp cifique Mots cl s Ozone peuplier d toxication conductance stomatique Role of stomatal regulation and capacity of foliar detoxification in the estimation of ozone critical level for vegetation Abstract Tropospheric ozone is a major air pollutant that acts as a phytotoxin It enters the leaf through the stomata before being dissolved in the apoplast by generating reactive oxygen species ROS causing oxidative stress Two defenses exist to restrict the effects of ozone 1 the stomata which can limit ozone uptake by regulating stomatal conductance and ii the detoxification processes of ROS generated by ozone We studied the effects of ozone 120 ppb on these two mechanisms of defense in three euramerican poplar genotypes Populus deltoides x Populus nigra under controlled conditions in phytotronic chambers A direct effect of ozone on photosynthesis and stomatal movements in response to changes in environmental factors by slowing the stomatal opening and closure has been highlighted Models of stomatal conductance on which indicators of critical level of ozone for vegetation are based must take them into account In addition these studies have highlighted the role of constitutive concentrations of antioxidants in tolerance to ozone as well as the complexity of these detoxification mechanisms
79. enfin une diffusion passive des ions K Hopkins 2003 Lawson 2009 Les flux d ions permettant les mouvements stomatiques n cessitent la participation de nombreux canaux plasmiques et vacuolaires tonoplastiques Figure 15 Pandey et al 2007 EFFET DU VPD VAPOR PRESSURE DEFICIT ET DE LA TEMPERATURE Les stomates sont sensibles au VPD et la temp rature deux param tres li s dont les effets sont difficilement s parables Si certaines esp ces apparaissent plus ou moins sensibles aux variations de VPD on observe cependant une r ponse commune la conductance stomatique baisse avec l augmentation du VPD Day 2000 Yong et aL 1997 Le m canisme de r ponse des stomates aux variations de VPD est peu connu alors que ce param tre environnemental est un facteur dominant par rapport aux variations de lumi re ou de concentrations de CO ce qui lui donne une grande importance dans la pr diction des mouvements stomatiques Aasamaa et al 2011 Certains pensent que les m canismes de r ponse au VPD pourraient tre ind pendants de l ABA Assmann ef al 2000 tandis que 10 Decreased C Inactivation of anion channel membrane hyperpolarization Vacuole Malate synthesis L gt Glu 1 P Malate Calvin cycle Triose phosphates Chloroplast Guard cell Figure 16 Sch ma de la cascade de r actions impliqu e dans la r ponse la
80. gamma glutamylcysteine synthase GSH 3 glutamate synthase NADH GOGAT 4 Glutamine synthetase GS 5 Cysteine synthase OAS 6 Serine o ac tyltransferase SATase 7 glycine hydroxymethyltransferase glyA 8 threonine aldolase THA 9 alanine glyoxylate transaminase AGT 10 glycolate oxydase Synth se bibliographique plus ou moins d taill es sont parfois d crites dans la litt rature mais sont pour certaines controvers es La GDP L galactose phosphorylase VTC2 produit le premier m tabolite d di la principale voie de biosynth se de l ascorbate le L galactose 1 P C est le point de r gulation le plus important dans le contr le transcriptionnel de la biosynth se de l ascorbate Linster and Clarke 2008 5 VOIE DE BIOSYNTHESE DU GLUTATHION Le glutathion est lui aussi un l ment cl du m canisme de d toxication des radicaux libres oxyg n s puisqu il sert renouveler le pool d ascorbate r duit dans la voie d Halliwell Asada Le glutathion est un cofacteur de nombreuses enzymes impliqu es dans la d toxication des ROS et joue donc un r le central comme antioxydant mais aussi comme composant de la signalisation cellulaire Galant et al 2011 Noctor et al 2012 Sa voie de biosynth se est tr s simple car rapidement reli e aux acides amin s Figure 26 Les deux principaux points de r gulation sont la disponibilit en cyst ine et l activit de la gamma glutamylcysteine syn
81. genes GOGAT6 UBL OST26 GLR2 and KAT3V2 for gene expression normalization Statistical analyses The data obtained from MEB analyses were fitted with a linear model with treatment side and genotype as factors Contrast analyses were performed to test for genotype and side effect before fumigation and treatment effect for each genotype and each side the porometer data were modelled as linear model with treatment and genotype as factors Normality and homoscedasticity of standardized residuals were graphically checked using quantile to quantile and residual vs predicted plots An ANOVA was used to test the differences between genotype and the treatment effect at each data for each leaf side In case of an interaction contrast analyses were performed to test for treatment effect within each genotype Data were analysed using R 2 15 2 R Core Team 2012 and multcomp package Hothorn et al 2008 Effects were considered significant when p 0 05 Cluster analysis was performed using Euclidean distance on the gene expression dataset with MeV Saeed et al 2003 2006 RESULTS Characterization of the effect of ozone on stomatal conductance of each leaf side Under filtered air conditions Carpaccio has a 2096 lower total stomatal conductance than Cima and Robusta Fig la b c The stomatal conductance of the upper side of the leaves represents 41 37 and 34 of the total stomatal conductance of Carpaccio Cima and Robusta respectively F
82. increased during the experimental period with higher values for Robusta compared to Carpaccio Fig 4 D Ozone treatment reduced the total Chl content of the two genotypes The decrease became significant on day 11 of the treatment for Robusta Fig 4D and on day 15 for Carpaccio Fig 4C Genotype effect between the O3 treated trees was only significant on day 11 Table 2 At the end of the O treatment Carpaccio retained only 45 of its total Chl content and Robusta 50 Carboxylase activities In response to fumigation we measured a significant increase in PEPC activity in the leaves of the two poplar genotypes Fig 5A B However the curves were slightly different for the two genotypes The differences between control and O3 treated leaves became highly significant from day 4 P 0 01 for Robusta but only from day 11 for Carpaccio P 0 001 Fig 5A B Maximum PEPC activity values were recorded on day 15 after the beginning of fumigation for the two genotypes Moreover on days 15 and 17 the activity levels in O3 treated leaves were slightly higher and statistically significant for Robusta compared to Carpaccio Table 2 Conversely to PEPC activity Rubisco activity gradually decreased in ozone treated leaves Fig 5C D That decrease was significant from day 11 of the treatment for the two genotypes Fig 5C D Rubisco activity inhibition reached a maximum on day 17 3096 and 3796 for Carpaccio and Robust
83. jours de traitement ozone ou control o 14 RAT 12 one AST 10 ey abe 2 agho AST MT e 30 20 259 6 e 2287 230 4 2486 20 cac a5T 50580 240 on 2 401 010 Q 315 ish amp 7 4880 SO szmar e 0 T 30 sT ME T 2 30 456 0 s2T _ ap 959 ae 4 To 24T 2037 3T 220 E N 210 40 T 8 IT 49 25 J N T 630510 10 w 12 20 14 16 14 12 10 8 6 4 2 0 2 4 6 8 10 12 14 16 PC1 24 SIMCA P 12 0 1 2013 05 14 12 57 42 UTC 1 Figure 49 Analyse en composantes principales bas e sur les concentrations des compos s ph noliques chez 3 g notypes de peuplier euram ricain Carpaccio en orange Cima en vert et Robusta en bleu soumis ou non un traitement ozone Les chantillons sont repr sent s par le temps de pr l vement 1 5 suivi d une lettre en fonction du traitement Ozone O et T moin T R sultats expliquent elles trois 57 de la variance La quatri me composante montre un effet ozone qui sur les trois g notypes d s la troisi me date et expliquant 8 de la variance Si nos analyses t moignent d un effet de l ozone sur les concentrations en compos s ph noliques avec principalement une hausse des concentrations cet effet reste mineur en face des diff rences constitutives entre g notypes Ceci semble indiquer que si les compos s ph noliques jouent un r le
84. le transfert en phytotrons 5 semaines apr s la plantation des boutures une tige unique a t gard e ils atteignaient alors entre 60 et 80 cm de hauteur Figure 29 La fumigation a d but apr s une semaine d acclimatation dans les phytotrons 4 PRELEVEMENT Durant les p riodes de fumigation des pr l vements de feuilles ont t effectu s toujours 15 h pour le dosage des m tabolites et l tude de l expression des g nes Pour 23 Figure 30 Restant d une feuille de peuplier apr s les pr l vements pour la microscopie lectronique Figure 31 Les Chambres phytotroniques Figure 32 Pr sentation d une chambre UMR1137 UL phytotronique contenant 6 peupliers Mat riel et M thodes r aliser ces pr l vements dans des conditions optimales toutes les feuilles pr lever ont t identifi es avec de la ficelle d s la mise en phytotrons Il s agissait des deux premi res feuilles matures en partant du haut 10779 et 11 feuille principale a t d coup e aux ciseaux et les feuilles pr lev es ont t imm diatement plong es dans des pochettes d aluminium identifi es et congel es dans de l azote liquide Une partie des chantillons d di e aux analyses m tabolomiques sauf ascorbate et glutathion et la micro dissection laser a t lyophilis e 40 C pendant 40 h une pression de 10 Pa dans un lyophilisateur FreeZone Plus freeze dryer Labconco Kansas City M
85. le vent plus limit dispersant moins les polluants les r actions d crites sont favoris es 3 EVOLUTION DES CONCENTRATIONS EN OZONE TROPOSPHERIQUE Les concentrations en ozone troposph rique ont t multipli es par 2 5 au cours du XX si cle Figure 5 et 6 Afin d viter que ces concentrations continuent de cro tre des mesures ont t prises pour limiter la production des pr curseurs de l ozone par l activit humaine conventions de Sophia 1988 et Gen ve 1991 des suivis de concentration d ozone ont t r alis s et des seuils de risque pour la sant et pour la v g tation ont t d finis ICP M amp M 2004 Journal Officiel des Communaut s Europ ennes directive 2002 3 CE du parlement europ en et du conseil Cependant malgr la baisse des missions de pr curseurs 3 lt 6000 pg m h 6000 2 000 pg m h 12 000 18 000 pg mr h 18 000 27 000 pg gt 27 000 pg mr h Ozone 40 for forests Reference year 2005 Combination with EMEP model sttude and solar radiation E 10000 ug m EM 22020 20000 ug min 20 000 30 000 ug min 0000 50000 ug a gt 50 000 ug EM countries Ca Poor data coverage Rural background station Figure 7 Cartes des valeurs d AOT40 indicateur de seuil de risque l ozone pour la v g tation en 2005 pour les cultures et pour les for ts EEA 2009 Synth se bibliographique en Europe les changem
86. les hypoth ses suivantes l humidit relative dans la feuille est gale 1 26 Figure 35 Mesure de la conductance stomatique sur la face adaxiale de la feuille r alis e l aide du porom tre SC1 Figure 36 Sch ma de fonctionnement de l appareil Li 6200 Mat riel et M thodes e toutes les valeurs de conductance sont en s rie le flux est donc constant entre deux points e temp rature de la feuille est gale la temp rature du premier capteur d humidit La conductance peut donc tre exprim e comme une fonction des distances entre les capteurs d humidit de la temp rature et des deux mesures d humidit relative 2 2 9 BDvapor hr1es Tas hr2es Taz l les Ta1 1 hr1 ld2 hr1es Ta1 hr2zes Ta2 ld avec f la densit molaire de l air Dy la diffusivit de la vapeur d eau hr l humidit relative e Ta la pression de vapeur satur e temp rature ambiante 41 la distance entre la feuille et le premier capteur et 42 la distance entre les deux capteurs UTILISATION Lors de la seconde exp rience de fumigation un suivi de la conductance stomatique sur la face abaxiale et adaxiale de la premi re feuille mature de tous les arbres a t r alis 3 fois par semaine deux heures apr s le d but la photop riode l aide d un porom tre SC 1 Decagon Devices Pullman USA Figure 35 2 ASSIMILATION NETTE DE ET CONDUCTANCE STOMATIQUE A LA VAPEUR D E
87. les processus de d toxication et sur la r g n ration m tabolique du pouvoir r ducteur Dizengremel et al 2009 Figure 23 Voie d Halliwell Asada Castagna et Ranieri 2009 Figure 24 Sch ma des syst mes de d toxication des ROS dans les diff rents organites des cellules v g tales Figure 25 Voies de synth se de l ascorbate Figure 26 Voie de biosynth se du glutathion Figure 27 Absorption d ozone et effets biologiques Tausz et al 2007 Figure 28 Plantation des boutures de peuplier Figure 29 D but de l acclimatation en phytotrons des peupliers g s de 5 semaines Figure 30 Restant d une feuille de peuplier apr s les pr l vements pour la microscopie lectronique Figure 31 Les Chambres phytotroniques 1137 UL Figure 32 Pr sentation d une chambre phytotronique contenant 6 peupliers Figure 33 Exemple de racines de peuplier apr s lavage pr tes pour le s chage Figure 34 Sch ma de principe du porom tre issu du manuel d utilisation Figure 35 Mesure de la conductance stomatique sur la face adaxiale de la feuille r alis e l aide du porom tre SC1 Figure 36 Sch ma de fonctionnement de l appareil Li 6200 Figure 37 Mesures r alis es l aide de l appareil de mesure d changes gazeux Licor 6200 Figure 38 Sch ma de fonctionnement de l appareil Li 6400 Figure 39 Param tres mesur s utilis s dans le calcul de la transpiration et de la conductance stomatique Figure 40 Mes
88. of Carpaccio C and Robusta R leaves Values n 24 Significant differences between O3 exposed and control extracts are indicated by P lt 0 001 Day 2 0 0 4 0 0 11 8 5 4 2 20 93 0 8 15 20 3 8 6 36 6 3 9 17 27 2 E 6 3 373 4 1 exposure severe significant leaf abscission occurred in the two genotypes Table 1 Leaf fall was located at the lower parts of the stems and went on toward the top Leaf fall was more severe in Robusta than in Carpaccio and its rate increased earlier Table 1 At the end of the treatment leaf abscission tended to stabilize for Robusta 36 to 37 while it was still increasing for Carpaccio 2796 Overall careful observation of leaf injuries and monitoring of leaf abscission strengthened our general visual conclusion of a higher O sensitivity of the Robusta genotype Net CO assimilation and total chlorophyll contents The two genotypes displayed comparable rates of CO assimilation in control conditions Fig 4A B Physiol Plant 148 2013 In response to treatment CO assimilation by the two genotypes gradually decreased Fig 4A B The decrease in assimilation in response to ozone was significant P 0 05 for the two genotypes at the end of exposure days 15 17 However no significant difference was detected between the two genotypes Table 2 In control conditions the Chl content of ML
89. ont t ajout s chaque plaque Comme pour les qPCR r alis es sur les feuilles des gammes ont t r alis es en triplicat pour chacun des g nes et chacun des g notypes L efficience de la PCR a ensuite t calcul e gr ce elles pour chaque g ne et chaque g notype Les valeurs de Ct ont t caract ris es pour un m me seuil L expression des g nes a ensuite t normalis e gr ce 2 g nes de r f rence GOGAT6 et UBL et trois g nes d int r t stable 05726 KAT3 et GLR2 d termin s par le logiciel GeneNorm VII TECHNIQUES DE MICROSCOPIE PREPARATION D ECHANTILLONS DE STOMATES MICRODISSEQUES Les techniques de microdissection laser permettent d isoler les stomates pour ensuite r aliser des mesures sur des chantillons de stomates purs Afin de microdiss quer ces derniers il convient d isoler l piderme des feuilles lyophilis es Ne pouvant d tacher 37 Mat riel et M thodes l piderme simplement des morceaux d piderme sont d croch s par grattage de la surface de la feuille avec une lame de rasoir au dessus d une lame de verre Les fragments d piderme sont fix s la lame par l ajout et l vaporation de quelques gouttes d thanol Les cellules de garde sont alors d coup es par microdissection laser et catapult es dans un tube Eppendorf gr ce au syst me PALM MicroBeam Zeiss Figure 45 A et B Les stomates se retrouvent isol s dans le tube Eppendorf tandis que sur la lame
90. pathway to produce pyruvate Asc ascorbate CW cell wall DHA dehydroascorbate GSH GSSG reduced oxidized glutathione GR glutathione reductase PPDK pyruvate phosphate dikinase refers to the NADPH produced by cytosolic NADP dehydrogenases 1 on carbon uptake by assessing net CO assimilation and carboxylase activities Rubisco and PEPC 2 on cytosolic NADP recycling enzymatic activities related to PEPC activity and 3 on pyridine nucleotide contents by associating their variations to the activity levels of the NADP dependent enzymes we assayed Material and methods Growth and experimental design Rooted cuttings of two Euramerican hybrid poplar Populus deltoides x Populus nigra genotypes Robusta and Carpaccio were transplanted to 5 1 plastic pots con taining optimized growth soil mixture N P K 14 16 18 1 2 Gramoflor SP1 Universel fertilized by 38 adding 20 g of slow release nutrient granules Nutricote T 100 N P K MgO 13 13 13 2 Fertil Boulogne Billancourt France Drainage was improved by a 2 cm thick layer of clay balls at the bottom of the pots and 1 mgl of magnesium limestone was added to buffer the pH The plants were left in growth chambers for 5 weeks Temperature was maintained at 23 C 1 C day and 20 C 1 C night with a relative humidity of 75 5 day and 85 night and with a 14 h light period Sun T Agro Philips Eindhoven The Netherlands 250 300 umol m s photon flux
91. r paration et la d toxication 125 CONCLUSION ET PERSPECTIVES 126 Conclusion et perspectives Dans les ann es venir une augmentation des concentrations de fond en ozone troposph rique est pr dite du fait de la production par l activit humaine d oxydes d azote NO et de compos s organiques volatils mais aussi du fait de l augmentation des temp ratures de la plan te qui favorise les r actions photochimiques Conscients des enjeux li s cette hausse les pouvoirs politiques dont les instances europ ennes tentent de limiter ces productions afin de limiter les cons quences conomiques et environnementales Pour ce faire un certain nombre d indicateurs de seuil de risque aident d terminer les concentrations en ozone ne pas d passer pour ne pas engendrer d effets d l t res pour la sant humaine mais aussi pour la v g tation N anmoins si des indicateurs de seuil de risque pour la v g tation bas s uniquement sur les concentrations en ozone troposph rique tels que l AOT40 ou le SUM60 donnent une indication du danger les indicateurs mesurant les flux d ozone par le calcul de la conductance stomatique tels que le CUO l AFstY ou le POD permettent quand eux de d terminer le risque d exposition En revanche pour valuer le risque r el encouru il faut mesurer le flux effectif d ozone qui prend en compte les deux moyens de d fense mis en uvre par les plantes savoir la r gulati
92. recyclant le NADP Cette tude a t r alis e en collaboration avec Dr Dghim dans le cadre de sa th se Toutes les analyses d activit ont t r alis es par Dr Dghim ma participation consistant principalement dans les mesures de chlorophylles d changes gazeux folaires et dans le calcul du POD Le manuscrit issu de cette coop ration met en avant les conclusions suivantes ozone entra ne une baisse de l assimilation nette de qui r sulte probablement d une diminution du contenu en chlorophylle ainsi que d un ralentissement de l activit de la Rubisco e ozone provoque en revanche une augmentation de l activit la PEPC probablement li e une fonction anapl rotique pour soutenir la production d interm diaire du cycle de Krebs ozone induit une hausse des activit s des enzymes r g n rant le NADPH et ce de mani re plus prononc e chez le g notype r sistant Carpaccio 107 R sultats e la disponibilit en NADPH est maintenue chez le g notype r sistant tandis qu elle chute chez le g notype sensible en r ponse l ozone Ces r sultats ont t publi s dans Physiologia Plantarum 2013 148 36 50 108 Physiologia Plantarum An International Journal for Plant Biology Physiologia Plantarum 148 36 50 2013 Copyright Physiologia Plantarum 2012 ISSN 0031 9317 Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity Ata Allah
93. relative to ambient relative to number of leaf 97 Carpaccio 27 20 Cima 34 41 Robusta 37 18 Effect of ozone on leaf fall and total biomass relative to ambient condition in three poplar genotypes Carpaccio Cima and Robusta after 18 days of treatment A Figure 1 Ozone induced leaf injury on three poplar genotypes Comparison of ozone induced leaf injury after 17 days when submitted to charcoal filtered air A B C or to 120 nmol mol D E F on three poplar genotypes differing in ozone sensitivity from the most resistant to the most sensitive Carpaccio A D Cima B E and Robusta C F R sultats variables Normality and homoscedasticity of standardized residuals were graphically checked using quantile to quantile and residual vs predicted plots Glutathione concentrations were log transformed to meet the assumptions of ANOVA Factorial ANOVA was used to test for significant differences among genotypes and treatments Student test was used to precise the genotype effect Data were analyzed using R 2 14 2 33 and the nlme package 34 Effects were considered significant when p lt 0 05 Principal component analysis PCA made with SIMCA P 12 0 1 Umetrics Ume Sweden was used for the amino acid data with log transformation when necessary and UV scaling to find out if there were any dominant patterns in the data regarding the treatment sampling time or genotype Heat map and clustering we
94. rentes esp ces qu ils s accumulaient en r ponse l ozone chez Betula pendula Roth Saleem et al 2001 chez Populus tremula x tremuloides H iki et al 2009 et chez Trifolium pratense L Saviranta et al 2009 Or nous avons pu voir pr c demment que la remobilisation des acides amin s tait favorable la voie de synth se de ces compos s Les carot no des quant eux peuvent agir comme antioxydants par oxydation du radical anion superoxyde et enrayer ainsi la production d autres ROS Galano et al 2010 Han et al 2012 Afin de confirmer chez nos trois g notypes de peuplier euram ricain s il existait la m me augmentation des concentrations en compos s ph noliques et en carot no des et afin d tudier leur r le potentiel dans leur diff rence de sensibilit nous avons conduit une analyse m tabolomique cibl e de ces compos s Lors de l extraction et l analyse des carot no des les chlorophylles a et b ainsi que des terp nols ont t extraits Comme le montre la figure 47 l ozone a un fort effet sur les carot no des les chlorophylles et les terp nols En effet une analyse en composante principale permet de mettre en avant l importance du traitement l ozone qui explique plus de 50 de la variance des chantillons PC1 D une mani re g n rale le g notype le plus r sistant Carpaccio se caract rise par des concentrations en chlorophylles et en carot no des l g rement plus faibles qu
95. the Os treatment with a significant difference on days 11 and 15 Fig 8B The maximum decrease was reached on day 15 6096 of the control value In contrast no significant effect of treatment was noticed on NADPH contents for Carpaccio Fig 8A Consequently genotype effect in O3 treated leaves with higher values for Carpaccio was significant on days 11 and 15 Table 2 NADH contents Fig 8E F followed the same pattern as Carpaccio NADPH contents without any difference between treatments Fig 8A E For Robusta the lower NADH content in O3 treated leaves was not significant Fig 8F For NADH no significant difference was found between the two genotypes all along the ozone treatment Table 2 Concerning the oxidative forms of pyridine nucleotides we observed a decrease in NADP contents for Carpaccio Fig 8C This decrease was significant on day 15 P lt 0 05 For Robusta few changes occurred in NADP pool in response to fumigation Moreover NAD curves showed similar patterns in the O3 treated leaves from the two genotypes nearly twice as high levels as in the control were found after 15 and 17 days exposure for the two genotypes Considering the changes in NAD or NADP contents in the O3 treated leaves no genotype effect was noticed Table 2 Taking these results into account we can consider that in response to exposure the main changes in the pyridine nucleotide forms occurred at the end of
96. the mitochondrial ascorbate synthesizing enzyme L Galactono 1 4 lactone dehydrogenase affects plant and fruit development in tomato Plant Physiol 145 1408 1422 51 Tsuyoshi I Mamiko N Yusuke B Masashi H Kazuko O et al 2009 Importance of the l galactonolactone pool for enhancing the ascorbate content revealed by l galactonolactone dehydrogenase overexpressing tobacco plants Plant Cell Tissue Organ Cult 96 105 112 52 Noctor G 2006 Metabolic signalling in defence and stress the central roles of soluble redox couples Plant Cell Environ 29 409 425 101 R sultats 53 Castro Rodriguez V Garcia Guti rrez A Canales J Avila C Kirby EG et al 2011 The glutamine synthetase gene family in Populus BMC Plant Biol 11 1 16 54 Masclaux Daubresse C Daniel Vedele F Dechorgnat J Chardon F Gaufichon L et al 2010 Nitrogen uptake assimilation and remobilization in plants challenges for sustainable and productive agriculture Ann Bot 105 1141 1157 55 Igarashi D Tsuchida H Miyao M Ohsumi C 2006 Glutamate glyoxylate aminotransferase modulates amino acid content during photorespiration Plant Physiol 142 901 910 56 Bagard M Le Thiec D Delacote E Hasenfrat Sauder MP Banvoy J et al 2008 Ozone induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves Physiol Plant 134 559 574 57 Mouillon JM Aubert S Bourguignon J
97. transduction to activate anion efflux The perturbation of calcium content observed previously may have an impact on the stomatal response to CO increase However calcium signaling is complex and involves various cellular compartments it is therefore difficult to interpret the higher calcium content in guard cell in response to ozone with no information on the 68 R sultats distribution of calcium in cellular compartments Dodd ef al 2010 In addition it has been shown that ozone induces a stress specific calcium signature leading to specific gene expression regulation and that it is not simply a ROS induced regulation Short et al 2012 In conclusion it appears that ozone impacts firstly stomatal conductance of the upper side probably due to greater exposure to ROS Modification of stomatal responses does not result from ultrastructural changes but more likely from a disturbance of ion fluxes and regulation of the expression of genes involved in signal transduction Expression of a majority of the studied genes coding for plasma membrane and vacuolar channels is inhibited by ozone especially the expression of genes coding for the vacuolar calcium channels CAXI and CAX3 Low constitutive expression of CAX1 and CAX3 genes in Robusta could be linked to its slower stomatal responses and thus could participate to its sensitivity Kinetics of mineral element content during 24 hours with measurements of stomatal conductance and calcium a
98. ultrastructure gene expression 57 R sultats INTRODUCTION Forest ecosystems covering 30 of land area of Earth FAO 2006 are ecologically crucial currently constituting the most important carbon sinks Ozone O3 is a tropospheric gaseous pollutant formed by photochemical reactions between sunlight and air containing nitrogen oxides emitted from human activities with biotic and abiotic VOCs Key player of global warming is a greenhouse gas which causes a major impact on plants growth and yield thus limiting the carbon sink possibilities Sitch et al 2007 ICP vegetation 2012 causes important economic losses and represents a threat to ecosystems Hayes et al 2007 Avnery et al 2011a Ainsworth et al 2012 As the tropospheric ozone concentrations are predict to increase in the coming decades the declines in agriculture and forestry productivity will become more important and the food security will be reduced Felzer et al 2007 Van Dingenen et al 2009 Avnery et al 2011b Ozone enters the leaf through stomata and is rapidly converted in the apoplast in reactive oxygen species ROS leading to an oxidative stress This results in visible and physiological damages in plants Wittig et al 2009 At leaf level ozone impairs photosynthesis reduces stomatal conductance and increases respiration Heath 1994 Dizengremel 2001 Ainsworth et al 2012 Stomatal conductance which modulates ozone entrance in the leaf
99. utilisation Mat riel et M thodes 3 MESURES DU CONTENU EN CHLOROPHYLLES PRINCIPE DE L APPAREIL Les mesures en chlorophylles sont importantes car elles sont directement li es aux capacit s photosynth tiques de la plante Les mesures rapides au chlorophylle m tre CCM 200 ont l avantage de ne pas tre destructives L absorbance optique est mesur e deux longueurs d onde diff rentes pour tenir compte de la transmission de la chlorophylle et de l paisseur de la feuille 653 nm chlorophylle et 931 nm pr s des infrarouges UTILISATION Le contenu en chlorophylle de la premi re feuille mature a t suivi 2 fois par semaine gr ce un chlorophylle m tre CCM 200 Opti Sciences Hudson NH USA Les valeurs obtenues Chlorophyll Content Index CCI ont ensuite t converties g m gr ce la relation suivante Bagard et al 2008 1994 1 Chl 0 0214 x CCI 0 0424 IV MESURES DES ECHANGES GAZEUX 1 CONDUCTANCE STOMATIQUE A LA VAPEUR D EAU POROMETRE SC1 PRINCIPE DE L APPAREIL Le porom tre permet de mesurer la conductance stomatique des feuilles sur chacune des faces s par ment Pour r aliser ces mesures la conductance de la feuille est mise en s rie avec deux l ments de conductance connue Figure 34 La diff rence d humidit mesur e entre les l ments permet d obtenir le flux de vapeur d eau La conductance stomatique peut ensuite tre calcul e en se basant sur
100. yeast Sigma dans 3 6 M NH4 SO4 pH7 0 1 mM DTT et bien m langer avant de pr lever Se conserve 1 mois 4 C Stock glutathion r duit GSH 10m 3 mg PM 307 3 g mol dans 1 mL d eau Se conserve 20 C Stock glutathion oxyd GSSG 4 mM 2 45 mg PM 612 6 g mol dans 1 mL d eau Se conserve 20 C 138 Annexes DTNB 5 5 Dithio 2 nitro benzoic acid 1 5 mg mL Pr parer frais au dernier moment dans DMSO Conserver pendant le dosage RT NADPH 50 mM 41 7 mg PM 833 4 Co1Ho6N7Na4O17P3 dans 1 mL NAOH 50 mM Se conserve 20 C PROTOCOLE D EXTRACTION D ANALYSE PAR HPLC DES CAROTENOIDES Toutes les tapes doivent tre r alis es dans l obscurit Iumi re rouge sombre Stocker les chantillons sous atmosph re azot e Peser environ 10 mg de materiel vegetal broy dans un tube Eppendorf Le poids pr cis doit tre connu Ajouter 1000 uL de m thanol contenant 0 01 de BHT butylated hydroxytoluene Ajouter 20 uL 1 mg mL B apo caroten 8 al dans de l hexane contenant 0 01 de BHT Vortexer 4 C 1400 rpm pendant 10 min Centrifuger 12000 rpm pendant 5 min 4 C Prendre le surnageant et commencer le s cher sous atmosph re azot e Ajouter 1000 uL d hexane contenant 0 01 de BHT Vortexer doucement 4 C 1400 rpm pendant 10 min Centrifuger 12000 rpm pendant 5 min 4 C Mettre en commun les surnageants
101. 00 g Vider le r servoir Ajouter 500 uL de tampon RPE Centrifuger 15 s 8000 g Vider le r servoir Ajouter 500 uL de tampon RPE Centrifuger 2 min 8000 g Jeter le tube collecteur et le remplacer par un nouveau Centrifuger 1 min vitesse max 14500 rpm Jeter le tube collecteur et le remplacer par un tube eppendorf 1 5 mL Ajouter sur la membrane directement sans la toucher 50 uL d eau on peut chauffer l eau 65 C au pr alable pour am liorer le rendement Centrifuger 1 min 100 g puis 1 min 8000 g Mettre les tubes dans la glace avant un dosage au Nanodrop Si besoin proc der une seconde lution de 30 uL dans un nouveau tube V PROTOCOLE DE TRAITEMENT DNASE I AMBION Calculer le volume d extrait n cessaire pour avoir 5 ug d ARN environ en se basant sur une mesure au nanodrop Compl ter avec de l eau pour avoir un volume final 44 uL 148 Annexes Ajouter 5 uL de DNA buffer Ajouter 1 uL de rDNAse I M langer doucement en retournant le tube Incuber pendant 20 30 min 37 C Ajouter 5 uL r actif d inactivation Dnase Incuber 2 min temp rature ambiante en m langeant occasionnellement Centrifuger 1 min 30 10000 g Transf rer le surnageant dans un nouveau tube Aliquoter sur barette 4 uL pour le dosage au Ribogreen et la v rification l exp rion VI PROTOCOLE DE VERIFICATION DE LA QUALITE DES ARNSAL EXPERION Pr pa
102. 000 su su su su ewp su su su su 100 0 gt 51 89900 0 su su su su su su su su oned ie 133JJ3 STO 580 LUO F 600 PTO SEO 200 581 6E0F S9 I 9C0 F 601 8 00 SFIE C T8C 100 t FOO 100 800 COO POOF 800 100 ILO 500 91020 800870 3SCO T6 0 6607600 970 801 820 F 8UI SPOT 690 6760 L tcCC 8 COO 900 COO 400 100 F 900 00 270 F 110 100 F 910 jusmury eJsnqoy TUO 990 810 F 080 LEO LEO LTO FISI ILOFSEC CVOTCSI VFIVE 9FEC 00 CIO 100 S00 COO CIO 1007 CLO 100 F 210 91020 800 600 ESOFLIO SSO FZEO SL O SOT EO FOSI LVOTLEI 6049 8765 TOO FIL0 200 9UO 600 F 610 200 600 200 ZOO 00 JU3Iquiy eun FCTEO 8VOFOLI 870 900 6671 EEO 660 LCOT 960 8750 002780 PH FO9 100 F 00 200 F S00 COO POO F 600 100110 200 F 900 91020 SLOT 5650 120 100 7 670 500 101 120 601 970 01 5108 87964 ELT 856 100 FELO CLO 100 FELO 1007 800 200 400 00 F 400 jusmqury 4 I t I T I t I T I oum usunean LV A 58 oss SI9jourPeIed adAjoua5y 7500 gt d JUPIYIUSIS paroprsuoo are uorperojur adAJOUSS pue adAjouas pue JUJUL 3430 Jaye uonejmursse 8 ourr Ly uorjerreA 1980 aniq UONeqruwuisse s jou oy paads Suruedo ejeurojs 5 A
103. 009 L ozone est hautement r actif et peut interagir avec les composants de la paroi cellulaire des stomates et du m sophylle Il est dissous en quasi totalit dans l apoplasme produisant des ROS Reactive Oxygen Species comme le peroxyde d hydrog ne H202 le radical le radical perhydroxyle HOO le radical superoxyde O27 le radical hyperoxyde ou l oxyg ne singulet O Langebartels et al 2002 Ashmore 2005 Wittig et al 2007 Wittig et al 2009 Ces compos s sont des mol cules tr s toxiques pouvant modifier ou d truire les constituants cellulaires comme les prot ines les lipides les pigments les acides nucl iques Ishida et al 1999 Cependant les ROS sont aussi des mol cules de signalisation produites naturellement par la plante Mittler et al 2004 Karuppanapandian et al 2011 Les ROS sont majoritairement produites au niveau des chloroplastes lors de la photosynth se et la photorespiration Asada 2006 des mitochondries par la chaine de transport des lectrons Rhoads et al 2006 des peroxysomes lors de la photorespiration Del Rio et al 2006 Les ROS interviennent notamment dans le contr le de la r ponse de d fense aux pathog nes et du programme de mort cellulaire Foyer and Noctor 2005 Le peroxyde d hydrog ne est relativement stable et son habilet traverser les membranes notamment par les aquaporines en fait un bon messager Bienert et al 2007 Il p
104. 06 61766 OL T 6C COO 200 00 800 100 T 200 500 120 900 F ZCO 000 620 quaiquiy omedreg T L T L T L T T L LV A 587 058 siajouueleq adAjouay G0 O0 gt d JULIYIUBIS 5 adAjous8 x pue PJ d zou 3 pue jusuneor uonerreA 1480 w joum Ly uorerreA 1480 uone rursse 5 ur OY peeds jejeurojs 5 ur ourur uonerreA o uejonpuoo 45 jo 5 ur ourur 58y syaam E 10 T J Joye pal Joye uonepusse s Sy _S 2 3U3W P 01JU09 JOU 10 EQ Jo our jowu 021 panrumqns eysnqoy pue ewn or ed re sadAjouas re dod 33147 ayy JO 45 F sueaut 5 ur 008 o3 002 W014 1480 pal e sasuodsai oSueuoxo ses ALILU Q z su su su su su su su su su su su su su su su t9 oro edie ejsnqoy su su su su su su su su su su LL 0 O su su su su t9 e1snqoy eui su su su su su su su su su su su su su su su 9 eurr oroed1e queues su su su su su su 1000 1000 gt su su su su su su su oroedae ejsnqoy su su su su su SU Z8600 0 6SZc0 0 su 98 00 0 LLVc0 O 10007 su su su ejsnqoy eu su su su su su su su su su su su su su su su eulr oroed1e 33yj sdAjous9 100 su su su su su su su su su su su su su su ejsnqoy su su su su su su su su su su 167
105. 08 Ozone risk assessment for plants Central role of metabolism dependent changes in reducing power Environ Pollut 156 11 15 97 R sultats 20 Matyssek R Wieser G Calfapietra C De Vries W Dizengremel P et al 2012 Forests under climate change and air pollution Gaps in understanding and future directions for research Environ Pollut 160 57 65 21 Di Baccio D Castagna A Paoletti E Sebastiani L Ranieri A 2008 Could the differences in sensitivity between two poplar clones be related to a difference in antioxidant defense and secondary metabolic response to O3 influx Tree Physiol 28 1761 1772 22 Brosch M Merilo E Mayer F Pechter P Puz rjova I et al 2010 Natural variation in ozone sensitivity among Arabidopsis thaliana accessions and its relation to stomatal conductance Plant Cell Environ 33 914 925 23 Dumont J Spicher F Montpied P Dizengremel P Jolivet Y et al 2013 Effects of ozone on stomatal responses to environmental parameters blue light red light CO and vapour pressure deficit in three Populus deltoides x nigra genotypes Environ Pollut 173 85 96 24 Rice Evans CA Miller NJ Paganga G 1997 Antioxidant properties of phenolic compounds Trends Plant Sci 2 152 159 25 Moller IM Jensen PE Hansson A 2007 Oxidative modifications to cellular components in plants Annu Rev Plant Biol 58 459 481 26 Wieser G Matyssek R 2007 Linking ozone uptake and defe
106. 1 Oud pysoussy ixguso VVOVIOLODOVVOOLOVDOLL 076015100 ULdOd 81269 2 20 GOT Sopoqo oo poy dna C6LOV T00 couosno DIVOLOVVOVOODOOLLLVV snjndod DAO H eN IIS 0 6006200 CIXHN2d VOVOLLVOVVOVODIVVOOD 006PE IDP T0 Od vILXHN 8 85 0 0 08 OTTEOSETOO ULdOd OSILZBCTV 20 ZJOZ 09 rayns 6620 lt 100 ODVVVODOLVOVIVOVVIOD DUO H eN 0 17955616000 INXBou VOLLLOLVOVIODIOOODVV OOLTEODE TO Od TIXHN L0000X DT Oud 81 69HVO JO JOT Sopoqo oo ponmuidno 042080100 ULdOd VVOVOODVIVOODVVOVDLL snndoJ O4O H eN IIS 7967616000 INXIOM U IXHN d WVDLOLODLLLLODDOLVVO 00STE0DO10 1304 OTIXHN L8tV8SVV 0 0s08c1V 0997085000 LLdOd OSILZBCTV Jo 7101 09 rayns 97100100 couosno IOLOLODODLLVVOVDDOLL 08D21paj tydO 1o10dnue H eN 0 pus l rescecoo INXBou VOLLOLOVOVOVOVVOOOOV 001SP0OS00 1304 IXHN 0 9 8 Jo S307 09 T THNNVHO WNISSVLOd 1191688000 WXPPA SSLOOOIA DT Oud pusouos 0 epus TINNVHO USI6 200 NX 07192859000 ULdOd DVVVVDDLVLODLVDOVVOD A PNVITVHL 51540 NS l 26680 700 INXIOM VIOMLVId VODVVVDOVOLLVODVVDVOO 00057209001 04 9CLVM IOLLOOOLIOOOLLLVIDOLVO 1 0 INXIoM 0160058100 ULdOd VOVIO
107. 10 En Europe les niveaux ambiants d ozone sont suffisamment lev s pour causer des blessures visibles chez les esp ces natives L valuation des l sions visibles est utilis e pour d tecter des zones de risques potentiels o les effets de l ozone sur les for ts sont les plus marqu s Le suivi de ces dommages foliaires est incorpor dans des programmes de surveillance une chelle europ enne dans le cadre des protocoles d ICP Forest et FutMon EU Life Les d g ts foliaires dus l ozone peuvent se voir l il nu et sont caract ristiques de ce polluant Ces sympt mes sont plus marqu s sur les feuilles g es o ils apparaissent en premier et moins importants sur les feuilles d ombre o ils sont presque inexistants Ils se limitent g n ralement la face sup rieure des feuilles et peuvent prendre la forme d une d coloration Figure 8 de rougeur ou de brunissage entre les nervures ou encore de plus ou moins grandes n croses Figure 8 B et C Vollenweider 2003 L ozone entraine une chute pr matur e des feuilles Chez les conif res comme pour les feuilles les sympt mes sont limit s la face sup rieure des aiguilles et on retrouve un effet ge et un effet lumi re Les marbrures chlorotiques sont le sympt me le plus commun d crit pour les aiguilles de conif res Elles peuvent tre d crites comme une alternance de jaune ou de vert p le de taille similaire sans fronti res nettes Figure 8D
108. 10 0 10 20 30 Percent change from CF air Figure 12 Variation en pourcentage de la photosynth se en lumi re saturante et de la conductance stomatique gs des arbres soumis des concentrations lev es en ozone par rapport aux t moins expos s l air filtr par charbon actif cf et l impact de diff rentes concentrations en ozone O sur la r ponse Les degr s de libert d f et les concentrations en ozone sont donn s le long de l axe y Wittig et al 2007 40 3 30 20 10 0 10 20 Photosynthesis HH 1 455 86 ppb Stomatal conductance 276 91 ppb Transpiration HOH 104 76 ppb Respiration o 56 75 ppb Total chlorophyll HH 176 76 ppb E Chlorophyll a 0 4 58 89 ppb Chlorophyll b _ 3 52 87 2 Chlorophyll a b ratio I CHH 68 82 ppb 8 Rubisco content i C 3 46 92 ppb Rubisco activity 0 4 58 154 Nitrogen content 184 78 ppb Suctose L 0 3 i 48 62 ppb Starch o 95 72 ppb 40 30 20 10 0 10 20 Percent change from charcoal filtered control Figure 13 Variation en pourcentage de la photosynth se de la conductance stomatique de la transpiration de la respiration de la chlorophylle totale de la chlorophylle a de la chlorophylle b du ratio entre chlorophylle a et b du contenu en rubisco de l activit de la rubisco du contenu en nitrog ne du sucrose et de l amidon des arbres soumis
109. 103 35 Saeed AI Sharov V White J Li J Liang W et al 2003 TM4 a free open source system for microarray data management and analysis Biotechniques 34 374 378 36 Saeed AL Bhagabati NK Braisted JC Liang W Sharov V et al 2006 TM4 microarray software suite Methods Enzymol 411 134 93 99 R sultats 37 D Haese D Vandermeiren K Asard H Horemans N 2005 Other factors than apoplastic ascorbate contribute to the differential ozone tolerance of two clones of Trifolium repens L Plant Cell Environ 28 623 632 38 Booker FL Burkey KO Jones AM 2012 Re evaluating the role of ascorbic acid and phenolic glycosides in ozone scavenging in the leaf apoplast of Arabidopsis thaliana L Plant Cell Environ 35 1456 1466 39 Munn Bosch S Queval G Foyer CH 2013 The impact of global change factors on redox signaling underpinning stress tolerance Plant Physiol 161 5 19 40 Ranieri A Castagna A Padu E Moldau H Rahi M et al 1999 The decay of O3 through direct reaction with cell wall ascorbate is not sufficient to explain the different degrees of O3 sensitivity in two poplar clones J Plant Physiol 154 250 255 41 Galant A Preuss ML Cameron JC Jez JM 2011 Plant glutathione biosynthesis diversity in biochemical regulation and reaction products Front Plant Sci 2 1 7 42 Noctor G Mhamdi A Chaouch S Han Y Neukermans J et al 2012 Glutathione in plants an integrated overview Plan
110. 16 AMBIENT OZONE o o Normalized expression o o gt o n 0 0 T T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day sampling dates day VTC211 AMBIENT OZONE 154 10 Normalized expression 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA VTC4162 AMBIENT OZONE 12 o o L Normalized expression o o 1 1 o 0 0 T T CARPACCIO CIMA ROBUSTA T CARPACCIO CIMA ROBUSTA sampling dates day sampling dates day 2 4 11 15 17 Figure S2 Normalized expression of genes involved in glutathione biosynthesis in leaves of three poplar genotypes Carpaccio Cima and Robusta exposed to ambient or ozone treatment at 2 4 11 15 and 17 days Mean SE GlyA1 AMBIENT OZONE o o o gt o L Normalized expression o d h 0 0 2 m 11 15 17 sampling dates day T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GlyA6 AMBIENT OZONE o 1 o 6 Normalized expression o 0 0 2 0 lul T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA GlyA10 sampling dates day 2 4 11 15 17 ROBUSTA AMBIENT OZONE o o o m o o 1 1 Normalized expression o 0 0 sampling dates d
111. 177 Provence Alpes C te d azur 900 418 Rh ne Alpes 7 700 10 836 Total 185 100 233 406 Tableau 6 R colte annuelle de grumes de peupliers m Ann e D roulage Sciage Total 1988 1 271 267 1 594 960 2 866 227 1989 1 459 434 1 798 902 3 258 336 1990 1 543 139 1 865 680 3 408 819 1991 1 514 798 1 722 431 3 237 229 1992 1 331 655 1 525 557 2 857 212 1993 1 254 010 1 223 380 2 477 390 1994 1 381 740 1 216 459 2 598 199 1995 1 334 250 1 225 594 2 559 844 1996 1 246 355 1 075 937 2 322 292 1997 1 267 212 1 059 475 2 326 687 1998 1 239 646 991 653 2 221 299 1999 1 196 186 966 518 2 162 704 2000 1 125 856 821 908 1 947 764 2001 1 086 718 710 237 1 796 955 2002 943 446 536 793 1 480 239 2003 902 922 528 433 1 431 355 2004 872 668 443 402 1 316 070 2005 847 124 478 151 1 325 275 2006 862 145 531 006 1 393 151 2007 993 568 581 716 1 575 284 2008 914 245 508 278 1 422 523 2009 885 706 407 043 1 292 749 Figure 29 D but de l acclimatation en phytotrons des peupliers g s de 5 semaines Mat riel et M thodes constitu es de cultivars issus de croisements entre deux esp ces nord am ricaines P deltoides x P trichocarpa peuplier interam ricain ou une esp ce nord am ricaine et une esp ce eurasiatique P deltoides x P nigra peuplier euram ricain 2 GENOTYPES UTILISES Trois g notypes euram ricains Populus deltoides x Populus nigra Carpaccio Cima et Robusta ont t s lectionn s dans l quipe pa
112. 2 Elsevier Ltd All rights reserved http dx doi org 10 1016 j envpol 2012 09 026 2010 and MEDDTL 2010 Such concentrations might be more frequent in the future stress leads to physiological Ainsworth et al 2012 Matyssek et al 2010 in beech biochemical Dizengremel 2001 Renaut et al 2009 in poplar and Wittig et al 2009 and molecular effects Castagna and Ranieri 2009 Heath 2008 Kangasj rvi et al 1994 This causes substantial declines in agriculture and forestry productivity linked to economic losses Avnery et al 2011 Felzer et al 2007 Van Dingenen et al 2009 Differences in sensitivity between tree species and even within species between genotypes are observed Betzelberger et al 2010 Hayes et al 2007 Sensitivity depends on the two defence mech anisms set up by plants i e regulation of stomatal conductance and detoxification of the reactive oxygen species generated during degradation Brosch et al 2010 Dizengremel et al 2008 As the present situation is becoming worrying it is urgent to improve the accuracy of the exposure indices used to assess risk Critical level indicators based only on atmospheric concentra tions as AOT40 Accumulated Ozone over a Threshold of 40 ug m 3 h and SUM60 sum of all hourly average concen trations over 60 nmol mol were previously used because they were easy to calculate but inaccurate Fuhrer et al 1997 Gerosa et al 2009 K
113. 25 0 06 0 03 0 Faire de m me avec une gamme talon GSSG mais partir 25 uL 655 4 mM dans 975 ul d eau 0 1 mM final Le robot va pr lever 10 uL d extrait pour chaque plaque Ajouter 1 uL de VPD 0 4 M la moiti des plaques Mettre toutes les plaques incuber 1 h RT 137 Annexes Pendant ce temps pr parer le tampon 1X pour 24 mL 5 2 mL de KH2 K2H 0 5 M pH7 0 24 mL EDTA 0 5 M 18 56 mL d eau Pr parer la GR 8 uL de GR dans 492 uL de Tp 1X Pr parer le NADPH 2 mM 200 uL de 50 mM dans 4 8 mL de Tp 1X Pr parer le DTNB Pr parer le MIX M langer 16mL de tampon 460 uL de GR 460 uL de DTNB Ne pas mettre le MIX dans la glace et faire au dernier moment Mettre une cuve avec le MIX et une plaque PCR avec du NADPH sur le robot Le robot va ajouter 150 uL de MIX en agitant Il ajoute ensuite 5 uL de NADPH plaque par plaque avec entre chaque plaque un message demandant s il passe la suivante Prendre chaque plaque l agiter et lire toutes les 30 sec pendant 10 min 405 nm Solution pr parer 0 5 M tampon phosphate pH7 a KoHPO 12H20 0 5 M b KH PO4 1H20 0 5 M Ajouter b a pour tre pH7 Se conserve 1 mois 4 C utiliser temp rature ambiante EDTA 0 5 M pH7 Se conserve 1 mois 4 C Acide m taphosphorique 5 5 g dans 100 mL d eau Se conserve 1 mois 4 C Glutathion R ductase 100 U 220uL Baker
114. 64 Ainsworth EA Yendrek CR Sitch S Collins WJ Emberson LD The effects of tropospheric ozone on net primary productivity and implications for climate change Annual Review of Plant Biology 2012 63 637 61 Asada K Production and scavenging of reactive oxygen species in chloroplasts and their functions Plant physiology 2006 141 391 396 Ashmore MR Assessing the future global impacts of ozone on vegetation Plant Cell amp Environnement 2005 28 949 964 Assmann SM Snyder JA Lee Y RJ ABA deficient abal and ABA insensitisve abil 1 abi 1 2 mutants of Arabidopsis have a wild type stomatal response to humidity Plant Cell amp Environment 2000 23 387 395 Avnery S Mauzerall D L Liu J Horowitz L W Global crop yield reductions due to surface ozone exposure 1 Year 2000 crop production losses and economic damage Atmospheric environment 2011 45 2284 2296 Bagard M Le Thiec D Delacote E Hazenfratz Sauder MP Banvoy J G rard J Dizengremel P Jolivet Y Ozone induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves Physiologia Plantarum 2008 134 559 574 Ball JT Woodrow IE Berry JA A model predicting stomatal conductance and its ontribution to the control of photosynthesis under different environmental conditions Progress in Photosynthesis Research ed I Biggins Martinus Nijhoff Publishers Netherlands 1987 221 224
115. 7 La d toxication constitutive est d j op rationnelle l entr e de l ozone dans les feuilles En effet les ROS tant produits naturellement dans diff rents compartiments cellulaires les acteurs de la d toxication sont toujours actifs Chaque compartiment cellulaire contient ses propres agents de d toxication L ascorbate pr sent dans l apoplasme sous forme r duite ASA constitue la premi re barri re s opposer l ozone et aux ROS Castagna and Ranieri 2009 Plus les concentrations en ozone et en ROS sont fortes plus l ascorbate r duit sera oxyd La d toxication inductive renforce la d toxication constitutive en produisant et en r g n rant les antioxydants mais n cessite beaucoup d nergie Musselman ef al 2006 alors que la photosynth se est limit e par l ozone Le m tabolisme primaire joue alors un r le 13 Synth se bibliographique important pour supporter ces processus Les sucres solubles en tant que source d nergie repr sentent de ce fait un facteur cl De plus ils associent un r le de signalisation impactant l expression de g nes Gupta et Kaur 2005 des capacit s d osmoprotection et de d toxication notamment le galactinol et le raffinose qui seraient capables de d toxiquer les radicaux hydroxyles Nishizawa et al 2008 Le pouvoir r ducteur des syst mes de d toxication d rive directement ou indirectement du NAD P H ou de la ferr doxine sauf la SuperOxyde Dismutase SOD et la
116. 7 02 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Normalized expression GT8 AMBIENT OZONE 1 5 7 5 sampling dates day 21 0 2 a Fh N 11 c 15 17 5 205 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GT16 OZONE o L o Normalized expression o o o o 0 0 Normalized expression o o o o 2 11 15 17 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA THA19 AMBIENT 4 o Normalized expression o a 1 0 0 CARPACCIO ROBUSTA CARPACCIO ROBUSTA i sampling dates day 2 4 11 15 17 0 0 sampling dates day 2 E 4 11 15 17 T CARPACCIO CIMA T T ROBUSTA CARPACCIO CIMA ROBUSTA Figure S3 Amino acids and polyamines content in leaves of three poplar genotypes Carpaccio Cima and Robusta exposed to ambient or ozone treatment at 2 4 11 15 and 17 days Mean SE umol g DW with alanine d4 as the internal standard Total amino acids Alanine ALA AMBIENT OZONE AMBIENT 020 200000 T 6000 gt 150000 TT L sampling dates sampling dates day day 2 2 2 2 8 4000 4 100000 11 Z 11
117. 98 Guidi L Degl Innocenti E Martinelli F Piras M 2009 Ozone effects on carbon metabolism in sensitive and insensitive Phaseolus vulgaris cultivars Environ Exp Bot 66 117 125 Gupta VK Singh R 1988 Partial purification and characterization of NADPC isocitrate dehydrogenase from immature pod walls of chickpea Cicer arietinum L Plant Physiol 87 741 744 Haikio E Freiwald V Julkunen Tiitto R Beuker E Holopainen T Oksanen E 2009 Differences in leaf characteristics between ozone sensitive and tolerant hybrid aspen Populus tremula x P tremuloides clones Tree Physiol 29 53 66 Hayashi M Takahashi H Tamura K Huang J Yu LH Kawai Yamada M Tezuka T Uchimiya H 2005 Enhanced dihydroflavonol 4 reductase activity and NAD homeostasis leading to cell death tolerance in transgenic rice Proc Natl Acad Sci USA 102 7020 7025 Heineke D Riens B Grosse H Hoferichter P Peter U Fl gge U I Heldt HW 1991 Redox transfer across the inner chloroplast envelope membrane Plant Physiol 95 1131 1137 Hodges M Flesch V Galvez S Bismuth E 2003 Higher plant NADP dependent isocitrate dehydrogenases ammonium assimilation and NADPH production Plant Physiol Biochem 41 577 585 Igamberdiev AU Gardestr m P 2003 Regulation of NAD and NADP dependent isocitrate dehydrogenases by Physiol Plant 148 2013 reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves Biochim Biophys Acta 1606 117 125
118. AU LI 6200 PRINCIPE DE L APPAREIL L appareil Li 6200 est un syst me ferm portable de mesure de photosynth se permettant de r aliser des mesures d changes gazeux pas de distinction des faces La feuille est plac e dans une chambre laissant passer la lumi re dans laquelle sont mesur es la temp rature de l air et de la feuille et l humidit relative Une pompe aspire l air de la chambre pour le faire passer par un analyseur de avant d tre r inject dans la chambre Figure 36 27 Figure 37 Mesures r alis es l aide de l appareil de mesure d changes gazeux Licor 6200 Mat riel et M thodes Lorsque la feuille respire elle d gage du CO et lorsqu elle photosynth tise elle consomme du L change de CO entre la feuille et l atmosph re est calcul en mesurant le changement de concentration de CO dans la chambre ferm e contenant la feuille dans un intervalle de temps court 30 s L assimilation nette de CO est alors calcul e en utilisant le taux de changement de mesur et d autres facteurs comme la surface de feuille enferm e le volume de la chambre la temp rature et la pression atmosph rique Lorsque les stomates sont ouverts la plante transpire entrainant une hausse de l humidit de l air dans le circuit ferm L utilisation de dessicant perchlorate de magn sium permet de maintenir une humidit constante 596 Le taux de transpiration est calcul par le c
119. BIOMASSE ET LA CROISSANCE Les concentrations actuelles en ozone entrainent d j une baisse de la croissance en diam tre et en biomasse chez de nombreuses esp ces dont une perte de biomasse totale de 7 chez les esp ces foresti res Wittig et al 2009 On estime une baisse de rendement d environ 4 pour le mais 9 pour le bl et 11 pour le soja Avnery et al 2011 Wilkinson et al 2011 Les baisses de croissance et les pertes en biomasse des concentrations un peu plus lev es Figure 9 laissent envisager que dans les ann es venir l impact de la pollution l ozone sur la croissance et la biomasse sera pour les arbres 10 plus important qu aujourd hui Wittig et al 2009 De plus les angiospermes sont plus sensibles que les gymnospermes Figure 10 EFFETS SUR LES ECHANGES GAZEUX FOLIAIRES Les concentrations actuelles en ozone 50 ppb sont suffisantes pour r duire les capacit s photosynth tiques des angiospermes Figure 11 Wittig et al 2007 Si les concentrations en ozone continuent de croitre la photosynth se des gymnospermes moins sensibles l ozone pourrait elle aussi tre r duite Dans le cas d une exposition aig e mais ponctuelle l ozone entraine une baisse r versible de la conductance stomatique et donc de la photosynth se tandis que dans le cas d une exposition chronique m me des concentrations d ozone plus faibles la r duction de la conductance stomatique et de la photosynth se est
120. Cima with a polynomial regression of order 2 Fig 6E Finally A gy was about the same for all three genotypes Fig 5D F and it increased with treatment during the first week only After 10 days the difference disappeared ns ns 0 001 0 001 0 013 ns ns 3 3 Effects of ozone on the responses to variations in blue light ns In control air conditions dark to blue light transition induced a significant genotypic effect on the amplitude of stomatal opening Table 3 Cima displayed the biggest amplitude more than double compared to Robusta which displayed the lowest In addition there was also a significant genotypic effect on the opening speed Robusta was the slowest with reaction rates 2 3 times lower than Carpaccio and Cima had no effect on the opening rate or speed of Robusta whereas the amplitude and the speed of the response were reduced for the other two genotypes Table 3 Under O3 treatment the amplitude of the response decreased in Carpaccio by around 75 and was around 40 lower for Cima Opening speed was reduced by around 65 and 55 for Carpaccio and Cima respectively from the second week onward The effects of O3 on stomatal conductance were not linked to Ci variations whose values remained stable data not shown In control air conditions there was no genotype effect on net CO assimilation in the dark or in blue light conditions Table 3 In the dark the respiration of the three
121. DHA GSH NADP B evn oD Figure 22 Double effet des ROS g n r s par l ozone sur les processus de d toxication et sur la r g n ration m tabolique du pouvoir r ducteur Dizengremel et al 2009 Abr viations CW Paroi cellulaire P Membrame plasmique Asc Ascorbate r duit DHA Ascorbate oxyd GSSG Glutathion oxyd GSH Glutathion r duit gt DHA apoplast o AS cytosol and chloroplast Figure 23 Voie d Halliwell A sada Castagna et Ranieri 2009 Tableau 2 M canismes de d toxication des ROS dans les cellules v g tales Moller et al 2007 M canismes Consomm s produits Localisation cellulaire SOD H202 Chl Cyt Mit Per Catalase H202 H20 Mit Per Peroxydases PODs H20 H20 Nombreuses localisations Ascorbate Cycle Glutathion H202 H20 Chl Cyt Mit Per H20 Glutathion peroxydases Lipides hydroperoxydes Chl Cyt RE Mit Autres hydroperoxydes H202 H20 Syst me peroxyredoxine Alkyle hydroperoxydes Chl Cyt Mit Noy Peroxynitrite Syst me Thioredoxine HO H20 Chl Cyt Mit H202 20 Syst me glutaredoxine Chl Cyt Mit Sec Hydroperoxydes Carot nes et tocoph rol 10 O2 Chl Chl Chloroplastes Cyt Cytosol RE R ticulum Endoplasmique Mit Mitochondrie Noy Noyau Per Peroxysomes Sec Voie s cr toire Synth se bibliographique La d hydroascorbate r ductase r duit le DHA obt
122. Kleist E Wildt J 2008 Stomatal uptake and stomatal deposition of ozone in isoprene and monoterpene emitting plants Plant Biol 10 44 54 Fontaine V Pelloux J Podor M Afif D G rant D Grieu P Dizengremel P 1999 Carbon fixation in Pinus 48 halepensis submitted to ozone Opposite response of ribulose 1 5 bisphosphate carboxylase oxygenase and phosphoenolpyruvate carboxylase Physiol Plant 105 187 192 Fontaine V Caban M Dizengremel P 2003 Regulation of phosphoenolpyruvate carboxylase in Pinus halepensis needles submitted to ozone and water stress Physiol Plant 117 445 452 Foyer CH Noctor G 2011 Ascorbate and Glutathione The Heart of the Redox Hub Plant Physiol 155 2 18 Fuhrer J Skarby L Ashmore MR 1997 Critical levels for ozone effects on vegetation in Europe Environ Pollut 97 91 106 Fukayama H Hatch MD Tamai T Tsuchida H Sudoh S Furbank RT Miyao M 2003 Activity regulation and physiological impacts of maize C4 specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants Photosynth Res 77 227 239 Gaucher C Costanzo N Afif D Mauffette Y Chevrier N Dizengremel P 2003 The impact of elevated ozone and carbon dioxide on young Acer saccharum seedlings Physiol Plant 117 392 402 Gerosa G Finco A Mereu S Marzuoli R Ballarin Denti A 2009 Interactions among vegetation and ozone water and nitrogen fluxes in a coastal Mediterranean maquis ecosystem Biogeosciences 6 1783 17
123. LOY merus 19q 0 puns oye AXOA S OUTUR e 0 9016200 IT SSSOT TOO IT 6 T60 700 NX IT 6IT O TO0 IWXI W T LSHPOETOO INXI W 166646500 WNXI W 9LI8000 ous8no T ILVOV 1d 0074810800 1104 16661 IA TMS TTLOV Id 009580000 1304 T 696 TA IMS 0089019900 10d 009cL0D 00 1104 0018 0D 00 1104 0089T09 10 104 0069c10 00 1104 L 68I OT OTA IOSIMOUOD jxgiso 00570101007 04 LLLOODVVOLLOVODDD LIV VIVOODLLLOOODLIVVOOD L VVODIODVDLVOLODVIVOD ILVOLVVOLOLLLOVOODDDO VOOIOOVOVLLVVDVOLLLLOL VIOLLOOLVOOLVOVVIODVV VVOLLVOODOLLLOVOJIVOD VOVVVDDVODVOVVVDIODO VDLIVOLIOLLLVIODVOOOOD VVVVDODV IVOOVVVDODDD L VDLIVOLIOLLLOLOLIVOIOOD DVDLVVOLLV LOILODDOOLV OLLOLIOODVVIOIODDLLL DVOLVOVOOVV IODIDIOOV VDLLIOIOVODIODDOOLLLV 0916198000 ULdOd 0619051000 ULdOd OtL0TS9000 ULdOd OTOLOSEOOO ULdOd 070708000 ULdOd O8LTOSETOO ULdOd 00LTIS 000 ULdOd 076081000 ULdOd 810 LID 910 ELD STHSD ITHSO THSO ITHSO L9EE8HV Jo c Jo ojox opre 0j 06298 10 ds esoonj 0 ets 06 6 69 0 sisdopiqpay
124. M A control without PEP was prepared for each assay All the protocols used to measure cytosolic NADP dependent enzymatic activities were based on NADPH formation at 340nm NADP G6PDH EC 1 1 1 49 activity was determined following Pitel and Cheliak 1986 who used a 50 mM HEPES buffer pH 7 6 The pH value was optimized for assaying the activity of the cytosolic isoform the optimal pH for the chloroplastic isoform is 8 5 NADP ME activity 1 1 1 40 was determined according to Casati et al 1997 NADP ICDH EC 1 1 1 42 activity was measured according to Gupta and Singh 1988 Cytosolic NADP GAPDH 1 2 1 13 activity was assayed following Bustos and Iglesias 2003 Pyridine pool determination The determination of NAD H and NADP H contents was based on the reduction of dichlorophenolindophe nol by phenazine methosulfate enzymatically cou pled with alcohol dehydrogenase and NADP G6PDH respectively According to the protocol described by Queval and Noctor 2007 slightly modified 100mg of leaf powder was homogenized 2 min with 2 tung sten balls using a Retsch MM301 mixer with 1 ml of 2N NAD or 1 ml of NaOH 0 2M NADH NADPH The mixture was centrifuged at 16000 g for 10 min and the supernatant was neutralized Absorbance reading at 600 nm was initiated after adding 10 ul of the appropriate enzyme in the presence of 20 pl of the assay extract and using a microplate spectropho tometer Biotek P
125. Nature 406 731 734 Pinheiro J Bates D DebRoy S Deepayan S R Development Core Team 2012 nlme Linear and Nonlinear Mixed Effects Models In R Package Version 3 1 103 Pleijel H Danielsson H Vandermeiren K Blum C Colls J K 2002 Stomatal conductance and ozone exposure in relation to potato tuber yield results from the European CHIP programme European Journal of Agronomy 17 303 317 R Development Core Team 2012 R a Language and Environment for Statistical Computing R Foundation for Statistical Computing Vienna Austria ISBN 3 900051 07 0 URL http www R project org Renaut J Bohler S Hausman J F Hoffmann L Sergeant K Ahsan N Jolivet Y Dizengremel P 2009 The impact of atmospheric composition on plants a case study of ozone and poplar Mass Spectrometry Reviews 28 495 516 Shimazaki K I Doi M Assmann S M Kinoshita T 2007 Light regulation of stomatal movement Annual Review of Plant Biology 58 219 247 Sitch S Cox P M Collins W J Huntingford G 2007 Indirect radiating forcing of climate change through ozone effects on the land carbon sink Nature 448 791 795 Ton J Flors Mauch Mani B 2009 The multifaceted role of ABA in disease resistance Trends in Plant Science 14 310 317 Torsethaugen G Pell E J Assmann S M 1999 Ozone inhibits guard cell K channels implicated in stomatal opening Proceedings o
126. O CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Isoleucine ILE AMBIENT OZONE quantity 2000 1500 1000 500 0 T T CARPACCIO CIMA ROBUSTA i sampling dates day sampling dates day 2 4 11 15 17 CARPACCIO CIMA ROBUSTA Leucine LEU Lysine LYS AMBIENT OZONE 1500 1000 quantity sampling dates day 2 F1 4 11 15 17 T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Methionine_MET AMBIENT OZONE 200 150 5 100 5 c 50 E CARPACCIO CIMA ROBUSTA CARPACCIO CIMA sampling dates day 2 11 15 17 ROBUSTA AMBIENT OZONE 1000 800 il T T CARPACCIO CIMA ROBUSTA quantity 8 a 8 T CARPACCIO CIMA Ornithine ORN ROBUSTA OZONE quantity 604 504 40 304 20 10 0 T T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day 2 4 11 15 17 sampling dates day 2 4 11 15 17 Phenylalanine_PHE AMBIENT OZONE quantity 5 8 1 6000 2000 CARPACCIO CIMA ROBUSTA T T T CARPACCIO CIMA ROBUSTA Putrescine EUT AMBIENT 600 500 200 100 0 T T CARPACCIO CIMA ROBUSTA 8 quantity 8 CARPACCIO CIMA ROBUSTA Spermidine SPE AMBIENT
127. ODIV 0089901001204 IDIdL 6L791CAVV p301 Sojoquo oo upums DAUDUDS DYO TMOOdS uinissejod 10 1100001 DT Oud pysoussy LLOOLOLVOOOOLLOLV LL Figure S1 o Normalized expression o a 0 0 2 0 Normalized expression o a L 0 0 2 5 2 0 1 0 Normalized expression 0 5 0 0 AHA11 Control Ozone Face Abaxial Adaxial Carpaccio Robusta Carpaccio Robusta AKT2 Control Ozone Face Abaxial Adaxial Carpaccio Cima Robusta Carpaccio Robusta ALMT68 Control Ozone Face Abaxial Adaxial Carpaccio Cima Robusta Carpaccio Robusta e Normalized expression 30 N N in o a Normalized expression o 0 57 0 0 Normalized expression AHA1115 _ Control Ozone Face Abaxial Adaxial QE Carpaccio Cima Robusta Carpaccio Robusta ALMT6 Control Ozone Face Abaxial Adaxial Carpaccio Robusta Carpaccio Robusta ALMT610 Control Ozone Face Abaxial Adaxial Carpaccio Robusta Carpaccio Robusta 2 0 7 o in Normalized expression o in 0 0 3 0 E in o a Normalized expression o 0 5 0 0 Normalized expression N Normalized expression N CA1
128. ODVOLVOLVOOLLVO 00SS 0DST0 Od ELVA TT L8DVI061818HV 0PTIPSSIV 016 15 000 ULdOd Jo Sojoquo sapropnwau snjndoq x omua V 69pb AT TMS LLOLOVVODVDLOLLVVODD snndod DO Wod ouueqo 0 repris 1106506200 INXIos LLODVODIVOVOOLVIOVOL 00ccETOFO0 Od CTLVM 08208 cv Jo 1301 870100 OT Ouid pusouosj So oq10 09 puue Sur4gnoo1 premyo OEHTOSLIOO ULdOd VVOVVOVVDDVODVODIVOD INS ouueqo wnissejod oj repris 609106000 INXBost v AOMS 3d LOVVDVOODDLODLVDLLVD O0PSETDLIO Hd AAOI 0 8208 Jo 06 00070502100 ULdOd jouueyo 8 snurATnd oj repus 1 060ECET00 0680059100 ULdOd LVDVOLOODVOVOLDLVVVD 002 009910104 DIdL 6000771 O ud 09508 OLVEISETOO ALdOd LLLODIOLLVODVODLLVOD Jo q ouueqo oj IIS 1 L6SOEETOO INXBost 4 VVOLOVVODVVDODVVDDVVO 0O1TETDETO Od 1041 VVDOVODIDODVVVVOVVVOO 0828051000 ULdOd DVVIOLLOLODIOOOLOLVV O0 t 110100 1304 IOVIS DVIVOOLVIVOOOODVIVID 119906000 INXBou 00LZ0S1000 ULdOd LODVDLVDVDDOOOVVV LLL 00EPr1D100 1304 0814156000 ULdOd 0612186000 ULdOd LLOLVOLLOOLODDLLOOLO OLILIS6000 ULdOd WVVDLOODVVD LLOVDLDOO 009010600 1304 6CLOHd T TST8TETOO INXUOU IVVOOLLLLOLVODOLLOOD T IST8TETOO JNXBou Or617SP000 ULdOd VVIODLODIODVVVVOLVDOO 00L60COT00 1304 620 Ira vv osLsrSciv ecTIPH 41690 VV 608901 0895 lt 81000
129. Polym re PrxR Peroxiredoxine SOD Superoxyde dismutase Trx Thioredoxine Polysaccharides LLL LLL D gradation galacturonate ES 7 D galacturonate L galactose 6 L Galactono 1 4 lactone Salvage pathway L Galactose 1 P GDP L Galactose L Galactose L Ascorbate GDP D mannose L gulono 1 4 lactone GDP L gulose L gulose 1 P L gulose L gulonate 12 Animal like pathway Myo inositol L gulose pathway Figure 25 Voies de synth se l ascorbate 1 GDP D mannose 3 5 epimerase GME gt GME 2 GDP L galactose phosphorylase VTC2 VTC5 3 L galactose 1 P phosphatase gt VTC4 4 L galactose dehydrogenase gt L GalDH 5 aldonolactonase 6 D galacturonase reductase gt GalUR 7 methylesterase 8 L galactono 1 4 lactone dehydrogenase GLDH gt GLDH 9 nucleotide pyrophosphatase ou sugar 1 P guanylyltransferase 10 sugar phosphatase 11 sugar dehydrogenase 12 uronate reductase 13 myo inositol oxygenase gt 4 14 L gulono 1 4 lactone d hydrogenase oxydase glycolate 10 glyoxylate s rmm 9 assimilation 2 9 RE 5 eM 2 oxo glutarate NAD 2 Gamma NADH glutamate glutamylcysteine Glutamine EE Glutamate 4 ADP Pi ATP NH3 N assimilation Figure 26 Voie de biosynth se du glutathion 1 glutathione synthase GSH2 2
130. TSESTIV ESLSTVVE O98TSBEIV 109620 lt 200 ETUIS 064 T08SSIY Jo TJOT Sopouo oo D7DIPDA 0800159000 ULdOd IVIOLLOOVOODIVIVOODL DUBLAQYO 1o8ueuoxo H T8 0 eug ODIVVVDIODVVVOLLOOOL 0066600900 H10d 9IXVO OLLLLOODOOVOLVIOOLVV 1 8LT86TT00 INXIou 08551000 ULdOd DLVLLOLLVODVODVDLOVO 0071570100 1304 XVO IZ8LOL OLTSESTIV ESLSTVVE 098168e1Vv TOSS TOSCOO ETUIS 96 10853 Jo ZJOT Sopouo oo 069159100 ULdOd VOOLVOLOVDLVVODIODVV DUBLAQYO H T8 01 1e urs CEXVOJd LLLLODIDOODDDDLLV LLOL 00 110910 1304 IXVO 100X DT 294 pysoussy 1xgnso VIOOLOVVVOVOLLOOOLLO ETOO INXBou 080S0S0100 ULdOd LOLOLVVLLDLODDVDDVVD 0011700010294 vo la HAT Cc8s9d V V L6L601 L90L1d V MAN E6L 601 VrScoLLI TELIEV VO ce9ccv v v d DII 9L8LCIV VD PXHTLOSSLAVV OrLVTSSIV D CHAT S8L6HIV VH MAL 00201S 00S108E1V OT jxgiso Jo Sojoquo ryofiaid sd DAO 08651000 ULdOd IVOVODVVODDOVVIVOLVOO 1 uJojosr ose1p que NUOLI oj 1 88H86TTOO ETV d LLOVODIODOVIOVVIOVOV 0068701001204 Ivo So oq10 09 puue Sur4moo1 premmno A 11000100 cSua8na VOLLODVDOVIVVOOVOOLL INS ouueqo wnissejod oj repris 699 16000 INXBost TAOMS Id IODLLVOVODLLLOOVOVVDO 0008700101204 11090 08208 27 JO 1020 011805000 ULdOd So oq10 09 puue Sur4goo1 premmno T OTLE AT TMS VIDLODVVDDLOLVOOVVDV INS ouueqo wnis
131. The notion of effective ozone flux must consider these two aspects to better characterize the intra and inter specific differences in sensitivity to ozone Keywords Ozone poplar detoxification stomatal conductance Cette th se cofinanc e par VULNOZ et la r gion Lorraine a t effectu e au de l UMR 1137 Ecologie et Ecophysiologie Foresti res INRA 1137 EEF F 54280 Champenoux Universit de Lorraine UMR1137 EEF F 54500 Vandoeuvre l s Nancy
132. UNIVERSIT DE LORRAINE AVERTISSEMENT Ce document est le fruit d un long travail approuv par le jury de soutenance et mis disposition de l ensemble de la communaut universitaire largie est soumis la propri t intellectuelle de l auteur Ceci implique une obligation de citation et de r f rencement lors de l utilisation de ce document D autre part toute contrefacon plagiat reproduction illicite encourt une poursuite p nale Contact ddoc theses contact univ lorraine fr LIENS Code de la Propri t Intellectuelle articles L 122 4 Code de la Propri t Intellectuelle articles L 335 2 L 335 10 http www cfcopies com V2 leg leg_droi php http www culture gouv fr culture infos pratiques droits protection htm UNIVERSIT INA SCIENCE amp IMPACT FACULTE DES SCIENCES amp TECHNOLOGIES Th se cofinanc e par la r gion Lorraine et le projet ANR Vulnoz Collegium Sciences amp Technologies UMR1137 Ecologie et Ecophysiologie Foresti res Ecole Doctorale RP2E D partement de Formation Doctorale Sciences agronomiques et foresti res biologie et cologie biotechnologies Th se pr sent e pour l obtention du titre de Docteur de l Universit de Lorraine en biologie v g tale et foresti re par Jennifer DUMONT R le de la r gulation stomatique et de la capacit de d toxication foliaire dans l estimation d un seuil de risque l ozone pour la v g
133. VIII PROTOCOLE DE RETROTRANSCRIPTION AVEC LE KIT ISCRIPT 151 IX PROTOCOLE D INCLUSION EN RESINE POUR OBSERVATIONS AU MET 151 R F RENCES nn 156 SYNTHESE BIBLIOGRAPHIQUE 1 278 AA a 116 8 Figure 1 Structure de la mol cule d ozone 35 Contains 90 of Atmospheric Ozone Beneficial Role Acts as Primary UV Radiation Shield Current Issues Stratospheric Ozone Long Term Global Downward Trends Springtime Antarctic Ozone Hole The Ozone Layer Each Year Springtime Arctic Ozone Losses inSeveral Recent Years o o S A 15 Contains 10 of Atmospheric Ozone Harmful Impact Toxic Effects on Humans and Vegetation 0 Tropospheric Ozone Current Issues Episodes of High Surface Ozone 5 7 in Urban and Rural Areas Altitude kilometers O 5 IO 5 2025 Ozone Amount pressure milli Pascals Figure 2 R partition de l ozone atmosph rique selon l altitude Document mis par la NASA Solar radiation E h f shorter longer J g 60 5 85 pue KG Ozone cycle photon of UVC f Step 1 o gt 20 photon of UVB Step 2 j gt Step 3 Chapman cycle Ozone generation Step 4 and destruction 20 slow Figure 3 Cycle de g n ration destruction de l ozone ou cycle de Chapman Yip 2000 I OZONE 1 GENERALITES SUR L OZONE L
134. Y SULIOS O puns oArjejnd 92 000A DT Ouid O oaned ose1ojsuenAqiourAxodp Aq OL 9ISS000 ALdOd ours oj re rurs SLINHS LLLOVOLLODDOODLLLOVOO eseTopsuemjAqourAxorpA g repris 696 06200 JNXUOW 0080419500159 IOODLLOLLLLODDOOVOVOV 098918000 ULdOd SVAID VOLVIDVIDOLVILLVIVOL 116906144148 007001001004 LLOVIOLLOLODLLOLIOOO OLLTESI000 HLdOd IV IO USLEVIAX IMS 086 8 1 Jo 8 VIODLIVIOLLODOODLIVIOVO ouojo p oj TEUS ouojoe e3 T EPS T 6097 C00 JNXUO 008009910109 VVLLOOLLODLOOLOLDDLV 066089100 ALdOd 9THGIO 203 39U39 19J91 GON SUI V lt S SULA TTA ourozoj ud SISUILId orgroeds PoIpNys 59108 Jo 1571 TS QLL 0682 18777 OL6ET SHV Jo c Jo c 8010120 02 tp oseJojsuem AgjoulAXOJp Q Ie Ius 0 6 8 Jo Jo 1 So oquo 09 LINHS 9seu1sjsuex Aujoui amp xodpAu euroA D ese amp qiour ouues LZ LC 10s1nooud eupuouoojrur 1e rurs toseJo suem AgjoulAXOJp Q 0 lt 6 69 2 Jo c JO c BO OULO 09 LINHS 9se1sjsuexmAujoui amp xodpAu euroA D ose AYJoUL ouues 10s1nooud eupuouoojrur 1e rurs toseJo suem AujoulAxoJp Q 1e rurs T ITETOETOO
135. a respectively The differences between the two genotypes in O3 treated leaves were not significant Table 2 NADP dependent enzymatic activities NADP ME NADP ICDH NADP G6PDH and NADP GAPDH We hypothesized thatthe results presented in this section were predominantly related to the activity of cytosolic NADP dehydrogenases Such a hypothesis could be justified by several works on Arabidopsis thaliana which show that cytosolic isoform mutants lose most of the total activity in crude leaf extracts Wakao et al 2008 Mhamdi et al 2010 Voll et al 2012 Concerning NADP GAPDH no significant change occurred in response to treatment in either genotype Fig 6A B In contrast NADP G6PDH activity clearly increased 41 CO assimilation umol m 7 5 1 N lt 0 8 0 6 2 04 0 2 Total chlorophyll Fig 4 Effect of O3 treatment on CO assimilation A B n 6 4 A Carpaccio control m Carpaccio ozone B Rob A Robusta ozone 4 kk 6 12 18 Time d usta control 7 1 6 12 18 and total chlorophyll contents C D 20 st respectively Os treated and control ML leaves of Carpaccio A and Robusta B D genotypes Type Ill anova tests performed with R stat 2011 considering Os treatment and genotypes as vari
136. a Robusta Carpaccio Robusta Control Ozone 3 0 2 5 c 9 2 2 0 a x o 154 N E o 1 0 z 0 5 0 0 Carpaccio Cima Robusta Carpaccio Robusta TPK1 Control Ozone 0 84 TA c 06 2 9 x 504 N 5 2 0 2 0 0 Ld Carpaccio Cima Robusta Carpaccio Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial 3 0 in o a Normalized expression o 0 5 0 0 o o Normalized expression o 0 0 2 0 in Normalized expression o o a 0 0 QUAC1 Control Ozone Carpaccio Robusta Carpaccio Robusta TPC1 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta TPK11 Control Ozone Carpaccio Cima Robusta Carpaccio Robusta Face Abaxial Adaxial Face Abaxial Adaxial Face Abaxial Adaxial R sultats II DETOXICATION FOLIAIRE 1 EFFETS DE L OZONE SUR LES VOIES DE SYNTHESE DE L ASCORBATE ET DU GLUTATHION EN RELATION AVEC LES CONTENUS EN ACIDE AMINES CHEZ TROIS GENOTYPES DE PEUPLIER UNE COMBINAISON D APPROCHES MOLECULAIRE ET METABOLOMIQUE Nous avons vu que la r gulation des mouvements stomatiques permet de limiter l entr e d ozone dans les feuilles notamment par l piderme sup rieur N anmoins l ozone alt re les mouvements stomatiques ce qui peu
137. a mol cule d ozone est une vari t allotropique de l oxyg ne compos e de trois atomes pr sente naturellement dans l atmosph re Figure 1 La d couverte de l ozone est attribu e au chimiste suisse Christian Friedrich Sch nbein qui lui donne le nom d ozone en 1840 en se basant sur le mot grec ozein signifiant exhaler une odeur Cependant l odeur cre caract ristique de l ozone avait d j t mise en vidence par le chimiste Hollandais Martin Van Marum en 1789 Contenu 90 dans la stratosph re entre 15 et 35 km d altitude constituant la couche d ozone l ozone prot ge la vie terrestre des irradiations aux Ultra violets provenant du soleil tandis que les 10 d ozone compris dans la entre O et 15 km d altitude repr sentent une menace pour la vie terrestre Figure 2 En effet c est un puissant polluant gaz effet de serre nocif pour la sant qui agit comme une phytotoxine Sa toxicit pour la v g tation se traduit surtout par un stress oxydatif 2 CYCLE DE L OZONE L ozone stratosph rique constituant la couche d ozone est produit et d grad naturellement par le cycle de formation destruction ou cycle de Chapman Figure 3 ce qui dans une atmosph re non pollu e lui conf re une concentration stable Ce cycle se base sur la succession de plusieurs r actions commengant par la photolyse de mol cules d oxyg ne sous l action des UV 1 longueurs d onde inf rieures 242 n
138. a r ponse la lumi re rouge serait la chlorophylle contenue dans les chloroplastes des cellules de garde et du m sophylle L importance respective de la photosynth se dans les cellules de garde et dans le m sophylle dans la r ponse la lumi re rouge fait actuellement d bat Des tudes r centes ont montr que la r ponse la lumi re rouge n est cependant pas due la baisse de la concentration intracellulaire CO par la photosynth se Wang et al 2008 De plus Wang et al 2010 montrent que le phytochrome B interviendrait dans la r ponse la lumi re rouge Un mutant d Arabidopsis thaliana surexprimant PhyB a une sursensibilit la lumi re rouge tandis qu inversement le mutant n gatif PhyB a une sensibilit la lumi re rouge fortement r duite La lumi re bleue provoque une ouverture rapide des stomates Cette r ponse est principalement ind pendante de la photosynth se Plusieurs photor cepteurs la lumi re bleue ont t candidats la z axanthine et les phototropines Kinoshita et 2001 Il est maintenant admis que les phototropines sont les r cepteurs la lumi re bleue et qu elles induisent une phosphorylation et une activation de l ATPase protons membranaire ce qui entra ne une hyperpolarisation de la membrane et une entr e massive d ions Figure 17 Enfin Chez Arabidopsis thaliana COP1 agit comme un r gulateur n gatif des signaux mis par les cryptochromes et les photo
139. a s paration entre les deux traitements est d termin e d s le premier pr l vement 277 jour de traitement Un effet temps au sein des chantillons soumis au traitement ozone est responsable de 16 5 de la variance des chantillons PC2 Parmi les modifications observ es on note une augmentation de la plupart des m tabolites impliqu s dans le cycle de Krebs Fig 51 ce qui est coh rent avec une forte activit respiratoire Une des modifications majeures des m tabolites primaires en r ponse l ozone est l accumulation de sucres glucose fructose galactose raffinose mannose sucrose Fig 52 Les sucres solubles sont tr s sensibles aux stress environnementaux qui agissent sur l apport de glucides des organes sources vers les organes puits Rosa et al 2009 La hausse des concentrations en sucre solubles dans les feuilles en r ponse l ozone peut tre due une inhibition de la mise en r serve du carbone et une inhibition de l export en carbone nouvellement assimil Einig et al 1997 Dizengremel 2001 Les sucres solubles en plus de leur r le en tant que ressource m tabolique et constituants cellulaires agissent comme signaux de r gulation du m tabolisme en r ponse aux facteurs environnementaux L ozone 124 A Citrate C Fumarate AMBIANT OZONE AMBIANT OZONE 150000 2000 7 1500 4 100000 Temps Temps jour jour E 2 E 2 4 t 4
140. ability factors Significant differences between Os treated and control leaves are indicated by lt 0 05 P lt 0 01 and P lt 0 001 Table 2 Determination of genotype dependent responses to Os using t test The test used the means of PEPC and Rubisco activity levels net CO assimilation rates A Chl contents NADP dependent dehydrogenase activity and pyridine nucleotide pools of control and O3 treated Carpaccio C O3 and R O3 At all sampling dates for all the tested parameters data not shown in the Table Carpaccio and Robusta control means displayed no statistical difference at the 5 probability level P values are given when Carpaccio C O3 values were significantly higher or lower than Robusta R O3 values at the 5 probability level ne no genotype effect Day 2 Day 4 Day 11 Day 15 Day 17 Day 18 Genotypes P value P value P value P value P value P value A C O3 R O3 ne ne ne ne ne Chl C 03 R O3 ne ne 0 023 ne ne ne PEPC C 03 R O3 Ms 0 005 0 002 Rubisco R O3 ne ne ne ne ne NADP G6PDH CO R O3 ne ne lt 0 001 0 016 ne ADP ME CO R O3 ne bi 0 001 0 05 0 006 NADP ICDH C 03 E R O3 ne ne ne 0 028 ne NADPH CO n R O3 ne me 0 026 0 044 ne ADH C O R O3 ne ne ne ne ne NADP C 03 R O3 ne ne ne ne ne NAD C 03 R O3 ne ne ne ne ne in ozone exposed leaves but the sequence of events of the stimulation process slightly differed between
141. ades of experiments Plant Cell amp Environment 2007 30 1150 1162 Wittig VE Ainsworth EA Naidu SL Karnosky DF Long SP Quantifying the impact of current and future tropospheric ozone on tree biomass growth physiology and biochemistry a quantitative meta analysis Global Change Biology 2009 15 396 424 Wojtaszek P Oxidative burst an early plant response to pathogen infection Biochemistry journal 1997 322 681 692 Xie X Wang Y Williamson L Holroyd GH Tagliavia C Murchie E Theobald J Knight MR Davies WJ Ottoline Leyser HM et al The identification of genes involved in the stomatal response to reduced atmospheric relative humidity Current Biology 2006 16 882 887 Xue S Hu H Ries A Merilo E Kollist H Schroeder JI Central functions of bicarbonate in S type anion channel activation and OSTI protein kinase in CO 2 signal transduction in guard cell The Embo Journal 2011 1 14 Yip M Ozone in the atmosphere Environmental Physics 2000 Lettner VO 437 503 Yong JWH Wong SC Farquhar GD Stomatal responses to changes in vapour pressure difference between leaf and air Plant cell amp Environment 1997 20 1213 1216 168 R f rences Zeiger E Talbott LD Frechilla S Srivastava A Zhu JX The guard cell chloroplast a perspective for the twenty first century New Phytologist 2002 153 415 424 169 R le de la r gulation stomatique et de la capacit de d toxication foliaire dans
142. agard M Oufir M Planchon S Hoffmann L Jolivet Y Hausman JF Dizengremel P Renaut J 2007 A DIGE analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism Proteomics 7 1584 1599 Bortier K Ceulemans R de Temmerman L 2000 Effects of tropospheric ozone on woody plants In Agrawal M ed Environmental Pollution and Plant Responses Lewis Publishers Boca Raton FL pp 153 182 Brendley BW Pell EJ 1998 Ozone induced changes in biosynthesis of Rubisco and associated compensation to stress in foliage of hybrid poplar Tree Physiol 18 81 90 47 Bustos DM Iglesias AA 2003 Phosphorylated non phosphorylating glyceraldehyde 3 phosphate dehydrogenase from heterotrophic cells of wheat interacts with 14 3 3 proteins Plant Physiol 133 2081 2088 Bustos DM Bustamante CA Iglesias AA 2008 Involvement of non phosphorylating glyceraldehyde 3 phosphate dehydrogenase in response to oxidative stress J Plant Physiol 165 456 461 Caban M Pireaux JC Leger E Weber E Dizengremel P Pollet B Lapierre C 2004 Condensed lignins are synthesized in poplar leaves exposed to ozone Plant Physiol 134 586 594 Casati P Spampinato CP Andreo CS 1997 Characteristics and physiological function of NADP malic enzyme from wheat Plant Cell Physiol 38 928 934 Casati P Drincovich MF Edwards GE Andreo CS 1999 Malate metabolism by NADP malic enzyme in plant defense Photosynth Res 61 99 105 Cas
143. al atoire L utilisation d azote liquide n cessite le port de gants de protection au dessus des gants normaux Apr s avoir pes le MAHC bien nettoyer la balance car il est corrosif GC MS Volume d injection 1 ul injection debit divis avec un ratio sup rieur 1 50 colonne Restek USA Rxi 5Sil MS 30 m 0 25 mmID 0 25 um df avec 10 m Integra Guard Cat 13623 127 serial 870087 temp rature d injection 260 C temp rature d interface 280 C temp rature de la source ionique 230 C MS Quad 150 C programme de temp rature du four 2 min isothermique 60 C puis 10 C min jusqu a 325 C 6 min a 325 C et 7 min pour quilibrer a 60 C entre chaque analyse Flux d h lium 1 mL min plage de d tection m z 50 550 d lai du solvent Ac tate d thyle bouteille 1 et hexane bouteille 2 sont utilis s pour nettoyer l aiguille plus fois avant et apr s injection Les chantillons de peuplier sont sale La gaine doit tre chang e tous les deux jours L aiguille et le canon doivent tre lav s a l ac tone tous les jours Laisser s cher l air libre 142 Annexes 3 ANALYSE DES COMPOS S PH NOLIQUES Diluer les aliquots dans 250 uL de MeOH et 250 uL d eau lancer l analyse Ajouter le PDA et le Corona dans la configuration v rifier la taille de la boucle de l chantillon C 0 1 d acide formique dans de l eau D 0 1 d acide formique dans de l ac tonitrile Flux 1 1 mL min Ent
144. al parameters e Stomatal conductance at the final state gs1 means of the last 10 measurements after reaching a steady state after changing environmental parameters J Dumont et al Environmental Pollution 173 2013 85 96 87 Table 1 Environmental conditions at the initial state and at the final state inside the Li 6400 chamber Environmental parameters Parameter studied Blue light Red light VPD CO2 pmol CO mol Initial 400 2 0 400 3 0 100 1 0 400 3 0 Final 400 2 0 400 3 0 100 1 0 1200 3 0 Temperature C Initial 25 03 25 03 25 0 3 25 0 4 Final 25 0 3 25 0 3 25 0 3 25 0 4 Red 630 nm blue light 470 nm Initial 0 0 200 30 5 0 0 0 800 30 5 0 umol m 571 Final 0 30 1 800 30 5 0 0 0 800 30 5 0 VPD kPa Initial 0 8 0 2 0 8 0 2 0 8 0 2 0 8 0 2 Final 0 8 0 2 0 8 0 2 3 0 0 3 0 0 0 0 8 0 2 e The evolution of stomatal conductance between the two states Ags 251 250 e The time taken for stomata to close for CO and VPD or to open for red or blue light T Finally we calculated closing or opening speed values v Ag T 2 6 Statistical analyses The data obtained from Li 6400 measurements were fitted with a linear mixed effect model with individuals as random variables and genotype treatment and time as fixed variables To cope with heteroscedasticity variances were modelled as different for each genotype Contrast analyses were perfor
145. aleem A Loponen J Pihlaja K Oksanen E Effects of long term open field ozone exposure on leaf phenolics of European silver birch Betula pendula Roth Journal of Chemical Ecology 2001 27 1049 1062 Saviranta NMM Julkunen Tiitto R Oksanen E Karjalainen RO Leaf phenolic compounds in red clover Trifolium pratense L induced by exposure to moderately elevated ozone Environmental Pollution 2010 158 440 446 Sitch S Cox PM Collins WJ Huntingford C Indirect radiative forcing of climate change through ozone effects on the land carbon sink Nature 2007 448 791 794 Stevenson DS Dentener FJ Schultz MG Ellingsen K Van Noije TPC Wild O Zeng G Amann M Atherton CS Bell N et al Multimodel ensemble simulations of present day and near future tropospheric ozone Journal of Geophysical Research 2006 111 D08301 Tausz M Grulke NE Wieser G Defense and avoidance of ozone under global change Environmental Pollution 2007 147 525 531 Tuzet A Perrier A Loubet B Cellier P Modelling ozone deposition fluxes The relative roles of deposition and detoxification processes Agricultural and Forest Meteorology 2011 151 480 492 Untergasser A Cutcutache I Koressaar T Ye J Faircloth BC Remm M and Rozen SG Primer3 new capabilities and interfaces Nucleic Acids Research 2012 40 1 12 U S EPA Air quality criteria for ozone and related photochemical oxidants 2006 FINAL U S Environmental Protection Agency Wa
146. alling 41 42 The glutathione redox ratio remained stable under ozone preserving cell signalling which suggests that the activity of glutathione reductase GR was sufficient to maintain the glutathione pool in O3 treated leaves 21 Carpaccio had higher glutathione concentrations in its two forms and their increases under ozone are stronger than in other genotypes The higher de novo synthesis of glutathione and therefore the higher availability of glutathione in Carpaccio could be an important tolerance trait as found in another tolerant poplar genotype 21 Ascorbate de novo synthesis in response to ozone correlated with VTC2 gene expression regulation in poplar There are several routes of ascorbate biosynthesis in plants although it occurs mainly via the L galactose pathway 43 GDP D mannose 3 5 epimerase GME catalyzes the first step of two ascorbate biosynthesis pathways the L galactose and the L gulose pathways 44 GME plays a key role at the intersection of ascorbate and non cellulosic cell wall biosynthesis 45 Even if its regulation at the gene expression level was weak it could be completed by an induced activity of GME caused by an increase in NAD P due to oxidative stress 46 90 R sultats GDP L galactose phosphorylase VTC2 produces the first metabolite dedicated to ascorbate biosynthesis pathway and has been shown to represent the major controlling step at the transcriptional level of ascorbate s
147. and Robusta respectively at the last sampling date Figure 3A The concentration of GSSG follows the same trend in all three genotypes in response to ozone with the strongest response of 2 fold 1 7 fold and 1 5 fold at the last sampling date in Carpaccio Cima and Robusta respectively Figure 3B Thereby the concentration of total glutathione increased progressively and significantly in all three genotypes in response to ozone with the strongest response at the last sampling date with a 2 fold 1 7 fold and 1 7 fold increase in Carpaccio Cima and Robusta respectively Figure 3C The higher increase in GSH in Carpaccio 86 Total ascorbate relative to Oxidised ascorbate relative to Reduced ascorbate relative Redox ratio relative to ambient ambient to ambient ambient 200 150 100 100 120 100 2 4 11 17 Robusta 2 4 11 17 Robusta 2 4 mm Robusta 2 ahah7 Carpaccio 2 4 11 17 Carpaccio 2 4 11 17 Carpaccio 2 411117 Cima 2 4 11 17 Cima 2 4 11 17 Cima Figure 2 Ozone effects on ascorbate concentration and redox state in poplar leaf Effects of ozone treatment on ascorbate concentrations percentage relative to ambient with reduced ascorbate ASA A oxidised ascorbate DHA B total ascorbate ASA DHA C and ascorbate redox ratio reduced form total D on three poplar genotypes Carpaccio Cima
148. and Grulke 2010 a slower stomatal response could play a major role in sensitivity Carpaccio and Cima reacted quite similarly and appeared less sensitive than Robusta Nevertheless Carpaccio which seemed the less sensitive closed its stomata more than Cima under treatment reducing entrance more than Cima and reacted more quickly in adjusting its stomatal conduc tance to environmental parameters While Robusta sensitivity can be explained by slower stomatal conductance responses Cima sensitivity was not found linked to stomatal behaviour and may thus be explained by the second type of defence reactions i e detoxification processes Finally we can hypothesize that in natural conditions Robusta may take more time than Cima and Carpaccio to reach a steady state due to slower stomatal opening and higher final conductance Fig 7A At midday if the VPD rises too much Carpaccio and Cima may partially close their stomata to save water but Robusta is less reactive and then will lose more water Finally at the end of the day Robusta may take more time to close its stomata In the morning the stomatal conductance of Robusta may limit its photosynthesis Fig 7A spotted area Rapidly Robusta stomatal conductance may become higher leading to higher water losses especially in the 93 J Dumont et al Environmental Pollution 173 2013 85 96 su su su su su su su su su su
149. ant la fin que 9 arbres par chambre Lors de la seconde exp rience nous n avions mis que 6 arbres par chambre Figure 32 L exp rience a t r alis e sur Carpaccio et Cima dans un premier temps puis elle a t dupliqu e sur Robusta 24 Figure 33 Exemple de racines de peuplier apr s lavage pr tes pour le s chage Mat riel et M thodes 2 CONDITIONS ENVIRONNEMENTALES Les conditions environnementales dans les phytotrons sont similaires pour les deux exp rimentations de fumigation afin de faciliter la synth se des r sultats Pour les 4 chambres t moin l air est filtr travers du charbon actif tandis que pour les 4 chambres du traitement ozone 120 ppb d ozone ont t inject s de 9h 22 h directement dans l air entrant dans les chambres L ozone a t produit partir d O par deux g n rateurs d ozone 07500 Fischer Bonn Germany and CMG3 3 Innovatec II Rheinbach Germany Les concentrations en ozone de chaque chambre ont t enregistr es sur ordinateur par un syst me automatique li un analyseur O341M Environnement S A Paris France La photop riode durait de 8 h 22 h avec 5 lampes par chambre le taux d humidit relative tait maintenu 85 la nuit et 75 le jour et la temp rature tait stabilis e 20 C la nuit et 23 C le jour III SUIVI DE CROISSANCE BIOMASSES ET CHLOROPHYLLES 1 MESURES DE HAUTEUR DE DIAMETRE ET CHUTES DE FEUILLES Afin de r aliser un su
150. arlsson et al 2007 They were progressively replaced by indicators of the uptake through stomata CUO 86 J Dumont et al Environmental Pollution 173 2013 85 96 Cumulative Uptake of Ozone or Accumulated stomatal flux above a threshold of Y nmol m 571 recently renamed PODy Phytotoxic Ozone Dose over a threshold of Y nmol m s This latter indicator takes into account the flux by modelling stomatal conductance with models such as the DO3SE model B ker et al 2012 Emberson et al 2000 which computes stomatal conductance using the additive algorithm of Jarvis 1976 That model calculates conductance with a maximum conductance value and reducing functions that depend on phenology and environmental factors such as irradiance Vapour Pressure Deficit VPD temperature and soil water potential Nevertheless it does not integrate the effect of on those factors although is known to impair stomatal functioning Fagerli et al 2011 Danielsson et al 2003 and Pleijel et al 2002 added the effect of O3 to the phenology function in potato and wheat That new function is being increasingly used but more knowledge is needed to determine if the effects of on trees should really be integrated in the same way because no attempt has been made to link the function to another function than phenology so far Stomatal movements are due to the regulation of guard cell turgor via the control of osm
151. as been linked to several mechanisms such as 1 an increase in its proteolysis Pe arrubia and Moreno 1990 2 protein and or mRNA synthesis inhibition Brendley and Pell 1998 Pelloux et al 2001 Bohler et al 2007 and 3 protein fragmentation or structure modification due to ROS production Ishida et al 1997 Pell et al 1999 Junqua et al 2000 Abat et al 2008 An increase in PEPC activity was distinctly observed for the two genotypes that kind of O3 induced effect has been previously mentioned for poplar trees Landolt et al 1997 Dizengremel 2001 PEPC Physiol Plant 148 2013 activation among other carbon metabolism changes is believed to provide carbon intermediates used for repair processes Dizengremel 2001 The increase in PEPC activity is generally linked to an increase in both the amounts of PEPC protein and transcripts Dizengremel 2001 Fontaine et al 2003 Matyssek et al 2006 In our work we showed that for the two genotypes the increase in PEPC activity was quite well correlated to the increase in NADP ICDH activity This relation supports the hypothesis of an anapleurotic function of PEPC to fuel the Krebs cycle by providing precursors for amino acid synthesis and of its contribution to cell maintenance in case of severe oxidative stress Hodges et al 2003 45 5 Carpaccio control m Carpaccio ozone NADPH nmol 0 1 FW Robusta control 1 A Robusta ozone
152. asted effect on amino acid contents The major effects of ozone appeared mainly from the third sampling date As illustrated by the PCA the variance in concentrations of the amino acids was explained at 4096 1796 and 11 by three components PCI PC2 and respectively Figure 7 PCI and PC2 show the ozone effect by separating ambient and ozone samples and stronger separation in later time points especially in Carpaccio Figure 7A PC2 and PC3 show the genotype effect in addition to the ozone effects by separating Carpaccio and Robusta samples whereas Cima is not separated from the two genotypes Figure 7C The variations in concentrations of Val Asn Thr Ser and Leu explain the time effect Figure 7B The ozone effect was mainly accounted for by variations in Trp Ile His Tyr and Asp concentrations and the genotype effect especially the difference between Carpaccio and Robusta by a difference in Put Lys and Phe concentrations 88 A GSH13 AMBIENT OZONE 1 0 sampling dates day 2 4 11 15 17 00 ill CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o o o gt o Normalized expression o B GSH25 AMBIENT OZONE t 1 0 sampling dates day 2 la 11 15 17 CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o o o gt o Normalized expression o THA13 C AMBIENT OZONE 12 sampling dates day 2 11 15 17 o Gica CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA No
153. at leaf height The plants were irrigated daily using tap water During the growth period one terminal shoot was kept When the trees were approximately 60 cm high selected uniform plants were transferred into phytotronic chambers for ozone exposure In order to obtain a Physiol Plant 148 2013 statistically relevant biological replicate 40 individuals of each genotype were distributed in eight chambers half of which four chambers were set for treatment Fumigation and cumulative ozone uptake calculation Phytotronic chambers were constantly ventilated with charcoal filtered air and growth conditions were main tained identical to those in the growth chamber with 400 umol m7 57 photon flux floor level treatment started at the end of a 7 day long acclimation period was produced from pure O2 with two ozone gen erators 02500 Fischer Bonn Germany and CMG3 3 Innovatec Il Rheinbach Germany and injected directly along with the filtered air entering the chambers Con trol trees were exposed to ambient Os concentrations 4ppb throughout the treatment period A set of automated systems and analyzers O341M Environment S A Paris France were used to monitor the concen trations and the length of Os exposure The plants were exposed to 120 ppb 10 of for 13 h 09 00 to 22 00 h daily for 17 days Fumigation started 1h after the beginning of the photoperiod and ran until its end The plants were sc
154. attered and spaced daily to avoid shading The ozone flux was estimated based on the measurement of stomatal conductance to ozone which is related to stomatal conductance to water vapor gs by the following formula 0 668 2 Fares et al 2008 Measurements were performed every 2 days For days when no measurement was performed was estimated from the adjacent values Bagard et al 2008 POD mmol m was estimated by summing up hourly ozone uptakes throughout the whole experiment In this context our data provide indicative PODo estimates Measurement of gas exchange parameters and of total chlorophyll contents Net assimilation A CO 72 5 1 and stomatal conductance to water vapor g mol H O m 2s were measured using two intercalibrated portable photosynthesis Systems LI COR 6200 LI COR Inc Lincoln NE USA Measurements were only performed on the last fully expanded mature leaves ML corresponding to the 8th 10th leaves down from the first apical leaf Three trees per genotype per chamber were kept from destructive sampling and only used for gas exchange Relative chlorophyll contents in CCI unit were estimated using a CCM 200 Chlorophyll Content Meter Opti Sciences Hudson NH USA Total Chl per leaf area g m was determined using a known relationship established for tree leaves Chl 20 0214 Physiol Plant 148 2013 CCI unit as mentioned in Bagard et al 2008
155. ay 2 4 11 15 17 T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o e o gt _ Normalized expression o o 02 0 0 e Normalized expression o o o 1 o h 0 0 Normalized expression o o o gt o o N 0 0 GlyA5 AMBIENT OZONE T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GIyA8 AMBIENT OZONE i T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GIyA17 AMBIENT OZONE T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day 2 4 11 15 17 sampling dates day 2 4 11 15 17 sampling dates day 2 4 11 15 17 GIyA19 AMBIENT OZONE sampling dates 10 day 0 5 0 0 T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA AMBIENT OZONE 12 sampling dates day 2 4 j 11 15 17 0 0 T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA Normalized expression GlyA24 o o L Normalized expression o o 1 o Dd GOGAT15 AMBIENT OZONE 15 sampling dates 2 4 11 15 17 0 0 T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA o Normalized expression o i GS4 AMBIENT OZONE c sampling dates 8 810 day a 2 g _ o 4 8 11 15
156. bH Berlin Germany 0 3 mg mL D glucose C13 Campro Scientific GmbH Berlin Germany 0 25 mg mL glycerol d8 Campro Scientific GmbH Berlin Germany 0 15 mg mL biochanin A Extrasynthese Genay France and 0 015 mg mL alanine d4 Aldrich Steinheim Germany in 6 4 MeOH H20 Samples were extracted for 15 min at 4 C with 1400 rpm of agitation using a thermomixer Eppendorf Germany After a centrifugation for 2 min at 10 C with 13500 g the supernatant was taken A second extraction in 1300 uL of 0 005 w v BHT in 100 MeOH was done with the same protocol and the supernatants were combined One aliquot of 100 uL was dedicated to amino acid and polyamines analysis performed the same day using the EZ faast kit Phenomenex Torrance CA USA as described in the kit except that 50 uL of the internal standard solution was used The samples were run with EZ faast AAA column by HPLC MS Thermo LTQ Thermo Finnigan with electrospray ionization The areas for the amino acids and polyamines were integrated using Xcalibur software Thermo Electron Corporation Waltham MA USA and alanine d4 was used as internal standard Statistical analyses The data obtained from ascorbate and glutathione assays were fitted with a linear mixed effect model with individuals as random variables and treatment genotype and time as fixed 84 Table 1 Ozone effect on leaf fall and total biomass Genotypes Leaf fall relative to ambient Total biomass
157. basent donc sur les concentrations en ascorbate pour essayer de repr senter la d toxication Tuzet et al 2011 N anmoins ces mod les restent limit s puisque l ascorbate apoplastique n est pas le seul m tabolite impliqu dans les processus de d toxication Les acteurs de la d toxication sont nombreux et nous avons mis en vidence que la capacit de d toxication r sulte de la combinaison de facteurs multiples tels que les concentrations de diff rents antioxydants leur r g n ration l activit d enzymes d toxifiantes Il faut cependant rappeler que la sensibilit l ozone si elle peut tre due une mauvaise r gulation de la conductance stomatique des conductances maximales plus importantes ou des capacit s de d toxication trop faibles elle peut aussi tre limit e par une forte disponibilit en ressources nerg tiques et une bonne remobilisation des ressources Dans l absolu l ensemble de ces param tres devraient tre pris en compte afin d obtenir un mod le le plus proche possible de la r alit physiologique des v g taux mais l int gration de multiples m canismes ne semble pas 129 Conclusion et perspectives tre applicable de grandes chelles de temps et d espace car n cessitant trop de param trages Les diff rences de tol rance sont difficiles appr hender du fait de la complexit des m canismes mis en jeu Robusta le g notype de peuplier le plus sensible parmi les troi
158. c enzyme 2 in Arabidopsis thaliana is associated with enhanced susceptibility to Colletotrichum higginsianum New Phytol 195 189 202 Vollenweider P Woodcock H Kelty MJ Hofer RM 2003 Reduction of stem growth and site dependency of leaf injury in Massachusetts black cherries exhibiting ozone symptoms Environ Pollut 125 467 480 Wakao S Andre C Benning C 2008 Functional analyses of cytosolic glucose 6 phosphate dehydrogenases and their contribution to seed oil accumulation in Arabidopsis Plant Physiol 146 277 288 Wildhagen H Durr J Ehlting B Rennenberg H 2010 Seasonal nitrogen cycling in the bark of field grown Grey poplar is correlated with meteorological factors and gene expression of bark storage proteins Tree Physiol 30 1096 1110 Wittig VE Ainsworth EA Naidu SL Karnoski DF Long SP 2009 Quantifying the impact of current and future tropospheric ozone on tree biomass growth physiology and biochemistry a quantitative meta analysis Glob Change Biol 15 396 424 Yun S C Laurence J 1999 The response of clones of Populus tremuloides differing in sensitivity to ozone in the fields New Phytol 141 411 421 Physiol Plant 148 2013 22714 3 PC2 16 o PCI 26 Figure 50 Analyse en composantes principales bas e sur les concentrations des m tabolites primaires chez trois g notypes de peuplier Carpaccio en rouge et rose Cima en jaune et beige et Robusta en bleu fonc et b
159. ch 5 20 Noctor et al 2011 Moreover the NADH NAD ratio undergoes important variations that depend on leaf cellular compartments and light dark conditions Heineke et al 1991 Igamberdiev and Gardestr m 2003 Szal et al 2008 In the cell NADP is produced from NAD by NAD H kinases Turner et al 2004 Noctor et al 2006 which are suggested to play an important role in providing cellular NADPH in response to a wide range of oxidative related stresses Hayashi et al 2005 Chai et al 2006 Noctor 2006 In our experimental conditions the total amounts of phosphorylated pyridine nucleotides NADP H decreased in the two genotypes probably in favor of the non phosphorylated pool Therefore the NAD H phosphorylation process did not seem to be involved in response to ozone treatment To conclude our work showed that treatment caused a significant increase in the activity of all key cytosolic NADP dependent enzymes we assayed except Physiol Plant 148 2013 NADP GAPDH That increase may be justified by an increased NADPH demand for detoxification and repair processes In fact we determined that only Carpaccio qualified in our work as the more tolerant genotype was able to maintain its NADPH pool during treatment while Robusta was not Differential responses could be associated to a higher stimulation of both NADP ME and NADP G6PDH in the more tolerant genotype Using mutants with a low activity level for these enzyme
160. chantillon inconnu Gerlier et al 2007 Mat riel et M thodes VI ANALYSES TRANSCRIPTOMIQUES 1 PRINCIPES DES ANALYSES Des analyses transcriptomiques ont t r alis es sur des chantillons de feuilles fraiches broy es et aussi sur des stomates microdiss qu s partir de feuilles lyophilis es Ces deux types d chantillons n cessitent des traitements diff rents Lors d un stress comme l ozone l expression de g nes peut tre modifi e c est cette r ponse que l on mesure par les analyses transcriptomiques Dans un premier temps les transcrits des g nes sont extraits ARNs totaux et ils sont r trotranscrits en ADNc Dans le cas des stomates microdiss qu s une amplification des ADNc est n cessaire pour obtenir un signal suffisant Les mesures sont r alis es sur cet ADNc par PCR quantitative ou PCR en temps r el C est une m thode de r actions en chaine par polym rase permettant d amplifier une portion d ADN cibl e et de mesurer la quantit initiale d ADN gr ce au suivi d un marqueur fluorescent Figure 44 Elle n cessite de dessiner des amorces morceaux d ADN simple brin compl mentaires et sp cifiques de la s quence du g ne tudier 2 ANALYSES TRANSCRIPTOMIQUES A PARTIR DE FEUILLES BROYEES PREPARATION DES MATRICES L extraction des ARNS totaux a t r alis e partir de 100 mg de poudre de feuilles fraiches grace au kit RNeasy Plant Mini Qiagen selon le protocole du fabricant
161. concentrations des m tabolites primaires chez trois g notypes de peuplier Carpaccio en rouge et rose Cima en jaune et beige et Robusta en bleu fonc et bleu clair soumis un traitement ozone couleurs vives ou ambiant couleurs pastelles cinq dates de pr l vements 1 2 3 4 5 pour 2 4 11 15 et 17 jours de traitement respectivement Figure 51 Concentration en A citrate B succinate C fumarate D malate dans les feuilles de trois g notypes de peupliers Carpaccio Cima et Robusta apr s 2 4 11 15 et 17 jours de traitement ozone ou ambiant Figure 52 Concentration en A fructose B glucose C raffinose D galactinol E galactose F mannose et G sucrose dans les feuilles de trois g notypes de peuplier Carpaccio Cima et Robusta apr s 2 4 11 15 et 17 jours de traitement ozone ou ambiant TABLEAUX Tableau 1 Equations des principaux mod les de calcul de conductance stomatique issu de Damour et al 2010 Tableau 2 M canismes de d toxication des ROS dans les cellules v g tales Moller et al 2007 Tableau 3 Principaux indicateurs d valuation de l impact de l ozone sur la v g tation Tableau 4 Caract ristiques des 6 sections du genre Populus Tableau 5 Les surfaces en ha de peuplier de production de bois en France et par r gion Tableau 6 R colte annuelle de grumes de peupliers m Tableau 7 Conditions environnementales avant et apr s variation d un des param tres
162. converted to g m thanks to a known relation Bagard et al 2008 Wellburn 1994 chl 0 0214 CCI 0 0424 2 4 CUO and POD calculations CUO and PODy with a threshold of 1 nmol m s recommended by Fagerli et al 2011 are modelled as the flux of from the atmosphere through stomata Fst pmol m s 1 POD and CUO are calculated from hourly values of Fst PODy Y O JAt i 1 i 1 n CUO FsAt i 2 i 1 n being the number of hours and At 1 h 1 nmol m s The flux was estimated based on the measurements of stomatal conductance to o which is related to stomatal conductance to water vapour gs by the following formula go gs 1 51 Massman 1998 gy measurements were per formed three times a week As our experiment was conducted in phytotronic chambers where environmental conditions were stable we applied a constant value of gw all day long When g measurements were not available we estimated them from the values of the flanking days Bagard et al 2008 2 5 Gas exchange measurements Gas exchange A net CO assimilation and gw stomatal conductance to water vapour was monitored using a portable photosynthesis system Li 6200 LiCor Lincoln NE USA in phytotronic environmental conditions Measurements were carried out 3 times a week 2 h after lights were switched on on the first fully expanded mature leaf at the beginning of the experiment Gas exchange res
163. cteurs Photo Diode Array Finnigan Surveyor PDA Detector Corona Ultra a rosol charg Esa Corona Ultra et spectrom tre de masse 5 ACIDES AMINES L extraction des chantillons pour l analyse des acides amin s est la m me que pour les compos s ph noliques Protocole d taill en annexe 3 Le m me jour un aliquot de 100 uL d extrait a t pr par avec le kit EZ FAAST Phenomenex Les prescriptions du fournisseur ont t suivies avec un ajustement du volume de r actif 1 50 uL au lieu de 100 uL La proc dure consiste en une extraction en phase solide suivie d une d rivatisation et d une extraction liquide liquide L analyse a t r alis e par chromatographie liquide haute performance coupl e un spectrom tre de masse MS Finnigan LTQ detector HPLC MS 6 SUCRES L extraction des chantillons est la m me que pour les compos s ph noliques Protocole d taill en annexe 3 Un aliquot de 150 uL est compl tement s ch et stock 70 C pour ensuite servir aux analyses des sucres Les flacons une fois sortis du cong lateur doivent rester 1h temp rature ambiante avant ouverture Une solution de standard C8 C20 alcane est ajout e 80 uL et aussit t vapor e Pour la d rivatisation nous ajoutons 80 uL de 20 mg mL de m thoxyamine hydrochoride dans de la pyridine et laissons 90 min 37 C Une autre tape de d rivatisation suit dans 80 uL de MSTFA N m thyle N trimethyl
164. d une conductance maximale fixe est inappropri e car celle ci est r duite par l ozone et qu il faudrait param trer chaque fonction r ductrice avec une fonction ozone pour traduire les ralentissements d ouverture et de fermeture observ s De plus nous avons mis en vidence que chez Robusta le g notype le plus sensible l ozone n induit pas de ralentissement suppl mentaire celui ci ayant des vitesses initiales de r ponse tr s lentes Il faut donc poursuivre les tudes visant caract riser plus finement ces effets pour d finir ces nouvelles fonctions et v rifier que notre tude soit transposable sur des arbres plus g s en conditions naturelles et sur d autres esp ces Afin de d finir pr cis ment l effet de l ozone sur les mouvements stomatiques il convient de comprendre les tapes alt r es en amont qui entrainent le ralentissement de l ouverture et de la fermeture des stomates en r ponse diff rents stimuli Nous avons montr que les flux ioniques dans les cellules de garde sont modifi s sous ozone et que l expression des g nes des canaux calciques vacuolaires tait fortement inhib e Ainsi il est important de v rifier si les perturbations des flux ioniques concernent majoritairement 128 Conclusion et perspectives certains compartiments cellulaires en mesurant le contenu min ral de chaque compartiment s par ment gr ce l utilisation d un microscope lectronique balayage haute r solution
165. d with the protein assay dye Bio rad Laboratories Inc Munich Germany Bovine serum albumin Sigma Aldrich Munich Germany was used as a calibration standard Enzymatic activity assays All enzymatic activities were assayed using a microplate spectrophotometer Biotek PowerWave HT USA Four different leaf extracts from either control or O3 treated trees were used for each sampling date Activities in each extract were measured in duplicates at 30 C Only linear segments were used to calculate the maximum rates of the enzymatic reactions Rubisco EC 4 1 1 39 activity was determined by monitoring NADH extinction at 340 nm according to the protocol described by Fontaine et al 1999 The reaction mixture was made of Bicine pH 8 0 100 mM NaHCO 25mM NADH 0 3 mM MgCl 39 20 mM phosphocreatine 5 mM ATP 5 mM glyceralde hyde 3 phosphodehydrogenase G3PDH EC 1 2 1 12 10 units ml 3 phosphoglycerate kinase 3 PGK 2 7 2 3 10unitsml and creatine phosphokinase EC 2 7 3 2 5 units ml The reaction was started by adding 1 mM ribulose 1 5 bisphosphate RuBP A control without RuBP was prepared for each assay The PEPC EC 4 1 1 31 assay followed the same principle as in Fukayama et al 2003 The assay medium contained Tris HCI pH 8 0 100 mM NaHCO 5 mM NADH 0 3 mM MgCl 20mM glucose 6 phosphate 2mM malate dehydrogenase MDH EC 1 1 1 37 15 unitsml The reaction was initiated by adding PEP 4m
166. de perception de l ozone et la cascade signal tique associ e sont toujours inconnus N anmoins il a t montr que les mutants de Nicotiana tabacum et Arabidopsis thaliana NtMPK4 0511 abil 1 abi2 1 et ont une sensibilit l ozone accrue due une conductance stomatique sup rieure et une r gulation de la conductance stomatique perturb e en r ponse l ozone Gomi et al 2005 Marten et al 2008 Vahisalu et al 2010 Ces travaux indiquent l implication des canaux anioniques et de ph nom nes de phosphorylation d phosphorylation de prot ines dans la r ponse des cellules de garde l ozone De plus suite un stress ozone la r ponse des stomates aux autres facteurs environnementaux est modifi e En effet apr s un traitement ozone les stomates ont une vitesse d ouverture et de fermeture plus lente en r ponse aux variations de lumi re Paoletti 2005 Paoletti and Grulke 2010 Le m me effet est observ pour la r ponse une variation de VPD Grulke et al 2007 SYSTEMES DE DETOXICATION 1 SYSTEME DE DEFENSE ANTIOXYDATIF Les d fenses de la plante comprennent la capacit restreindre les flux d ozone entrants dans les tissus refl t e par les modifications de la conductance stomatique mais aussi d toxiquer l ozone une fois les stomates franchis Un syst me de d toxication constitutif puis inductif agit sur les ROS pour limiter le stress oxydatif Wieser and Matyssek 200
167. demi disque Les panalyses X ont t accomplies gr ce un dispersion d nergie EDS avec les r glages suivants EHT 15 kV ISonde 800 pA WD 15 mm grandissement 1000X De plus toutes les deux heures et au d but et la fin de chaque portoir analys une recalibration de l nergie des spectres avec un standard mangan se tait effectu e 4 ANALYSE ULTRASTRUCTURALE DES CELLULES DE GARDE MICROSCOPIE ELECTRONIQUE A TRANSMISSION PRINCIPE DE L APPAREIL Le Microscope Electronique Transmission MET permet de visualiser des objets de taille inf rieure une cellule avec une haute r solution et donc d tudier les structures cellulaires Un faisceau d lectrons haute tension focalis l aide de lentilles lectromagn tiques traverse un chantillon tr s mince 10 100 nm Les lectrons vont alors r agir avec les atomes de l chantillon Figure 46 En MET seuls les lectrons traversant l chantillon sont analys s La distinction entre les lectrons transmis n ayant pas interagit avec l chantillon et les lectrons diffus s r sultant de l interaction avec l chantillon permet de recr er les images UTILISATION Des observations au MET de coupes tangentielles ultrafines 100 nm de feuilles incluses en r sine Protocole d taill en annexe 9 ont permis d tudier l ultrastructure des stomates et d appr cier l tat et la pr sence d organites tels que les chlo
168. duced different responses on two poplar genotypes in terms of symptoms and acceleration of foliar senescence We visualized different types of leaf injuries They first appeared within a few days on Robusta and consisted in large mottle necrosis first on ML and subsequently on younger leaves For Carpaccio Os necroses were mainly located on ML and consisted in brownish stippling Acceleration of leaf abscission another characteristic of sensitivity on aspen Karnosky et al 1996 Yun 44 and Laurence 1999 Ribas et al 2005 H iki et al 2009 was also more noticeable on Robusta and started a few days after the beginning of fumigation We interpreted all these visual features as different levels of sensitivity to O3 and concluded that Robusta was more sensitive to O3 than Carpaccio All along the treatment no major difference in POD was observed between the two genotypes Considering this fact the differential sensitivity of the two genotypes could be attributed to biochemical responses rather than to differential stomatal Os uptake As already shown on different poplar species a decrease in CO assimilation is one of the effects of exposure Reich 1983 Yun and Laurence 1999 Bortier et al 2000 Bagard et al 2008 In this study the decrease in CO assimilation occurred to the same extent for the two genotypes The evolution of chlorophyll contents and of rubisco activity could partly account for th
169. e PCR avec les conditions suivantes 5 min 25 C 45 min 42 C 5 min 85 C et maintenir 8 C IX PROTOCOLE D INCLUSION EN RESINE POUR OBSERVATIONS AU MET Mat riel n cessaire prouvettes b chers barreaux aimant s sorbonne gants 151 Annexes pH m tre pipettes automatiques pipettes pasteur capsules d inclusion et moules tuve thermostat e Produits Na2HPO anhydrous Acros Organics r f temp rature ambiante NaH PO 2 H20 Prolabo r f 28015 294 temp rature ambiante Glutarald hyde 25 EM grade distillation purified EMS r f 16210 r frig rateur et cong lateur http www emsdiasum com microscopy technical msds 16200 pdf Ac tone Sigma armoire solvant Osmium tetroxide 4 aqueous solution EMS r f 19150 R frig rateur http www emsdiasum com microscopy technical msds 19150 pdf R sine Epon Kit Embed 812 DER73 EMS r f 14130 temp rature ambiante Pr cautions a prendre pour la manipulation et l utilisation de ses diff rents produits Ces produits sont dangereux ils sont nocifs pour les voies respiratoires et sont irritants pour la peau et peuvent causer de graves maladies voir les fiches de s curit des produits Toutes les manipulations effectu es ci dessous ainsi que l utilisation des diff rents produits sont faire sous sorbonne le port de la blouse et des gants est obligatoire ainsi que les l
170. e de Bordeaux et le LABEX Arbre pour leur soutien financier et mat riel Mes remerciements s adressent d abord toi Didier Le Thiec pour m avoir offert l opportunit de r aliser cette th se Tout au long de ces 3 ans et demi tu m as toujours guid e efficacement Toujours l coute nous avons pu construire ensemble cette tude gr ce tes conseils et ta r activit m auras permis de voyager pour exposer mes r sultats d acqu rir des comp tences vari es et ce jusqu au pays du p re No l que demander de plus Un grand merci Pierre Dizengremel qui aura t l pour me pr senter avec passion la probl matique de l ozone lors de mes premiers pas Nancy et qui a accept de participer mon jury de th se pour voir l aboutissement de cette belle histoire qu il aura suivi tout du long Yves Jolivet tu m as soutenue toujours avec calme et s rieux tu as accept d tre mon pr sident de jury et surtout mon tuteur de monitorat Gr ce toi j ai pu enseigner comme je le souhaitais merci a repr sentait beaucoup pour moi Marie No lle Vaultier merci d avoir relu mon manuscrit et d avoir toujours accept de mettre la main la p te pour m aider Garde la bonne humeur qui te caract rise si bien Je remercie vivement Nathalie Leonhardt et Daniel Laffray pour avoir accept de juger mon manuscrit de th se en tant que rapporteurs Elina Oksanen et Markku Keindnen je vous suis tr s reconnaissante d avo
171. e decrease in assimilation capacity For these two parameters exposure had the same effect or nearly the same effect on the two genotypes depending on sampling times Physiol Plant 148 2013 1 T T T y 1 53x 0 45 TO 41 R2 0 87 7 Os 3L D A 2 Q T 1 A gt gt C RB y 0 84 0 14 82 0 66 B y2030x2 0 Ig4 R2 0 38 Da lt 3L 8 _ d rs I 2 oA 5 00 26 R 0 001 1 4 _ C y 0 86x 0 03 54 R 0 95 4 E a y 0 37x 0 35 d 3 R 0 90 4 lt 2 Z 1 D y 1 45x 0 54 4 0 974 3 4 d QE 2 lt AC A 1 1 49 0 86 7 1 2 3 4 PEPC nkat mg prot Carpaccio control Robusta control m Carpaccio ozone a Robusta ozone E T T T T a _ 0 60x 0 28 47 R2 0 99 2 Qao y 0 26x40 55 3F R 0 98 7 a D Of 2 4 lt Zx Eir 7 1 2 3 4 NADP ME nkat mg prot Fig 7 Linear regression of NADP G6PDH activity A NADP GAPDH activity B NADP ME activity C and NADP ICDH D activity in relation to PEPC activity and NADP ICDH activity versus NADP ME activity E of Carpaccio and Robusta genotypes The reduction in Rubisco content and activity has been recorded in leaves of many species undergoing an oxida tive stress and h
172. e les deux autres g notypes Fig 48a b N anmoins en r ponse l ozone on observe une baisse de ces compos s chez les trois g notypes Fig 48 Les diff rences de sensibilit l ozone entre nos g notypes ne sont donc pas li es aux concentrations en carot no des en chlorophylles ou en terp nols L analyse des compos s ph noliques a mis en avant une diff rence tr s nette dans le profil de concentrations en compos s ph noliques entre g notype en particulier chez Cima Fig 49 En effet les trois premi res composantes s parent nettement les trois g notypes et 105 Carot noides CONTROL OZONE 34 4 h Temps 27 jour 2 4 5 11 17 4d 04 r r x Carpaccio Robusta Carpaccio Robusta B Chlorophylies CONTROL OZONE 204 157 Temps jour 2 n 2 t 4 3107 rs 17 od Carpaccio Cima Robusta Carpaccio Cima Robusta C Terp nols CONTROL OZONE 307 Temps 204 11 H 17 104 07 Quantit _ a 5 Carpaccio Cima Robusta Carpaccio Cima Robusta Figure 48 Concentration en carot no des A en chlorophylles B et en terp nols C dans trois g notypes de peuplier Carpaccio Cima et Robusta apr s 2 4 11 15 et 17
173. e on tree biomass growth physiology and biochemistry a quantitative meta analysis Glob Chang Biol 15 396 424 96 R sultats 11 Matyssek R Le Thiec D L w M Dizengremel P Nunn AJ et al 2006 Interactions between drought and O stress in forest trees Plant Biol 8 11 17 12 Heath RL 2008 Modification of the biochemical pathways of plants induced by ozone What are the varied routes to change Environ Pollut 155 453 463 13 Ainsworth EA Yendrek CR Sitch S Collins WJ Emberson LD 2012 The effects of tropospheric ozone on net primary productivity and implications for climate change Annu Rev Plant Biol 63 15 1 15 25 14 Castagna A Ranieri A 2009 Detoxification and repair process of ozone injury From uptake to gene expression adjustment Environ Pollut 157 1461 1469 15 Ishida H Makino A Mae T 1999 Fragmentation of the large subunit of ribulose 1 5 bisphosphate carboxylase by reactive oxygen species occurs near Gly 329 J Biol Chem 274 5222 5226 16 Apel K Hirt H 2004 Reactive oxygen species Metabolism oxidative stress and signal transduction Annu Rev Plant Biol 55 373 399 17 Mittler R Vanderauwera S Gollery M Van Breusegem F 2004 Reactive oxygen gene network in plant Trends Plant Sci 9 490 498 18 Foyer CH Noctor G 2011 Ascorbate and Glutathione the heart of the redox hub Plant Physiol 155 2 18 19 Dizengremel P Le Thiec D Bagard M Jolivet Y 20
174. e que PhyB agisse de concert avec les phototropines les cryptochromes et le phytochrome A dans la r ponse la lumi re blanche Wang et al 2010 EFFET DU CO Une forte concentration en entraine une fermeture des stomates tandis que des concentrations faibles vont avoir l effet inverse En effet le CO va activer les canaux anioniques et provoquer un fort efflux d ions K entra nant une sortie de et de malate et une d polarisation de la membrane Figure 18 Kim et al 2010 Cependant les tapes du signal pr c dent l activation des canaux anioniques restent encore mal connues bien que des avanc es aient permis l laboration d un nouveau mod le de r ponse au CO Figure 19 Xue et al 2011 La forte concentration en CO va par le biais des anhydrases carboniques BCA1 et BCA4 entra ner une hausse de la concentration en HCO Deux cascades de signaux se mettent en place Ind pendamment des concentrations en Ca la forte concentration en et HCO va activer les canaux ioniques de type S Hu et al 2010 inhiber les canaux d entr e de K inhiber les ATPases entra nant ainsi la fermeture des stomates et l inhibition de l ouverture stomatique En parall le les canaux anioniques SLACI d pendants vont aussi tre activ s par l amor age de la sensibilit au Figure 20 Les signaux de fermeture des stomates dus au CO et l ABA deviennent les m mes partir de cet instant bien que l
175. e stagiaire telle que l on esp re toujours en avoir Ton encadrement aura toujours t facile et agr able En compl ment de mes recherches j ai pu transmettre un peu de mes connaissances enfin j esp re par l interm diaire de l enseignement Ceci n aurait pas t possible sans Jean Claude Dany Val rie Pierrick et bien stir Yves Gr ce vous l exp rience aura t une r ussite Enfin j ai une pens e particuli re pour les autres doctorants ayant d j soutenu en particulier Ata mon bin me de fumigation mais aussi Nicolas Rana R my Pauline Cyril Sacha Fahad Cl ment Nianguiri Daniel ou en train de s en rapprocher Julien Silv re et Florian courage c est la derni re ligne droite Emilie j esp re que tu as trouv ta voie Morgane m me si tu manques d assurance parfois tu iras loin si tu gardes confiance en toi Tu es capable de tout si tu le veux vraiment Fran ois et Charlotte vous avez encore un peu de temps profitez en bien ca passe vite et longue vie a journ e des doctorants Nicolas sans avoir travaill directement avec toi tu m auras marqu e par ta gentillesse Je me souviendrai toujours de toi qui avais toujours un mot agr able accompagn d un sourire Pour finir je remercie tous ceux qui m ont apport de la bonne humeur au quotidien En premier lieu Christian sans qui la pause caf serait moins agr able mais aussi Marie B atrice Oliver Patrick B atrice Audrey Andr Jacq
176. e x treatment x time 10 2 5310 0 0107 Oxidized ascorbate DHA Genotype 2 76 9992 lt 0 0001 Treatment 1 3 7923 0 0554 Time 4 8 3456 lt 0 0001 Genotype x treatment 2 5 5870 0 0056 Genotype x time 8 5 0058 0 0001 Genotype x treatment x time 12 2 3456 0 0134 Redox ratio ASA ASA DHA Genotype 2 23 4853 lt 0 0001 Treatment 1 0 8656 0 3553 Time 4 6 3915 0 0002 Genotype x time 8 3 7001 0 0012 Treatment x time 4 2 8703 0 0290 Genotype x treatment x time 10 2 5797 0 0100 Table S3 ANOVA the main effects and significant interactions of genotype treatment and sampling date on the concentrations of glutathione total reduced form oxidized form and redox ratio DF Degrees of Freedom Metabolite name DF F value p value Total glutathione GSSG GSH Genotype 2 54 474 lt 0 0001 Treatment 1 279 081 lt 0 0001 Time 4 14 622 lt 0 0001 Treatment x time 4 10 790 lt 0 0001 Reduced glutathione GSH Genotype 2 20 348 lt 0 0001 Treatment 1 99 539 lt 0 0001 Time 4 4 349 0 0029 Treatment x time 4 4 777 0 0015 Oxidized glutathione GSSG Genotype 2 34 803 lt 0 0001 Treatment 1 158 216 lt 0 0001 Time 4 8 115 lt 0 0001 Treatment x time 4 7 447 lt 0 0001 Redox ratio GSH GSH GSSG Genotype 2 1 2317 0 2963 Treatment 1 2 7861 0 0983 Time 4 0 1552 0 9602 Figure S1 Normalized expression of genes involved in Ascorbate biosynthesis in leaves of three poplar genotypes Carpaccio Cima and Robusta ex
177. eants ont ensuite t s ch s sous un flux de nitrog ne par 400 uL et redissouts dans des bouteilles opaques dans un m lange de 600 uL de dichlorom thane DCM 1200 uL d ac tonitrile ACN et 200 uL de m thanol MeOH Les analyses de chlorophylles et de carot noides ont t r alis es par chromatographie liquide haute performance Ionisation Chimique Pression Atmosph rique Spectrom tre de masse HPLC APCI MS 4 COMPOSES PHENOLIQUES Les extractions des chantillons ont t r alis es dans du m thanol 85 avec 0 005 de butylated hydroxytolu ne BHT Protocole d taill en annexe 3 Durant l extraction 200 uL de solution de standard interne la solution est faite partir de 2 mL d acide benzoic d5 1 mg mL dans un m lange 1 1 MeOH H20 2 mL de D glucose C13 1 mg mL dans un m lange 1 1 MeOH H0 1 mL de biochanin A 1 mg mL dans du m thanol 100 600 uL de glyc rol d8 2 8 mg mL dans un m lange 1 1 et 1 mL d alanine d3 a 0 1 mg mL dans un m lange 1 1 MeOH H20 sont ajout s Les surnageants sont divis s en aliquots d di s diff rentes analyses Un aliquot de 500 uL est s ch et redilu dans 250 uL 32 Mat riel et M thodes de MeOH et 250 ul d eau pour ensuite servir aux analyses de compos s ph noliques Nous avons analys les compos s ph noliques par chromatographie liquide haute performance HPLC avec ionisation par lectron bulisation EST quip de d te
178. eatment 37 Chloroplast _ AA synthesis Rubisco Glu N 7 Glu GS GOGAT Gin 206 Triose P PEP OAA y NADP M Asc DHA GSH lt Halliwell Asada Foyer cycle Glycolysis NPK za NADP GAPDH Pyruvate Cytosol HCO _ PEPC PPDK cw i LI Triose P VADP G6PDH HMP cycle Citrate isocitrate NADPH ICDH 20G Fig 1 Simplified scheme showing the NADP dependent enzymes associated with cytosolic NADPH production and its use for detoxification reactions Halliwell Asada Foyer cycle NADP isocitrate dehydrogenase NADP ICDH activity is linked to oxoglutarate 2 OG production in the cytosol which is then imported into plastids to generate glutamate and other amino acids AA NADP glucose 6 phosphate dehydrogenase NADP G6PDH activity is linked to the functioning of the hexose monophosphate pathway HMP The substrate of the NADP malic enzyme NADP ME is malate which can be the product of the successive reactions catalyzed by phosphoenolpyruvate carboxylase PEPC and cytosolic malate dehydrogenase MDH Phosphoenolpyruvate PEP and oxaloacetate OAA are respectively the substrates for these latter enzymatic reactions NADP glyceraldehyde 3 phosphate dehydrogenase NADP GAPDH is considered as a shunt in the glycolytic pathway which bypasses the production of NADH and ATP Pyruvate kinase PK catalyzes the last step of the glycolytic
179. ed LV T TOTI AX M3 IVOOVIOVOLLVOVOOVVOO H 9ue1quiour ewsejd oaned oj repris 16020 lt 000 INXBos CCVH d IOVOVIOLLLLOLLODJIODO 0009909S10 10d SIIIVHV EESLLOVE ICCSCOVO 92869 2 416 lt lt 90pp9VVO PCCSCOVO CEV6TIVI TESLLOVE 0L9c08e1Vv rc869d VO OS6LPSEIV L6rs8g VO JO GOT OSELOSTIOO ALdOd vo1s42d sNUNIJ OYO 9584 LV 1 886 I8 OVVVDLVOVDLVDVOVVODD H oaned oj repris 1 8LS8TEZOO INXBost LVVOODVODDLDOLVOLVVL 0091400101204 uondrnosop 4 JON sey lt S pue q syour iq A 070 544 9UIEN orroeds 1194 15 52098 JO 871 TS 91981 lt urojoid OTO exir jouueqo oj repris 88606 lt 000 INXBost 0829059000 ULdOd VOLLVIVIODDOLDLLOLOOO 0001900900 1304 92010 LO680P8SIVOLILTSEIV VC60I OT Od jxqiso Jo Sojoquo v y TOV OT6 EST000 ULdOd LLOVVOIOVOLVVIOLODOO 8 19 ur0joid ouueqo opuopu serwis T S9000E700 LVOLOOLLLOODLLOOLLOL OOLIEED 100 Od 2010 LT6EEAVO OcSccava 911718 nv ocersovd 080118000 ULdOd 6HSTEAVO Jo 00 7215124 66806000 IODIODIOVOIOVOLLLLOL snunig QAO uodsuen IIS 19 606200 INXIIoW T ILUNdd id VLLODIOVOVVDVOODLLOV 00 TTIO 00 1304 THO IZ8LOL OL
180. em F Reactive oxygen gene network of plants Trends in Plant Science 2004 9 490 498 Moller IM Jensen PE Hansson A Oxidative modifications to cellular components in plants Annual Review of Plant Biology 2007 58 459 481 Mott KA Opinion Stomatal responses to light and CO2 depend on the mesophyll Plant Cell amp Environment 2009 32 1479 1486 164 R f rences Musselman RC Lefohn AS Massman WJ Heath RL A critical review and analysis of the use of exposure and flux based ozone indices for predicting vegetation effects Atmospheric Environment 2006 40 1869 1888 Nishizawa A Yabuta Y Shigeoka S Galactinol and raffinose constitute a novel function to protect plants from oxidative damage Plant Physiology 2008 147 1251 1263 Noctor G 2006 Metabolic signalling in defence and stress the central roles of soluble redox couples Plant Cell amp Environment 29 409 425 Noctor G Mhamdi A Chaouch S Han Y Neukermans J Marquez Garcia B Queval G Foyer CH Glutathione in plants an integrated overview Plant Cell amp Environment 2012 35 454 484 Noe SM Giersch C A simple dynamic model of photosynthesis in oak leaves coupling leaf conductance and photosynthetic carbon fixation by a variable intracellular CO pool Functional Plant Biology 2004 31 1196 1204 Outlaw WH Manchester J Guard cell starch concentration quantitatively related to stomatal aperture Plant Physiology 1979
181. ent 33 914 925 B ker P Morrissey T Briolat A Fall R Simpson D Tuovinen J P Alonso R Barth S Baumgarten M Grulke N Karlsson P E King J Lagergren F Matyssek R Nunn A Ogaya R Penuelas J Rhea L Schaub M Uddling J Werner W Emberson L D 2012 DO3SE modelling of soil moisture to determine ozone flux to forest trees Atmospheric Chemistry and Physics 12 5537 5562 Castagna A Ranieri A 2009 Detoxification and repair process of ozone injury from Os uptake to gene expression adjustment Environmental Pollution 157 1461 1469 Castillo F J Heath R L 1990 Ca transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure Plant Physiology 94 788 795 Danielsson H Karlsson G P Karlsson P E Pleijel H 2003 Ozone uptake modeling and flux response relationships an assessment of ozone induced yield loss in spring wheat Atmospheric Environment 37 475 485 Dghim A A Dumont J Hasenfratz Sauder M P Dizengremel P Le Thiec D Jolivet Y Capacity for NADPH regeneration in the leaves of two poplar geno types differing in ozone sensitivity Physiologia Plantarum in press Dizengremel P 2001 Effects of ozone on the carbon metabolism of forest trees Plant Physiology and Biochemistry 39 729 742 Dizengremel P Le Thiec D Bagard M Jolivet Y 2008 Ozone risk assessment for plants central role of metabo
182. ent responses of stomata to blue red and green monochromatic light differences between the normally oriented and inverted leaves of sunflower Plant amp Cell Physiology 2011 52 479 489 Wellburn A R The spectral determination of chlorophylls a and b as well as total carotenoids using various solvents with spectrophotometers of different resolution Journal of Plant Physiology 1994 144 307 313 White DA Beadle C Sands PJ Worledge D Honeysett JL Quantifying the effect of cumulative water stress on stomatal conductance of Eucalyptus globulus and Eucalyptus nitens a phenomenological approach Australian Journal of Plant Physiology 1999 26 17 27 167 R f rences Wieser G Matyssek R Linking ozone uptake and defense towards a mechanistic risk assessment for forest trees New Phytologist 2007 174 7 9 Wilkinson S Mills G Illidge R Davies W J How is ozone pollution reducing our food supply Journal of Experimental Botany 2011 63 527 536 Wilson RC Fleming ZL Monks PS Clain G Henne S Konovalov IB Szopa S Menut L Have primary emission reduction measures reduced ozone across Europe An analysis of European rural background ozone trends 1996 2005 Atmospheric Chemistry and Physics 2012 12 437 454 Wittig VE Ainsworth EA Long SP To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees A meta analytic review of the last 3 dec
183. entr e de la chambre en comparaison avec la sortie de la chambre Figure 39 gt UelWo We 8 s 1 wo 1 9 Isw Rp Itw avec E la transpiration nette ue le flux net Wo et We les concentrations en eau entrante et sortante s la surface gs la conductance la vapeur d eau gm la conductance totale de la feuille calcul e partir de la pression de vapeur saturante la temp rature de la feuille et de l air la pression atmosph rique et la transpiration Zp la conductance de couche limite et kr facteur d duit du ratio stomatique UTILISATION Lors de la premi re exp rience de fumigation des mesures de conductance stomatique la vapeur d eau et de photosynth se Asat en condition de lumi re saturante 1200 s de lumi re dont environ 10 de bleu et 90 de rouge temp rature foliaire 25 C concentration en CO 400 VPD 0 9 kPa ont t r alis es avec l appareil de mesure d changes gazeux Li 6400 LI COR Inc Lincoln NE USA aux temps t0 t14 et t18 en jour sur 1 arbre de chaque g notype dans chaque chambre Figure 40 Lors de la seconde exp rience de fumigation des mesures de conductance stomatique la vapeur d eau et de photosynth se ont t r alis es l aide de 3 appareils Li 6400 LI Lincoln NE USA intercalibr s Figure 41 Les mesures ont t r alis es sur 3 arbres par g notypes d di s ces mesures Apr s
184. ents climatiques notamment les augmentations de temp rature et la hausse des productions de pr curseurs due en partie l augmentation du trafic automobile sur d autres continents semblent estomper la baisse attendue des concentrations en ozone en Europe Wilson et al 2012 Sur la base de 26 mod les de pr diction diff rents le changement de concentration moyenne en ozone troposph rique devrait tre compris entre 5 et 15 pour 2030 Stevenson al 2006 L application mondiale plus ou moins stricte des limitations de production de pr curseurs et les pr dictions variables concernant les changements climatiques entrainent une forte impr cision des mod les de pr diction des concentrations en ozone IPCC 2007 Cape 2008 Fuhrer 2009 4 IMPACT DE L OZONE SUR LA VEGETATION L exposition des plantes de grandes cultures est valu e comme d finie l chelle de l Europe par la Directive Ozone selon les valeurs 40 Accumulated Ozone over a Threshold of 40 ppb for global radiations gt 50 de mai juin tandis que celle des for ts s tend sur toute la p riode de v g tation d avril septembre Selon les connaissances actuelles un niveau critique d ozone partir duquel des effets directs nocifs pour la v g tation peuvent se produire est fix pour les esp ces de grandes cultures et pour les for ts dans le Mapping Manual de M amp M 2004 6000 ug m et 10000 ug m respectivement
185. enu par dismutation spontan e 18 du MDHA en utilisant le glutathion r duit GSH produisant du glutathion oxyd GSSG 20 20 DHA 2 GSH ASA 6556 Le glutathion oxyd est ensuite r g n r son tour par la glutathion r ductase GR en utilisant du NADPH 21 Puis l ascorbate retourne dans l apoplasme 21 GSSG NADPH 2 GSH NADP 3 AUTRES ENZYMES ET MOLECULES ANTIOXYDANTES D autres m canismes moins importants ou moins connus servent la d toxication des ROS issus de l ozone et viennent en compl ment de la voie d Halliwell Asada On les retrouve r partis dans la plupart des organites des cellules Tableau 2 et Figure 24 Le tocoph rol stabilise les radicaux produits lors de la peroxydation des lipides stoppant ainsi les r actions en chaine Les carot noides dont la z axanthine permettent de neutraliser l oxyg ne singulet et de reconvertir le tocoph rol porteur d un radical tocoph rol Les composants ph noliques ont g n ralement un fort pouvoir antioxydant notamment les flavonoides querc tine anthocyanidines cyanidine et delphinidine Rice Evans et al 1997 Les anthocyanines peuvent d toxiquer 620 Il se peut donc qu ils agissent dans les m canismes de d toxication de l ozone Les superoxydes dismutases SOD sont des enzymes qui lient deux ions superoxydes deux ions hydrog ne pour produire de l oxyg ne mol culaire et de l eau oxyg n e neutres et moins toxiques La ca
186. eration of reducing power de novo synthesis of antioxidants and repair processes of oxidized proteins participate to limit ozone effects these processes being costly in energy 25 26 Amino acids are key elements in the balance between catabolism and biosynthesis of various compounds Amino acids of the aspartate family can be used as substrate for alternative way of respiration during stress and thus represent a source of energy 27 Some amino acids are directly linked to antioxidants biosynthesis such as aromatic amino acids for phenolic compounds biosynthesis 28 or cysteine which appears as a rate limiting factor in glutathione biosynthesis 29 The purpose of this study was to characterize the impacts of ozone on the ascorbate and glutathione content and particularly on their biosynthetic pathways in three Euramerican poplar genotypes differing in ozone sensitivity Based on visible injuries and number of abscised leaves Robusta was the most sensitive Cima showed an intermediate sensitivity whereas Carpaccio was the least sensitive to ozone Two complementary approaches were combined i the expression of genes encoding enzymes involved in the main ascorbate and glutathione biosynthesis pathways was investigated in response to ozone and ii the ascorbate glutathione and amino acids contents were quantified Our work intends to provide a new insight on the effect of ozone on ascorbate and glutathione biosynthesis and attempts to
187. ers followed by the generation of a dissociation curve to check for specificity of amplification The reaction mixture contained 10uL Brillant III Ultrafast SYBR green QPCR Master mix 0 3uL Agilent technologies Massy France 250 nM of gene specific primers and 2 uL cDNA diluted 1 5 in each 20 uL reaction We studied genes coding for enzymes of the main biosynthesis pathway of ascorbate and glutathione based on homology with genes described in Arabidopsis thaliana Supporting Information Table S1 The study of the glutathione pathway was extended to the links with major amino acids Specificity of all primers has been checked by sequencing the products Beckman Coulter Genomics Essex UK No template control NTC reactions were prepared for each gene The Ct values were determined with the same threshold and PCR efficiency was estimated using a standard curve for each gene and each genotype Efficiency values were taken into account in all subsequent calculations The 83 R sultats expression values were normalized to two control genes the internal control alien and UBL and two stable genes of interest determined by the GeneNorm program GOGAT6 and VTC21 Amino acid and polyamines extraction and analysis Freeze dried leaf powder 30 mg was mixed with 1500 uL 0 005 w v butylated hydroxytoluene BHT in 80 v v MeOH and 200 uL of internal standard mixture containing 0 3 mg ml benzoic d5 acid Campro Scientific Gm
188. es amin s vers les processus de r paration et de d toxication tout en trouvant des ressources nerg tiques alternatives Cima quant lui se d finit par un niveau de sensibilit interm diaire entre Carpaccio et Robusta avec une r duction de biomasse toutefois importante Les facteurs justifiant sa sensibilit sont moins clairs que pour Robusta En effet contrairement Robusta il cumule certains facteurs de tol rance comme des concentrations constitutives en ascorbate importantes un maintien des formes r duites de l ascorbate et du glutathion des vitesses de r actions des stomates importantes N anmoins il semblerait que Cima se distingue de Carpaccio par une ressource primaire plus faible notamment au niveau des sucres et par un profil en compos s ph noliques particulier qui pourrait constituer un pool antioxydant moins performant bien que cette hypoth se n cessite d tre v rifi e Ainsi la notion de r sistance l ozone r sulte bien de la combinaison de marqueurs de tol rance multiples 130 Conclusion et perspectives En ne prenant pas ses facteurs dans leur globalit on ne peut donc pas pr dire la tol rance d un g notype Cette th se aura permis de mettre en vidence des pistes suivre pour l am lioration des seuils de risque l ozone pour la v g tation en approfondissant les connaissances concernant les effets de l ozone sur la r gulation stomatique et concernant les processus de d t
189. es expression combined with remobilization processes in poplar The glutathione biosynthesis pathway consists of a two step reaction sequence from Cys Glu and Gly 1 gamma glutamylcysteine synthetase y ECS catalyses the formation of gamma glutamylcysteine and 2 final step catalyzed by glutathione synthetase GSH S from Gly and gamma glutamylcysteine 42 The activity of y ECS is one of the controlling factors of glutathione synthesis whereas the activity of GSH S has less impact 52 Here Cys content and y ECS activity increased under ozone suggesting that GSH S activity could become limiting in turn justifying the increased gene expression of GSH S observed Higher expression of these genes in Carpaccio could in part explains the increase in its tolerance to ozone The glutamine synthetase GS family has been previously described by 53 The expression of the two genes coding for the chloroplastic GS isoenzyme in poplar GS8 and GS10 decreased in response to ozone whereas the expression of genes coding for the cytosolic isoenzyme increased Ozone led to the same responses as senescence and pathogen attack i e a decreased expression of chloroplastic gene proteins 058 GS10 GOGATI2 and whereas the expression of genes of the cytosolic isoforms is induced to allow a remobilization of N and to sustain glutamate availability 54 The major Gly biosynthesis pathway is linked to photorespiration 55 The fact that photores
190. es r cepteurs soient diff rents Xue et al 2011 Dans les deux cas la sensibilit est amorc e et les deux signaux deviennent commun avant ou partir de la prot ine kinase OSTI aussi appel e SnRK2 6 et SnRK2E Contrairement au CO l action de se base sur le mod le PYR RCAR PP2C SnRK2 d crit par Hubbard et al 2010 pour activer OSTI Figure 21 Des tudes suppl mentaires sont n cessaires pour lucider les m canismes par lesquels les stimuli au et ABA amorcent la sensibilit au Ca Enfin il faut rappeler que la prot ine kinase joue un r le important dans la r gulation du mouvement stomatique sous forte concentration en CO Elle agit comme un r gulateur n gatif majeur Hashimoto et al 2006 12 messenger production anions K transport Figure 20 Sch ma illustrant le mod le PYR RCAR PP2C SnRK2 de r ponse l ABA Hubbard et al 2010 Un primed Stimulus X Present Sensor activation threshold Stomate remains Stomate remains Stomate open open closes Figure 21 Mod le d amorgage de la sensibilit au Hubbard et al 2011 L amor age et l augmentation des Ca sont n cessaires pour l activation des canaux anioniques et potassiques de type S r sultant la fermeture des stomates Synth se bibliographique EFFETS DE L OZONE D une mani re g n rale l ozone entraine une r duction de la conductance stomatique Cependant les m canismes
191. es repr sentent la premi re barri re m canique de d fense permettant de r guler l entr e d ozone dans les plantes Ce sont les premi res cellules en contact avec l ozone et elles sont donc susceptibles d tre soumises un stress oxydatif important Les stomates ont un fonctionnement complexe qu il est important de rappeler et le calcul de la conductance stomatique voir ci apr s est par cons quent d licat et toujours am liorable GENERALITES Les stomates sont des pores situ s sur l piderme des parties a riennes des plantes Figure 14 Ils sont constitu s de deux cellules de gardes entourant un orifice appel ostiole La paroi des cellules stomatiques est dissym trique et plus paisse du c t interne Les stomates contr lent deux des plus importants processus des plantes la photosynthese et la transpiration Plus de 95 du dioxyde de carbone entrant pour la photosynth se et de la vapeur d eau sortante pour la transpiration et le refroidissement de la surface foliaire chang s entre la feuille et l atmosph re passent par les stomates De par ces fonctions ils sont essentiels pour la croissance des plantes et sont de premi re importance dans toutes les consid rations de rendements des cultures Tableau 1 Equations des principaux mod les de calcul de conductance stomatique issu de Damour et al 2010 P 12b 29 31 32 33 35 Jarvis 1976 White er al 1999 Noe
192. estdagh P Van Vlierberghe P De Weer A Muth D Westermann F Speleman F amp Vandesompele J 2009 A novel and universal method for microRNA RT qPCR data normalization Genome Biology 10 61 74 Outlaw W H amp Manchester J 1979 Guard cell starch concentration quantitatively related to stomatal aperture Plant Physiology 64 79 82 Pandey S Zhang W amp Assmann S M 2007 Roles of ion channels and transporters in guard cell signal transduction FEBS 581 2325 2336 Paoletti E 2005 Ozone slows stomatal response to light and leaf wounding in a Mediterranean evergreen broadleaf Arbutus unedo Environmental Pollution 134 439 445 Paoletti E amp Grulke N E 2010 Ozone exposure and stomatal sluggishness in different plant physiognomic classes Environmental Pollution 158 2664 2671 Pei Z M Murata Y Benning G Thomine S Klusener B Allen G J Grill E amp Schroeder J I 2000 Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells Nature 406 731 734 R Core Team 2012 R A language and environment for statistical computing R Foundation for Statistical Computing Vienna Austria ISBN 3 900051 07 0 URL http www R project org Saeed A I Sharov V White J et al 2003 TM4 a free open source system for microarray data management and analysis Biotechniques 34 374 378 Saeed A L Bhagabati N K Braisted J C Liang W Sharov V Howe E A Li J Thiagaraja
193. eut n anmoins tre transform en radical hydroxyle par la r action de Fenton catalys e par des ions m talliques libres Fe et Cu 9 10 et la r action de Haber Weiss 11 Les radicaux superoxyde et hyperoxyde sont en quilibre dans les cellules vivantes La forme hyperoxyde tant plus hydrophobe elle p n tre plus facilement les bicouches lipidiques des membranes Dans des conditions de pH neutre ou l g rement acide ces deux radicaux peuvent dans des milieux aqueux se dismuter H202 et spontan ment ou sous l action de la superoxyde dismutase SOD Wojtaszek 1997 12 13 14 Un quilibre entre production et d toxication r gule la toxicit et la signalisation des ROS Mittler et al 2004 R actions de Fenton 9 gt Fe OH OH 10 Fe QV gt Fe Cu Op Synth se bibliographique R actions de Haber Weiss 11 H0 OH Dismutation de superoxyde 12 2 OH gt O5 13 2 OH H gt HO 14 2 02 2 gt EFFETS VISIBLES SUR LES FEUILLES Tandis qu une exposition chronique la pollution l ozone peut induire une perte de biomasse sans signe visible une exposition aig e l ozone entra ne l apparition de d g ts visibles sur les feuilles ou les aiguilles des plantes ce qui peut repr senter des pertes conomiques sur des esp ces horticoles ICP forests 20
194. f biomass than Cima 1 3 times more The only significant effect of O3 on biomass was a 35 decrease of the root biomass of Carpaccio These results lead to a significantly higher root shoot ratio in Robusta twice as much than in the other genotypes treatment had no significant effect on that ratio even though a decrease in the ratio was observed for Carpaccio Although the effect of O3 on total biomass was not significant we observed a trend for a decrease in total biomass values in all three genotypes 5 in Carpaccio and Robusta and 12 in Cima 3 2 Effects of ozone on chlorophyll content net CO assimilation and stomatal conductance Robusta differed significantly from the other two genotypes with higher chlorophyll content about 1 5 times more Fig 4C An B 250 lt Ags 5 mmol m s Times s Fig 1 Examples of A light curve stomatal opening and B VPD or CO curve stomatal closure showing the parameters used for calculations i e 60 stomatal conductance before changing environmental parameters g 1 stomatal conductance at equilibrium after changing environmental parameters t the time needed to reach equilibrium Ag the difference between 2 0 and 2 1 88 J Dumont et al Environmental Pollution 173 2013 85 96 lt Ambient Ozone 160 140 e 2 5 120 2 100 8 3 80 60 T 40 H 0 5 10 15 20 160 4 B 140 4 120 8 s
195. f each treatment was sampled A square of 1cm was cut and immediately immersed in a fixative solution of 2 5 glutaraldehyde and 0 2 M phosphate buffer pH 7 2 at 4 C for Transmission Electron Microscopy observations Three discs of leaves were sampled by a punch put in an eppendorf tube and immediately flash frozen in liquid nitrogen for Scanning Electron Microscopy analysis They were freeze dried at 40 C for 40 h at a pressure of 10 Pa in a FreeZone Plus freeze dryer Labconco Kansas City Missouri and then the temperature was turned back progressively to 20 C within 10 h Transmission electronic microscopy observations One to 2 mm were cut using a razor blade from the square immersed in fixative solution of 2 5 v v glutaraldehyde fixative in 0 1 M phosphate buffer pH 7 2 Leaf samples were infiltrated under vacuum in fixative solution and were maintained at 8 C for 4h After being rinsed in the same phosphate buffer by 5 washes samples were post fixed in 2 osmium tetroxide in the rinsing buffer for 1 h at 4 C The samples were washed in rinsing buffer 3 times of 5min followed by distilled water prior to slow dehydration through a graded acetone series Samples were embedded in Epon 812 EMS Hatfield US according to the manufacturer s instructions These samples were orientated in moulds and the resin polymerised at 60 C for 48 hours The resultant resin blocks were trimmed before ultra thin sections 80nm were obtained on a RMC MT7 ult
196. f the National Academy of Sciences of the United States of America 96 13577 13582 Vahisalu T Puzorjova L Brosche M Valk E Lepiku M Moldau H Pechter P Wang Y S Lindgren O Salojarvi J Loog M Kangasjarvi J Kollist H 2010 Ozone triggered rapid stomatal response involves the production of reactive oxygen species and is controlled by SLAC1 and OST1 Plant Journal 62 442 453 Van Dingenen R Dentener Raes Krol M C Emberson L Cofala J 2009 The global impact of ozone on agricultural crop yields under current and future air quality legislation Atmospheric Environment 43 604 618 Vingarzan R 2004 A review of surface ozone background levels and trends Atmospheric Environment 38 3431 3442 Wang Y Nogushi K Terashima 1 2011 Photosynthesis dependent and inde pendent responses of stomata to blue red and green monochromatic light differences between the normally oriented and inverted leaves of sunflower Plant and Cell Physiology 52 479 489 Wellburn A R 1994 The spectral determination of chlorophylls a and b as well as total carotenoids using various solvents with spectrophotometers of different resolution Journal of Plant Physiology 144 307 313 Wilkinson S Davies W J 2010 Drought ozone ABA and ethylene new insights from cell to plant to community Plant Cell amp Environment 33 510 525 Wittig V E Ainsworth E A Naidu S L Karnosky
197. genase NADP ICDH Fig 1 NADP ME catalyzes the reversible oxidative decarboxylation of L malate into pyruvate which leads to the release of Physiol Plant 148 2013 NADPH in the cytosol Fig 1 An increase in its activity has been linked to plant metabolic responses to various biotic and abiotic stresses see Doubnerov and Ryslav 2011 for a review and interestingly to chronic and acute Os fumigation Fontaine et al 1999 Dizengremel et al 2009 Guidi et al 2009 NADP ICDH catalyzes the formation of 2 oxoglutarate from isocitrate using NADP as a cofactor It delivers NADPH directly into the cytosol Fig 1 Higher NADP ICDH activity has been linked to plant responses to oxidative stress Maller and Rasmusson 1998 Hodges et al 2003 NADPH pro duced by NADP ICDH contributes to redox homeostasis not only in response to abiotic stress Dizengremel 2001 Smeets et al 2005 but also in the case of pathogen attacks Mhamdi et al 2010 The increase in NADP ICDH activity can also be part of reactions involving amino acid replenishment for cell maintenance or repair processes Dizengremel et al 2009 Cytosolic NADPH can also be produced by other NADP recycling enzymes such as glucose 6 phosphate dehydrogenase NADP G6PDH and non phosphorylating NADP dependent glyceraldehyde 3 phosphate dehydrogenase NADP GAPDH Fig 1 Plant NADP G6PDH has mostly been studied for its role as an important NADPH source in fat produci
198. genotypes increased with treatment especially from the second week by around 90 30 and 55 for Carpaccio Cima and Robusta respectively In addition from the second week the three genotypes continued to respire under blue light when submitted to treatment whereas the controls began to photosynthesize ns ns ns ns 0 00643 0 01282 ns ns 0 0184 ns ns ns 3 4 Effects of ozone on the responses to variations in red light In control air conditions there was no significant genotypic effect on the amplitude or the speed of opening in response to an increase in red light Table 4 From the second week the amplitude of the reaction was significantly reduced by treatment in Carpaccio by around 85 and in Robusta by around 75 while it was reduced only during the last week in Cima by around 75 Finally opening speed was not altered by exposure except during the second week for Carpaccio and Robusta The effects of O3 on stomatal conductance were not linked to variations in Ci values which remained stable data not shown In control air conditions the photosynthesis rate under low or intense red light was more important in Robusta than in the other two genotypes around 1 3 times more Table 4 From the second week there was a decrease of photosynthesis under treatment It was significant from the second week for Robusta with around 40 decrease and from the last week with 55 and 45 dec
199. gest that in poplar the enhanced glutathione biosynthesis is mainly due to an increased GSH1 and GSH2 genes expression combined with remobilization processes Regulation of plant amino acid metabolism in response to ozone De novo synthesis of antioxidants and repair processes induced by ozone lead to increased needs for amino acids requiring remobilization processes The central position of amino acids in between anabolic and catabolic pathways complicates the interpretation of their concentrations Monitoring pools of amino acids at five measurement points allows to highlight the strongest trends characterizing the remobilization of amino acids under ozone treatment While concentrations of most of the amino acids decreased in response to ozone Cys Ile and the aromatic amino acids Phe Tyr Trp as well as the polyamine Put increased Cys content is one of the limiting factors of glutathione biosynthesis 52 and was increased in 93 R sultats response to ozone mainly in Carpaccio probably due to a redox regulation of APS reductase activity 60 The increase in aromatic amino acids may be due to an induction of shikimate pathway under ozone 58 61 62 Aromatic amino acids are precursors of various important metabolites 28 like lignins phenolic acids and flavonoids that have been shown to strongly increase in response to ozone in many studies 58 63 64 65 Under ozone an increase of putrescine content occurred ma
200. gure 10 Variation en pourcentage de la biomasse totale biomasse foliaire surface foliaire biomasse a rienne biomasse racinaire et ratio feuille racine pour les gymnospermes et angiospermes expos s des concentrations lev es d ozone par rapport aux t moins expos s l air filtr par charbon actif Les symboles sont encadr s par des intervalles de confiance 95 les degr s de libert et les moyennes des concentrations l ozone sont donn es le long de l axe Y Wittig et al 2009 Asat He i 347 47 ppb 9 HEH i 265 42 ppb 83 45 ppb Gymnosperms Angiosperms 263 48 ppb d f mean O Gymnosperms 57 44 ppb Angiosperms 207 42 ppb 30 20 10 0 10 20 Percent change from CF air Figure 11 Variation en pourcentage de la photosynth se en lumi re saturante et de la conductance stomatique gs des arbres soumis des concentrations ambiantes en ozone par rapport aux t moins expos s l air filtr par charbon actif cf en incluant les diff rences entre gymnospermes et angiospermes Les degr s de libert d f et les concentrations ambiantes moyennes en ozone O sont donn s le long de l axe y Wittig et al 2007 Asat e 455 86 ppb Is 276 91 ppb gt 120 48 143 ppb 90 119 154 106 ppb _ 60 89 155 74ppb 5 30 59 77 47 ppb 5 0 29 17 25ppb gt 120 29 152ppb 90 119 114 104 ppb 60 89 91 75 ppb 30 59 39 45 ppb 40 30 20
201. h noliques ses 32 Xe ALES AMINES A des cole Henin AAA 33 NAMA R rti a den IAE 33 VL ANALYSES 2 34 l andalhs s aes oed e ete ete tt e 34 2 Analyses transcriptomiques partir de feuilles broy es eee 34 Preparation des Matr ices Jue iter n her AER e REPE ees BER TR ERU ER RES 34 Dessin des amoreces sies de Be f eed e d eb t da Be ER eb ede 35 PCR n temps r el Cl49 p 36 3 Analyses transcriptomiques partir de stomates microdiss qu s sss 36 Pr paration des Matrices e 36 Dessin ES atrorCes ssl eee T A rs Mn 37 PGR en temps r el qPCR aee ette eee eit e e eres edt 37 TECHNIQUES DE MICROSCOPIE cesses then 37 1 Pr paration d chantillons de stomates microdiss qu s ss 37 2 D termination de la densit stomatique et de la taille des stomates Microscopie Electronique xt e ER ENT e e eite n ne E 38 Principe SE MEE 38 pie deme deed dnte ded er e 38 3 Microa
202. hangement d humidit dans le temps en tenant compte du flux d air sec traversant le dessicant Le taux de transpiration est alors utilis avec la temp rature de l air et de la feuille et la conductance de couche limite connue dans la chambre pour calculer la conductance stomatique gr ce la formule 3 1 3 Isw epe _ Ki _ pte 9bwo K 1 avec E la transpiration nette T la temp rature de feuille e la pression de vapeur dans la chambre Zsw la conductance la vapeur d eau Zbwo la conductance de couche limite d une face es lt gt la fonction de pression de vapeur saturante K le ratio stomatique P la pression UTILISATION Lors de chacune des deux exp riences un suivi de la conductance stomatique la vapeur d eau gw et de l assimilation nette de face adaxiale et abaxiale confondue a t r alis avec le syst me de mesure d changes gazeux foliaires 11 6200 LI COR Inc Lincoln NE USA Figure 37 3 fois par semaine deux heures apr s le d but de la photop riode En combinaison avec le suivi horaire des concentrations en ozone dans chaque chambre nous avons pu calculer 0 4 le PODy 5 et le CUO 6 L AOT40 Accumulated Ozone over a threshold of 40 ppb correspond l exposition horaire cumul e au del d une concentration limite de 40 ppb Le CUO Cumulative Uptake of Ozone et le PODy Phytotoxique Ozone Dose over a threshold of Y nmol m s correspondent aux flux d ozone ho
203. he French National Research Agency ANR project VMCS Vulnoz the Lorraine Region and the Academy of Finland project nro 138161 95 References 1 Sandermann H 1996 Ozone and plant health Annu Rev Phytopathol 34 347 366 2 Vingarzan R 2004 A review of surface ozone background levels and trends Atmos Environ 38 3431 3442 3 Sitch S Cox PM Collins WJ Huntingford G 2007 Indirect radiating forcing of climate change through ozone effects on the land carbon sink Nature 448 791 795 4 ASPA 2010 Bilan de qualit de l air en Alsace P riode 2000 2009 ASPA 10083002 ID 5 MEDDTL 2010 Bilan de la qualit de l air en France en 2010 6 Felzer BS Cronin T Reilly JM Melillo JM Wang X 2007 Impacts of ozone on trees and crops CR Geosci 39 11 12 7 Van Dingenen R Dentener FJ Raes F Krol MC Emberson L et al 2009 The global impact of ozone on agricultural crop yields under current and future air quality legislation Atmos Environ 43 604 618 8 Avnery S Mauzerall DL Liu J Horowitz LW 2011 Global crop yield reductions due to surface ozone exposure 1 Year 2000 crop production losses and economic damage Atmos Environ 45 2284 2296 9 Dizengremel P 2001 Effects of ozone on the carbon metabolism of forest trees Plant Physiol Biochem 39 729 742 10 Wittig VE Ainsworth EA Naidu SL Karnosky DF Long SP 2009 Quantifying the impact of current and future tropospheric ozon
204. he expression of putative genes of channels involved in stomatal movements in guard cells of the upper Upp and lower Low sides of leaves of three poplar genotypes Carpaccio C Cima I and Robusta R after 18 days of treatment Compounds are clustered using Euclidean distance R sultats observed Fig 3f In Cima the chlorine content is decreased only in guard cells of the lower side and is slightly increased after 18 days of treatment in guard cells of the upper side Ozone increases significantly potassium content in guard cells of both sides of leaves in Robusta from 11 days of treatment whereas in Carpaccio and Cima it is increased only from 18 days of treatment Fig 3g The phosphorus content in guard cells of both sides of the leaf is significantly decreased from 11 days of ozone treatment especially on the lower side Fig 3h The decrease is more important in Cima in which it is almost halved than in Carpaccio or Robusta Effect of ozone on the expression in guard cells of genes involved in stomatal movements Most of the genes involved in stomatal movements exhibit a repressed expression under ozone treatment See Fig 51 for complete dataset Hierarchical clustering reveals four main groups of genes with different pattern of regulation of expression in response to ozone Fig 4 i The expression of CAX3 and CA is strongly repressed more than 10 times in guard cells of both side of leaf in all genot
205. he response to variations in environmental parameters appears as a determining factor of genotype 2012 Elsevier Ltd All rights reserved 1 Introduction Tropospheric ozone is a powerful greenhouse gas which acts as a phytotoxin IPCC 2007 Its toxicity results in oxidative stress in plants The concentration of tropospheric is currently still rising while there are already impacts on vegetation and ecosystems Sitch et al 2007 Vingarzan 2004 Annual concentrations are increasing but they are not homogeneous at a global scale Between 2008 and 2009 maximum O3 concentration decreased by 1 2 nmol in the south of Europe whereas France and Belgium it increased by 2 4 nmol mol of Fagerli et al 2011 Currently in France concentrations exceed 120 nmol several days a year depending on the area ASPA Corresponding author E mail addresses jdumont nancy inrafr J Dumont fabien spicher agroparistech fr Spicher montpied nancy inra fr Montpied pierre dizengremel univ lorraine fr P Dizengremel yves jolivet univ lorraine fr Y Jolivet lethiec nancy inra fr D Le Thiec Present address INRA UMR 1092 Laboratoire d tude des ressources for t bois Champenoux F 54280 France Present address AgroParisTech ENGREF UMR 1092 Laboratoire d tude des ressources for t bois Nancy F 54042 France 0269 7491 see front matter 201
206. ient 8 Ozone A 0 7 0 6 0 5 0 4 0 3 0 2 0 1 Tee Carpaccio 2 1 gs mol m s 0 7 0 6 0 5 0 4 0 3 0 2 0 1 4 Cima gs mol m s 15 10 0 7 0 6 0 5 0 4 0 3 4 0 2 0 1 Robusta gs mol m s 20 Time day Fig 5 Effects of treatment Ozone Ambient on stomatal conductance g mol m Pollution 173 2013 85 96 91 0 Ambient 8 Ozone 50 D 40 30 4 E 72 20 4 20 lt 10 0 H 0 5 10 15 20 597 E 404 2 30 E 5 20 04 4 0 4 A 0 5 10 15 20 50 F m 40 E 30 E 5 20 4 amp 101 4 0 T T T T 1 0 5 10 15 20 Time day 2 1 s A B and C and on the water use efficiency mol D E and F of Carpaccio A and D Cima B and E and Robusta C and F Significant differences between treatments T are indicated by p lt 0 05 p lt 0 01 or p lt 0 001 controls That effect can be due to the important decrease in chlorophyll content that results from exposure and can also be linked as suggested by Dghim et al in press Dizengremel 2001 Dizengremel et al 2008 and Wittig et al 2009 to lower rubisco content and activity The energy needs that remain unfulfilled by photosynthesis can be partly compensated for by respiration 4 2 Effects of ozone on stomatal conductance As previous
207. ier les autres r sultats une sensibilit l ozone Pour comprendre l importance des processus de d toxication des analyses m tabolomiques analyse de l ascorbate du glutathion des carot noides des sucres ont t r alis es au niveau foliaire Une approche g nomique par PCR quantitative en temps r el au niveau foliaire compl te les r sultats en analysant l expression de g nes impliqu s dans les processus de d toxication Ces r sultats viennent enrichir nos connaissances pour identifier un indicateur du niveau de d toxication ce qui permettra in fine de calculer des flux effectifs Parall lement afin d am liorer le mod le de Jarvis permettant de calculer la conductance stomatique nous avons caract ris l impact de l ozone sur la r ponse de la conductance stomatique diff rents param tres environnementaux lumi re bleue lumi re rouge CO et VPD De plus l expression de g nes impliqu s dans la r gulation des mouvements stomatiques a t tudi e par qPCR sur des chantillons de stomates microdiss qu s pour comprendre les m canismes impact s 20 MATERIEL ET METHODES 21 Tableau 4 Caract ristiques des 6 sections du genre Populus Crit res de diff rentiation Section Aire Principales Bourgeon Feuille Rameau Bract e Port habitat d origine esp ces florale Etroite Seconde Zone aride Est et ilicifolia Jaune
208. ig 1d e f Ozone decreases significantly the total stomatal conductance of the three genotypes Fig la b c It declines by around a quarter from the fifth day of treatment in Cima Fig 1b and Robusta Fig 1c and from the eighth day of treatment in Carpaccio Fig 1a Since the fifth day the stomatal conductance of the upper side is halved in all three genotypes Fig 1d e f whereas the stomatal conductance of the lower side is stable in Cima Fig 1h and shows a globally non significant diminution of around 2096 in Carpaccio and Robusta Fig 1g 1 Observation by microscopy On the lower side stomatal density is more than twice as important as on the upper side with 65 of the total number of stomata on the lower side in Carpaccio and 62 in the other two 63 Table 1 Stomatal density and size of stomata of adaxial and abaxial sides of leaf of three poplar genotypes under ozone or charcoal filtered air treatment mean SE Stomatal Genotype Treatment density e ies idi stomata mm width pu Control Lower 138 2 32 1 0 2 17 0 0 2 Carpaccio Upper 76 1 32 6 0 3 16 7 0 2 C Ores Lower 134 2 32 5 0 2 16 5 0 2 Upper 77 1 32 9 0 3 15 7 0 2 Control Lower 138 2 32 0 0 2 17 5 0 2 Cima Upper 85 1 29 6 0 2 15 7 0 2 1 Ozone Lower 132 2 32 8 0 2 17 2 0 2 Upper 85 1 30 9 0 2 14 6 0 2 Control Lower 146 1 31 6 0 2 17 4 0 2 Robusta
209. ight and vapour pressure deficit in three Populus deltoides x Populus nigra genotypes Jennifer Dumont Fabien Spicher Pierre Montpied S Pierre Dizengremel lt Yves Jolivet gt lt Didier Le NRA UMR 1137 Ecologie et Ecophysiologie Foresti res Champenoux F 54280 France P Universit de Lorraine UMR 1137 Ecologie et Ecophysiologie Foresti res Vandceuvre l s Nancy F 54506 France lt FR110 EFABA Vandoeuvre l s Nancy F 54500 France ARTICLE INFO ABSTRACT Article history Received 27 July 2012 Received in revised form 17 September 2012 Accepted 28 September 2012 Keywords Ozone Stomatal conductance Light related sensitivity CO concentration VPD The effect of ozone O3 on stomatal regulation was studied in three Euramerican poplar genotypes Populus deltoides x Populus nigra Carpaccio Cima and Robusta The impact of O3 on stomatal conductance responses to variations in blue light red light concentration and vapour pressure deficit VPD was studied Upon exposure sluggish response of stomatal movements was observed characterized by slower reactions to increases in blue light intensity concentration and VPD and lower amplitude of the response to variations in light intensity That sluggish response should be taken into account in stomatal conductance models for phytotoxic ozone dose PODy calculations The speed of t
210. inly in Carpaccio and elevated levels of putrescine are correlated with ozone resistance in various species 66 67 68 Aspartate family amino acids Ile Asn Lys Met Thr and Asp have been suggested to represent an alternative catabolic pathway as substrate for TCA cycle under various abiotic stresses that cause energy deprivation such as ozone 27 This could explain the final decrease of the aspartate family amino acid levels in response to ozone Our results suggest an important remobilization of amino acids in response to ozone in order to provide energy and antioxidants to limit the negative effects Ozone appeared to lead to energy deprivation and to induce regulation at the transcriptomic level linked with metabolomic changes The response to ozone is highly complex as it consists of many interactions at different scales and involves several metabolic and catabolic pathways Studies aiming to understand the resistance to ozone should incorporate several techniques to obtain a more comprehensive view Here we report that the higher resistance of Carpaccio could reside in its capability to increase and regenerate its antioxidants like ASA and GSH and to find energy resources by remobilizing its amino acids pool Figure 10 Although Cima shared some of these factors and responded even more rapidly than Carpaccio it is not enough to make it as tolerant as Carpaccio but allowed it to better withstand than Robusta Indeed it seems tha
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213. ion of phenolic compounds in birch leaves is changed by elevated carbon dioxide and ozone Glob Chang Biol 11 1305 1324 65 Kontunen Soppela S Ossipov V Ossipova S Oksanen E 2007 Shift in birch leaf metabolome and carbon allocation during long term open field ozone exposure Glob Chang Biol 13 1053 1067 66 Langebartels C Kerner K Leonardi S Schraudner M Trost M et al 1991 Biochemical plant responses to ozone 1 Differential induction of polyamine and ethylene biosynthesis in tobacco Plant Physiol 95 882 889 67 Ye B M ller HH Zhang J Gressel J 1997 Constitutively elevated levels of putrescine and putrescine generating enzymes correlated with oxidant stress resistance in Conyza bonariensis and wheat Plant Physiol 115 1443 1451 103 R sultats 68 Durmus N Kadioglu A 2005 Spermine and putrescine enhance oxidative stress tolerance in maize leaves Acta Physiol Plant 27 515 522 104 9VL990LT 1D ose1ojsuedAuoul amp xoJpAuy SULIOS epus 9SE OPIE O ejus ose1ojsuen AqiourAxodp Aq V8STIA OT 234 pusouos jxquso ours 1e rurs oAnejnd 8LWHS DLVOVLVOOVODLLOLLOIO ose1ojsuezjAqiour amp xompAu 8 oj epus 606200 MX 00 7 79900 1104 DLLOVLVOLLOVVIODODLO 0 88089000 ULdOd 9VAID 9VL990LT 1D oselozsueyAYJOWAXOIpA
214. ion of putative genes of enzymes involved in the ascorbate biosynthesis and more precisely the L galactose pathway on three poplar genotypes Carpaccio orange border Cima green border and Robusta blue border at five sampling dates 1 2 3 4 and 5 corresponding to 2 4 11 15 and 17 days respectively R sultats resulted in a non significant increase in the redox state of glutathione in this genotype Figure 3D For detailed statistical analyses see Supporting Information Table S2 Effects of ozone on the expression of genes involved in ascorbate biosynthesis Ozone had a noticeable effect on the expression of genes attributed to the major ascorbate biosynthesis pathway Figure 4 see also Supporting Information Figure S1 for complete dataset with major effects appearing at the third sampling date 11 days and often before in Cima Expression of all the putative genes encoding enzymes involved in the main ascorbate biosynthesis pathway were increased in response to ozone in the three genotypes except for the expression of LGalDH9 which decreased G DH16 and GMES which were almost stable in the three genotypes in response to ozone and 13 which was induced in Carpaccio and Robusta whereas it decreased in Cima The expression of VTC25 was the most enhanced in response to ozone with a 9 5 fold 43 3 fold and 1 9 fold increase in Carpaccio Cima and Robusta respectively Effects of ozone on the expression of genes involved i
215. ir fait le d placement Nancy pour faire partie de mon jury de th se Merci vous deux mais aussi Sari Sarita Inna Jenna Maarit sans oubli Matti pour m avoir tellement bien accueillie et m avoir tant aid e Je garderai toujours en m moire ces trois mois pass s avec vous tous en Finlande Je remercie aussi Yves Gibon qui m aura accueillie dans son quipe Bordeaux et Pierre Baldet qui m aura aid e venir bout de l ascorbate et du glutathion Je crois qu il convient ensuite de remercier comme il se doit ceux qui ont du me supporter durant quelques mois ou quelques ann es dans leur bureau me connaissant ca n a pas du tre facile tous les jours pour R my Silv re Inna et le petit dernier Fran ois Tous votre fa on vous m avez aid e au quotidien Et puis il y a aussi ceux qui ont du me supporter dans leur bureau malgr que ce ne soit pas le mien tout particuli rement David et Cyril David tu auras t d un grand soutien pour moi tant au niveau technique que moral Merci d avoir t toujours disponible pour moi de m avoir conseill e et cout e Sans toi ca n aurait d cid ment pas t pareil tu resteras toujours mon ma tre Jedi Cyril merci d avoir accept mes entr es intempestives et surement trop fr quentes dans ton bureau Je n oublie pas non plus ton aide technique et ton efficacit NathNath je n aurai pas compt les mots et tu es apr s David mais comme tu m auras to
216. ire surface foliaire par unit de surface biomasse ligneuse a rienne biomasse a rienne biomasse racinaire et ratio feuille racine hauteur et diam tre de tous les arbres expos s des concentrations lev es d ozone par rapport aux t moins expos s l air filtr par charbon actif Wittig et al 2009 Figure 10 Variation en pourcentage de la biomasse totale biomasse foliaire surface foliaire biomasse a rienne biomasse racinaire et ratio feuille racine pour les gymnospermes et angiospermes expos s des concentrations lev es d ozone par rapport aux t moins expos s l air filtr par charbon actif Wittig et al 2009 Figure 11 Variation en pourcentage de la photosynth se en lumi re saturante Asat et de la conductance stomatique gs des arbres soumis des concentrations ambiantes en ozone O3 par rapport aux t moins expos s l air filtr par charbon actif cf en incluant les diff rences entre gymnospermes et angiospermes Wittig et al 2007 Figure 12 Variation en pourcentage de la photosynth se en lumi re saturante Asat et de la conductance stomatique gs des arbres soumis des concentrations lev es en ozone O3 par rapport aux t moins expos s l air filtr par charbon actif cf et l impact de diff rentes concentrations en ozone sur la r ponse Wittig et al 2007 Figure 13 Variation en pourcentage de la photosynth se de la conductance stomatique de la transpiratio
217. issouri puis la temp rature a t remont e progressivement 20 C en 10 h Les autres chantillons ont t broy s dans l azote liquide avant analyse Les chantillons utilis s pour la microscopie lectronique ont t pr lev s diff remment Figure 30 Un emporte pi ce nous a servi pr lever le mat riel n cessaire nos observations et analyses au microscope lectronique balayage MEB avant de le d poser dans un cryotube plong imm diatement dans de l azote liquide Pour les chantillons d di s l observation au microscope lectronique transmission MET un petit carr de 1 cm a t d coup la lame de rasoir et plong dans un tube Eppendorf contenant du fixateur voir Analyse ultrastructurale des cellules de garde Microscopie Electronique Transmission pour la composition du fixateur FUMIGATION AL OZONE Les r sultats pr sents dans cette th se sont issus de mesures et de l analyse d chantillons pr lev s au cours de deux exp riences de fumigation distinctes 1 PLAN D EXPERIENCE Lors de chacune des exp rimentations de fumigation 8 chambres ont t utilis es 4 chambres t moin et 4 chambres avec 120 ppb d ozone Figure 31 Lors de la premi re exp rience nous avons dispos 15 arbres par chambre 5 de chaque g notype puis nous avons sacrifi un arbre de chaque g notype dans chaque chambre apr s 2 et 4 jours de fumigation pour des mesures de biomasse ne laiss
218. ity within the same species between genotypes were even observed indicating that the best adapted threshold can vary from one genotype to another from one species to another and that maybe for some parameters the best adapted relation is not linear 4 4 Suggested impact of ozone on the daily time course of stomatal conductance and its effects Based on our results we can infer the effect of on the daily course of stomatal conductance in natural conditions Fig 7B We showed that i in the early morning opening speed in response to the increase in blue light could be reduced ii the opening rate in response to an increase in light was also reduced and iii the closing speed in response to an increase in or VPD was reduced Thus such changes may lead to water saving in the morning and throughout the day Fig 7B spotted area but in the evening they may lead to a loss of water in the presence of O3 compared to the control Fig 7B undulated area The flux may be reduced by the A RE Stomatal conductance Stomatal conductance Time Fig 7 Hypothetical daily courses of stomatal conductance in natural conditions A Differences between genotypes with an ambient ozone concentration for Robusta dotted line Carpaccio and Cima solid line B Differences between low solid line and high dotted line ozone concentrations on Carpaccio and Cima impact of O3 on stomatal conductance during the da
219. ivi de croissance des mesures de hauteur et de diam tre la base de la tige ainsi que le d compte du nombre de feuilles et du nombre de feuilles tomb es ont t r alis es sur tous les arbres deux fois par semaine l aide d un m tre et d un pied coulisse 2 MESURES DE BIOMASSES Ces mesures permettent d valuer la croissance des plants et d identifier s il y eu une modification de l allocation des ressources carbon es vers les parties a riennes ou souterraines Les arbres ont t d coup s et les parties a riennes et souterraines ont t isol es Les feuilles et les tiges ont t s par es dans des pochettes de papier avant d tre mises s cher dans une tuve 60 C jusqu stabilisation du poids tandis que les racines ont n cessit pr alablement au s chage un lavage l eau afin de retirer toute la terre Figure 33 25 The parameters listed above represent the following C amp 22 d d The mole fraction of vapor inside the leaf The mole fraction of vapor at node 1 The mole fraction of vapor at node 2 Stomatal conductance of the leaf surface Vapor conductance of the diffusion path between leaf surface and node 1 Vapor conductance of the diffusion path between node 1 and node 2 Distance between the leaf surface and the first humidity sensor Distance between the two humidity sensors Figure 34 Sch ma de principe du porom tre issu du manuel d
220. l effet de l ozone sur la r ponse des stomates aux variations de lumi re bleue de lumi re rouge de CO et de VPD chez trois g notypes de peuplier variant en tol rance bas e sur des mesures de croissance de biomasse et de n croses Elle a permis de mettre en avant les r sultats suivants ozone entra ne une baisse de la conductance stomatique de l assimilation nette de des teneurs en chlorophylles et une perte de biomasse e lozone provoque un ralentissement de l ouverture des stomates lors d une augmentation de l intensit de lumi re bleue 41 R sultats l ozone conduit un ralentissement de la fermeture des stomates lors d une augmentation de la concentration en CO ou d une hausse du VPD lozone induit une baisse de l amplitude de l ouverture des stomates lors d une augmentation de l intensit de lumi re rouge le g notype le plus sensible Robusta est caract ris par des vitesses de r ponse plus lentes et par des conductances stomatiques initiales plus importantes Ces r sultats ont t publi s dans le journal Environmental Pollution 2013 173 85 96 42 Environmental Pollution 173 2013 85 96 di ELSEVIE Contents lists available at SciVerse ScienceDirect Environmental Pollution journal homepage www elsevier com locate envpol ENVIRONMENTAL POLLUTION Effects of ozone on stomatal responses to environmental parameters blue light red l
221. l estimation d un seuil de risque l ozone pour la v g tation R sum L ozone troposph rique est un polluant atmosph rique majeur qui agit comme une phytotoxine Il p n tre dans les feuilles par les stomates avant d tre dissout dans l apoplaste en g n rant des radicaux libres oxyg n s ROS provoquant ainsi un stress oxydatif Deux barri res existent pour restreindre les effets de l ozone 1 les stomates qui peuvent limiter les flux entrants par contr le de la conductance stomatique et ii le syst me de d toxication des ROS issus de la d gradation de l ozone Nous avons tudi les effets de l ozone 120 ppb sur ces deux moyens de d fense chez trois g notypes de peuplier euram ricain Populus deltoides x Populus nigra plac s en conditions contr l es dans des chambres phytotroniques Un effet direct de l ozone sur la photosynth se et sur les mouvements stomatiques en r ponse des variations de facteurs environnementaux ralentissement des ph nom nes d ouverture et de fermeture a t mis en vidence Les mod les de calcul de la conductance stomatique sur lesquels se basent les indicateurs de seuil de risque l ozone pour la v g tation doivent donc les prendre en compte De plus ces travaux ont mis en vidence le r le pr pond rant des concentrations constitutives en antioxidants dans la tol rance l ozone ainsi que la complexit de ces m canismes de d toxication La notion de flux effectif d ozone
222. leaves Treatment day Genotype T18 TO T18 T18 T18 T18 T18 TO T18 ooooo HHH Aowno 6565 883 o o o w A a vn m lt N ni oi H H H Inm MN H oH d o n N 38505 6 6 HHHHH 5 03 43 00 Soi ai Y O oH d m o lt N m 0 ose H H H H H ASS CN N r rm m m o o GB HoH o 4 lt moo HHHHH oot m m o o m 90 N a o ero HOHHH lt HHH O 0 15 n SO m o o N A N 2 2 c 59505 DESEES E NENE lt lt lt 2 5 3 a E LA 0 10 0 2 7 04 28 0 2 5 10 0 6 15 3 1 5 30 1 8 1 Ozone Treatment effect Carpaccio Cima lt 0 001 ns ns ns ns ns ns 0 00039 ns ns ns ns ns 0 03936 0 03937 J Dumont et al Environmental Pollution 173 2013 85 96 89 regression of order 2 did not improve the relation Fig For net CO assimilation a linear regression was sufficient for Carpaccio and Robusta Fig 6A and C although a polynomial regression of order 2 fitted better for Carpaccio On the other hand a real improvement was noted for
223. les carot noides et les chlorophylles qui jouent un r le dans les processus de d toxication ainsi que les acides amin s et les sucres afin de voir si le m tabolisme carbon est modifi par l ozone Les analyses ont t r alis es l aide d une chromatographie en phase liquide haute performance HPLC et d une chromatographie en phase gazeuse GC coupl es diff rents types de d tecteurs Ces outils de s paration analytique permettent de s parer les m tabolites inject s dans la phase mobile liquide ou gazeuse en les faisant passer dans une phase stationnaire en fonction de leurs propri t s chimiques et donc de leur affinit avec la phase stationnaire De plus dans le cadre d une collaboration avec le Plateau enzymatique de la plateforme M tabolome Fluxome de l INRA de Bordeaux sous la direction d Yves Gibon les formes r duite et oxyd e de l ascorbate et du glutathion ont t dos es en microplaques pr par es l aide d un robot de pipetage Figure 43 Ces mesures spectrom triques se basent sur le suivi spectrom trique d un compos color qui apparait ou disparait en fonction d une activit chimique d pendante de l l ment mesurer 2 ASCORBATE ET GLUTATHION L ascorbate et le glutathion ont t extraits partir de 20 mg de poudre de feuille dans 400 uL d acide m taphosphorique 5 4 C Protocole d taill en annexe 1 L homog nat a 31 Mat riel et M thodes ensuite t cen
224. leu clair soumis un traitement ozone couleurs vives ou ambiant couleurs pastelles cinq dates de pr l vements 1 2 3 4 5 pour 2 4 11 15 et 17 jours de traitement respectivement R sultats 4 CHANGEMENTS INDUITS PAR L OZONE DANS LES METABOLITES PRIMAIRES DE TROIS GENOTYPES DE PEUPLIER Malgr les barri res m caniques et biochimiques que repr sentent la r gulation de la conductance stomatique et le syst me complexe de d toxication l ozone entraine une privation d nergie En r ponse l ozone les flux de carbone du m tabolisme primaire vers le m tabolisme secondaire sont stimul s pour r pondre aux besoins de r paration et de d toxication Iriti et Faoro 2009 Afin de mieux caract riser les modifications du m tabolisme primaire en r ponse l ozone nous avons proc d une analyse des m tabolites primaires par GC MS chez trois g notypes de peuplier euram ricain Le m tabolisme carbon tant la base de la r ponse de d toxication et de r paration cette analyse a aussi pour but de mettre en vidence un marqueur potentiel de r sistance l ozone Comme le montre la figure 50 l ozone a un fort effet sur le m tabolisme primaire et explique 26 de la variance des chantillons PC1 De plus chez le g notype le plus r sistant Carpaccio l effet de l ozone n est visible qu partir de 39 date de pr l vement 11 jour de traitement tandis que chez Robusta et Cima l
225. lism dependent changes in reducing power Environmental Pollution 156 11 15 96 J Dumont et al Environmental Pollution 173 2013 85 96 Emberson L D Ashmore M R Cambridge H M Simpson D Tuovinen J P 2000 Modelling stomatal ozone flux around Europe Environmental Pollution 109 403 413 Fagerli H Gauss M Benedictow A Griesfeller J Jonson J E Nyiri A Schulz M Simpson D Steensen B M Tsyro S Valdebenito A Wind P Aas W Hjellbrekke A G Mareckova K Wankmiiller R Iversen T Kirkevag A Seland O Vieno M 2011 Transboundary Acidification Eutrophication and Ground Level Ozone in Europe in 2009 EMEP Status Report 1 2011 The Norwegian Meteorological Institute Oslo Norway Felzer B S Cronin T Reilly J M Melillo J M Wang X 2007 Impacts of ozone on trees and crops Comptes Rendus Geoscience 11 12 Fuhrer J Sk rby L Ashmorer M R 1997 Critical levels of ozone effects on vege tation in Europe Environmental Pollution 97 91 106 Gerosa G Marzuoli R Desotgiu R Bussotti F Ballarin Denti A 2009 Validation of stomatal flux approach for the assessment of ozone visible injury in young forest trees Results from the TOP transboundary ozone pollution experiment at Curno Italy Environmental Pollution 157 1497 1505 Grulke N E Neufeld H S Davison A W Roberts M Chappelka A H 2007 Stomatal behavior of ozone sensitive and
226. llement de d finir des seuils critiques ne pas d passer De mai juillet c est l indicateur d exposition des cultures et d avril septembre c est l indicateur d exposition des for ts Le SUMOO et le SUM60 Sum of hourly ozone concentrations equaling or exceeding 0 and 60 ppb sont aussi employ s et correspondent l exposition cumul e sans ou au del d une concentration limite de 60 ppb 1 AOT40 max O3 40 ppb 0 At 1 1 L AFsY Accumulated Stomatal Flux above a threshold of Y nmol m s ou le PODy 2 s J 2 accept en 2005 lors Phytotoxic Ozone Dose above a threshold flux of Y nmol m de la convention de Gen ve sur la pollution atmosph rique transfrontali re sont plus repr sentatifs de l impact de l ozone sur la v g tation car tant bas s sur le mod le de conductance stomatique multiplicatif de Jarvis Jarvis 1976 ils prennent en compte les conditions climatiques influence de la temp rature du d ficit de pression de vapeur d eau VPD de la lumi re du potentiel hydrique du sol de la concentration en ozone et la ph nologie des plantes Ils simulent l entr e d ozone dans les feuilles et repr sentent le flux moyen horaire accumul au dessus d un seuil Y par surface de feuille pendant la p riode de croissance de la plante 18 Exposition ext rieur Ozone Concentration AOT40 Entr e d ozone Flux CUO Effets biologiques sur les cellules d pendant de l activit
227. ls Torsethaugen et al 1999 Zhang et al 2001 as confirmed here at the gene expression level with the ozone inhibition of the expression of inward K channels genes However as we observed a lower opening with ozone we should notice lower potassium content in guard cell with lower counter ions concentrations and stronger calcium content In our study a decrease in chlorine is observed except in the sensitive genotype Robusta and an increase in potassium earlier in Robusta than in Carpaccio and Cima Chlorine is not the only potassium counter ions the anion influx may take the form of other anions like malate X ray microanalyses technique only gives the global element content of cells with no distinction between subcellular compartments The increase in potassium content in response to ozone compared to ambient treatment may be caused by potassium retention in the vacuole or in the cytosol due to a deregulation of outward potassium channels on tonopast or plasma membrane Indeed there is a time effect leading to a progressive increase of potassium content in guard cell earlier in the sensitive genotype This progressive increase should be linked to a wider opening it therefore needs to be counterbalanced by other factors to cause lower opening such as a lower sugar concentration in stomata In response to ozone the increased calcium content is correlated with the reduced stomatal conductance on the upper side in the three genotypes and
228. lumi re bleue et la lumi re rouge Lawson 2009 Blue light Increase of turgor pressure HO e Q Decrease in water potential 2 Malate2 Inwardly rectifying K channel ADP ATP y Stomatal opening lt 100mv e Plasma membrane hyperpolarization H ATPase 7 Figure 17 Processus d ouverture stomatique r ponse la lumi re bleue Shimazaki 2007 Synth se bibliographique d autres avancent l hypoth se que la r ponse au VPD soit li e aux concentrations en ABA Bunce 1998 Xie et al 2006 Pour la temp rature on observe un optimum et la conductance baisse lorsque l on s loigne de celui ci Jarvis 1976 Le contr le de la r ponse stomatique aux variations de temp rature semble li la respiration Lu et al 2000 EFFET DE LA LUMIERE La lumi re induit une ouverture des stomates Cependant cette ouverture d pend de l intensit et du type de longueur d onde de celle ci Figure 16 et implique plusieurs photor cepteurs tels que les cryptochromes les phytochromes et les phototropines De plus la r ponse la lumi re r sulte d au moins deux composantes une composante ind pendante et une autre d pendante de la photosynth se Wang et al 2011 La r ponse la lumi re rouge est d pendante de la photosynth se intervenant dans le m sophylle et les cellules de garde Lawson et al 2002 Mott 2009 Zeiger et al 2002 Le pigment impliqu dans l
229. ly higher p 0 001 in Carpaccio than in the other two genotypes especially in Robusta which was the smallest Fig 2 During the experiments we observed a similar increase in height and diameter for the three A gsl a E 2 Ags E of lt gt 250 t Time s genotypes in the absence of O3 treatment O3 treatment had no significant effect on those parameters in Cima and Robusta but in Carpaccio height significantly increased by 9 and diameter decreased by 10 The significant effects of O3 on Carpaccio height Fig 3A and diameter Fig 3B as related to POD were modelled by linear regression which explained a high percentage of the variance 94 and 97 respectively but a polynomial regression of order 2 was found to fit better for height 98 of the variance explained Carpaccio had also significantly more leaves than the other two genotypes Table 2 exposure significantly decreased the number ofleaves produced during the experimentation by all genotypes with 13 10 and 14 loss for Carpaccio Cima and Robusta respectively In parallel O3 increased leaf fall significantly in Carpaccio 10 times more and Robusta 8 times more and non significantly in Cima 4 times more Regarding biomass and in accordance with previous results Carpaccio had a significantly higher stem biomass than the other genotypes 1 7 times more than Cima and 2 6 times more than Robusta and a significantly higher lea
230. ly written flux net CO assimilation and transpi ration are tightly linked to stomatal conductance This requires a balance between entrance water loss and needs Trees have to adjust their stomatal conductance to each change in environmental parameters to take advantage of the best conditions Therefore stomatal opening and closing speed is a parameter of importance We showed that had a first effect on stomatal conductance so that decreased stomatal conductance reduced net CO assimilation That shift led to an increase in water use efficiency during the first week which vanished once stomatal conductance had stabilized In the early morning the increase in blue light opens the stomata and the increase in red light keeps them open Shimazaki et al 2007 slowed stomatal opening in response to an increase in blue light but reduced stomatal aperture Ag in response to an increase in red light or blue light These results suggest that the blue light specific signalling cascade was slowed down at some point The reduced amplitude of the response to variations in red or blue light may result from the alteration of the ion channels implied in stomatal opening Torsethaugen et al 1999 Decreased Ag led to a reduction of the flux but as the reaction to an increase in blue light under treatment was slower the trees may open their stomata more slowly in the morning when concentration is low although
231. m Les atomes d oxyg ne produits r agissent rapidement avec le dioxyg ne pour former une mol cule d ozone 2 L ozone peut alors lui m me tre rapidement photolys 3 entra nant un quilibre entre les r actions 2 et 3 L ozone peut aussi tre d truit par une r action lente en r agissant avec un atome d oxygene 4 1 O hv 0 0 2 O 0 M 0 M o M est un partenaire de collision non affect par la r action 3 O3 hv gt Oz 4 O gt 202 Stratosphere Non Polar gt Q3 Oxygen 4 Volatile Organic Compounds ee Peroxides Oxygen Emissions Other Organic of and Products Rainout VOCs Deposition Aerosol Troposphere Uptake Figure 4 Cycles de l ozone stratosph rique et troposph rique U S EPA 2006 Ozone ppb 80 4 704 1870 1890 1910 1930 1950 1970 1990 304 Pic du Midi 3 000 m A Pfander Mountain 1 064 m 1 1982 84 1940 H Zugspitze 3 000 m Mont Ventoux 1 900 m 1 4 1977 80 1938 Hohenpeiisenberg 1 000m Jungfraujoch 3 500 m 19 1971 76 1933 Arosa 1 860 m Pic du Midi 3 000m 1951 53 1874 1909 o 1840 1860 1880 1900 1920 1940 1960 1980 2000 2020 2040 2060 2080 2100 2120 Figure 5 Evolution des concentrations Figure 6 Concentrations annuelles pass es europ ennes en ozone ppb mesur es entre 1870 actuelles et pr dites en ozone trop
232. m me Tm gale 58 C ou 60 C gr ce la formule G C x 4 A T x 2 e Taille de l amplicon zone transcrite entre les amorces additionn e la taille de chacune des amorces comprise entre 100 et 150 pb La s quence de l amorce doit tre au moins diff rente de 3 b par rapport toute autre s quence dans le g nome Les amorces ne doivent pas avoir plus de 4 nucl otides identiques cons cutifs Dans les 5 derniers nucl otides de l extr mit 3 il ne doit pas y avoir plus de 2 C ou G e Les couples d amorces doivent tre v rifi s pour viter la formation de dim re ou d pingle cheveux gr ce aux logiciels en ligne primer3 Untergasser et al 2012 et clustalw2 Larkin et al 2007 e Les amorces doivent tre dessin es de pr f rence dans la seconde moiti du g ne Ont t choisis comme cibles des g nes codant pour les enzymes de la voie de biosynth se du glutathion et la principale voie de biosynth se de l ascorbate La sp cificit des couples d amorces a t test e par PCR sur de l ADNc de chacun des 3 g notypes tudi s 35 Mat riel et M thodes ainsi que de la r f rence trichocarpa suivie d un d p t et migration sur gel La sp cificit a ensuite t confirm e par s quen age de l amplicon par l entreprise Beckman coulter genomic PCR EN TEMPS REEL QPCR La PCR en temps r el a t r alis e par le syst me Mx3000P QPCR Agilent technologies
233. mL auquel on ajoute un embout adsorbant dans chaque flacon Aspirer doucement par tape jusqu ce que tout passe dans la seringue Mettre 200 uL de R2 washing solution dans le flacon Aspirer doucement D tacher l embout et le laisser dans le flacon Vider les seringues et les mettre laver Ajouter 200 uL du milieu d lution pr par le jour m me ns rer les embouts sur des seringues de 0 6 mL avec le piston moiti enfonc Aspirer jusqu au filtre et expulser Refaire jusqu ce que la totalit des billes soient expuls es Mettre les seringues laver jeter les embouts vident Mettre 50 uL de R4 avec le microdoseur sans toucher les flacons Vortexer 8 s et attendre 1 min Vortexer 5 s et attendre 1 min Ajouter 100 uL de RS avec le microdoseur sans toucher les flacons Vortexer 5 sec et attendre 1 min Pr lever la phase organique celle du dessus dans un flacon avec une pipette pasteur Mettre les flacons s cher moins de 10 min sous un jet d azote Redissoudre dans 90 uL de phase mobile HPLC Transf rer dans un insert mettre l insert dans le flacon et boucher Mettre du MeOH dans un flacon sans insert et boucher HPLC MS 145 Annexes Pas d utilisation de Corona ou PDA Flux 0 28 mL min C 10 mM de formiate d ammonium dans de l eau D 10 mM de formiate d ammonium dans du MeOH Chaque jour la fin des analyses V rifier que toutes les analyses aient r ussies
234. med to test for treatment effect at each time point for each genotype genotype effect at each time point on ambient values and the genotype x treatment interaction at each time point All the biomass data were fitted with a linear model with treatment and genotype as factors Contrast analyses were performed to test for genotype effect before fumi gation and treatment effect for each genotype Other data were modelled as linear time dependent responses so that the responses before fumigation time 0 i e intercepts were identical within each genotype and slopes depended on genotype and treatment factors and their interaction These models were fitted as linear mixed effect models with individuals as random variables Normality and homo scedasticity of standardized residuals were graphically checked using quantile to quantile and residual vs predicted plots An ANOVA was used to test the differ ences between genotypes on initial values before fumigation and treatment effect and genotype x treatment interaction on slopes In case of an interaction contrast analyses were performed to test for treatment effect on slopes within each genotype Data were analysed using R 2 14 2 R Development Core Team 2012 and the nlme Pinheiro et al 2012 and multcomp Hothorn et al 2008 packages Effects were considered significant when p 0 05 3 Results 3 1 Effects of ozone on growth and biomass Height and stem diameter appeared to be significant
235. min Il est important de supprimer les sels avant de changer de phase mobile IV PROTOCOLE D EXTRACTION DES ARNS AVEC LE KIT RNEASY PLANT MINI Sortir le nombre d chantillons extraire en une demis journ e Pr parer par chantillons Une colonne lilas une colonne rose 2 tubes eppendorf 2 mL 2 tubes collecteurs 1 5 mL V rifier que le tampon RPE est pr t ajout d thanol Pr lever 550 uL de tampon par chantillon et sous la hotte ajouter 10 uL de f mercapto thanol par mL de tampon RLT Mettre au maximum 100 mg de poudre dans un tube eppendorf 2 mL gard dans l azote liquide Disposer tous les chantillons dans un bloc froid tubes ouverts pour les garder congel s en manipulant Ajouter dans chaque tube 550 uL du m lange RLT Vortexer temp rature ambiante 30sec I min apr s avoir mixer manuellement Centrifuger 2 min vitesse max 14500 rpm Transf rer la totalit du surnageant sur une colonne lilas pour chaque chantillon Centrifuger 2 min vitesse max 14500 rpm 250 uL d thanol dans un nouveau tube eppendorf par chantillon vider y directement le lysat et jeter la colonne lilas Homog n iser en pipetant doucement avec une P1000 et transf rer au maximum 700 uL sur une colonne rose par chantillon 147 Annexes Centrifuger 15 s 8000 g Vider le r servoir Ajouter 700 uL de tampon RWI Centrifuger 15 s 80
236. montre que e la conductance stomatique baisse d abord sur la face sup rieure avant d tre l g rement r duite sur la face inf rieure ozone entra ne un accroissement du nombre de mitochondries dans les cellules de garde des deux faces ozone induit une augmentation du contenu en calcium dans les cellules de garde de la face sup rieure des feuilles Chez le g notype r sistant Carpaccio l augmentation a lieu sur les deux faces ozone provoque une diminution du contenu en chlore dans les cellules de garde sauf chez le g notype le plus sensible Robusta nous observons un accroissement 55 R sultats l ozone conduit une hausse du contenu en potassium dans les cellules de garde qui apparait plus t t chez le g notype le plus sensible Robusta l ozone engendre une baisse du contenu en phosphore dans les cellules de garde des deux faces avec un effet plus marqu sur la face inf rieure e l expression des g nes codant pour les canaux de calcium vacuolaires et les anhydrases carboniques impliqu es dans la r ponse des stomates au CO est fortement diminu e Ces r sultats ont t soumis dans le journal Plant Cell amp Environment 56 R sultats Distinct responses to ozone of abaxial and adaxial stomata in three euramerican poplar genotypes Running title Guard cells response for ozone tolerance Dumont Jennifer Cohen David G rard Jo lle Jolivet Yves Dizengremel Pierre
237. n de la respiration de la chlorophylle totale de la chlorophylle a de la chlorophylle b du ratio entre chlorophylle a et b du contenu en rubisco de l activit de la rubisco du contenu en nitrog ne du sucrose et de l amidon des arbres soumis des concentrations lev es en ozone par rapport aux t moins expos s l air filtr par charbon actif Wittig et al 2009 Figure 14 A Stomate et B piderme inf rieur de feuille de peuplier observ s au microscope lectronique balayage Figure 15 Canaux et transporteurs ioniques impliqu s dans les mouvements stomatiques Figure 16 Sch ma de la cascade de r actions impliqu e dans la r ponse la lumi re bleue et la lumi re rouge Lawson 2009 Figure 17 Processus d ouverture stomatique en r ponse la lumi re bleue Shimazaki 2007 Figure 18 Mod le simplifi illustrant les fonctions de g nes r cemment identifi s et de m canismes impliqu s dans le contr le des mouvements stomatiques en r ponse au CO2 Kim et al 2010 Figure 19 Nouveau mod le montrant la s quence d v nements induits par le entrainant l activation des canaux anioniques de type S et la fermeture des stomates Xue et al 2011 Figure 20 Sch ma illustrant le mod le PYR RCAR PP2C SnRK2 de r ponse l ABA Hubbard et al 2010 Figure 21 Mod le d amorcage de la sensibilit au Hubbard et al 2011 Figure 22 Double effet des ROS g n r s par l ozone sur
238. n M White J A amp Quackenbush J 2006 TM4 microarray software suite Methods in Enzymology 411 134 93 Scheibe R Backhausen J E Emmerlich V amp Holtgrefe S 2005 Strategies to maintain 72 R sultats redox homeostasis during photosynthesis under changing conditions Journal of Experimental Botany 56 1481 1489 Schroeder J I Allen G Hugouvieux V Kwak J amp Waner D 2001 Guard cell signal transduction Annual Reviews in Plant Biology 52 627 658 Schwarzl nder M Logan D C Johnston I G Jones N S Meyer A J Fricker M D amp Sweetlove L J 2012 Pulsing of membrane potential in individual mitochondria A stress induced mechanism to regulate respiratory bioenergetics in Arabidopsis The Plant Cell 24 1188 1201 Shimazaki K Doi M Assmann S M amp Kinoshita T 2007 Light regulation of stomatal movement Annual Reviews in Plant Biology 58 219 247 Short E F North K A Roberts M R Hetherington A M Shirras A D amp McAinsh M R 2012 A stress specific calcium signature regulating an ozone responsive gene expression network in Arabidopsis The Plant Journal 71 948 961 Sitch S Cox P M Collins W J amp Huntingford C 2007 Indirect radiative forcing of climate change through ozone effects on the land carbon sink Nature 448 791 794 Stael S Wurzinger B Mair A Mehlmer N Vothknecht U C amp Teige M 2012 Plant organellar calcium signalling an emerging field Journal of Ex
239. n effet non n gligeable du fait des variations du m tabolisme au fil des saisons pour r pondre des besoins saisonniers Deux stress en interaction peuvent en fonction des sc narios avoir des effets antagonistes additifs ou synerg tiques Si l on prend l exemple de l interaction ozone s cheresse au printemps nous observons souvent une hausse des concentrations en ozone du fait de l augmentation de l clairement des temp ratures et des missions biologiques des Compos s Organiques Volatils COV qui peut impacter le comportement stomatique et induire une faiblesse pour lutter contre les s cheresses de l t Les effets d une s cheresse apr s un stress ozone seraient donc amplifi s par celui ci A l inverse une s cheresse pr coce provoquerait une fermeture des stomates qui pourrait limiter les flux entrant d ozone et avoir ainsi un effet protecteur N anmoins ces deux stress induisent une perte de biomasse et auront donc au final quel 131 Conclusion et perspectives que soit le sc nario un effet d l t re Matyssek et al 2006 Comprendre les m canismes de chaque stress ind pendamment est primordial pour pouvoir ensuite interpr ter correctement les tudes de stress combin s ceci pour tre capable de pr dire la r ponse de la v g tation au changement climatique pour pouvoir prendre des mesures politiques dans le but de prot ger les cosyst mes et pour adapter les pratiques foresti re et agricole afin
240. n glutathione biosynthesis Ozone treatment had a strong effect on gene expression related to glutathione biosynthesis Figure 5 see also Supporting Information Figure S2 for complete dataset Major effects of ozone appeared mainly from the third sampling date 11 days and often slightly before in Cima Expression of the putative genes encoding enzymes involved in the direct glutathione biosynthesis pathway from Cys Glu and Gly were increased in response to ozone in the three genotypes Expression of most of the putative genes encoding enzymes involved in the synthesis of these three amino acids from other amino acids were also increased in response to ozone in the three genotypes except for the expression of GOGATI2 GOGAT16 GS10 688 GlyA8 GTI0 GTS and THA19 that decreased in response to ozone in the three genotypes 87 AJaanoadsa1 sAep pue TI H T 0 SUIPUOdsa1109 pue p T 4 sap Surdues ye on q eisnqoy pue 18 ew 19pioq asueio sodfjouss 3914 uo SISSUJU SOIq SUOIUJEMNIS ur sowAZUd JO sou98 oAnvjnd JO UOISSAIdXS uo ouozo JO Sunuosoudo1 sdew oH Sisauju sorq 8 sour amp zuo Surpoouo 59098 JO 1 94 uo SIIIJJI 020 IMZ Ol lt 07 L O gt Le uoisseJdxe pjoj x GvVETILGYVEZTILSHEZL eurejsAo AuuejnjB ELHSOL
241. nalyse X des l ments min raux Microscopie Electronique Balayage 999 39 4 Analyse ultrastructurale des cellules de garde Microscopie Electronique Transmission 39 Principe detTapparellz usi ane ee Ee AS Se See ie ide idee I 39 Utilisation oo de e eade t edes ee deed E 39 RESULTATS 40 I REGUEATION STOMATIQUE toe eet 41 1 Effets de l ozone sur la r ponse stomatique aux param tres environnementaux lumi re bleue lumi re rouge CO et d ficit de pression de vapeur d eau dans trois g notypes de Populus deltoides x 41 2 R ponses distinctes des stomates sur la face abaxiale et adaxiale en r ponse chez trois genotypes de peuplier euram ricain eese ener eene enne 55 H DETOXICATION FOLIA RE o Deren hen I E e RESO UH ER e eere te 75 1 Effets de l ozone sur les voies de synth se de l ascorbate et du glutathion en relation avec les contenus en acide amin s chez trois g notypes de peuplier Une combinaison d approches mol culaire et metabolomigue L sra A e 75 2 Effets de l ozone sur les concentrations en compos s ph noliques et en carot noides 105 3 Capacit de r g n ration du NADPH dans les feuilles de deux g notypes de peuplier pr sentant de
242. nalyses in each cellular compartment could help to understand the origin of the ionic disturbance caused by ozone ACKNOWLEDGEMENTS The authors are thankful to Cyndie Cl ment for microdissection of guard cells Aur lie Heinis and Fabien Spicher for gas exchanges measurements Nathalie Ningre for molecular analyses Christophe Rose for elements microanalyses and St phane Martin for technical assistance The research was supported by the French National Research Agency ANR project Vulnoz the 110 EFABA and Jennifer Dumont was supported by a PhD grant from the ANR and the Lorraine Region We thank the nursery of Gu m n Penfao for providing the cuttings 69 R sultats REFERENCES Ainsworth E A Yendrek C R Sitch S Collins W J amp Emberson L D 2012 The effects of tropospheric ozone on net primary productivity and implications for climate change Annual Review of Plant Biology 63 637 661 Apel K amp Hirt H 2004 Reactive oxygen species metabolism oxidative stress and signal transduction Annual Review of Plant Biology 55 373 399 Avnery S Mauzerall D L Liu J amp Horowitz L W 2011a Global crop yield reductions due to surface ozone exposure 1 Year 2000 crop production losses and economic damage Atmospheric Environment 45 2284 2296 Avnery S Mauzerall D L Liu J amp Horowitz L W 2011b Global crop yield reductions due to surface ozone exposure 2 Year 2030 potential crop produc
243. nd analysis of the effects of air pollution on forests Part VIII Assessment of Ozone Injury 2010 IPCC In Solomon S Qin D Manning M Chen Z Marquis M Averyt KB Tignor M Miller HL eds Climate change 2007 the physical science basis Contribution of working Group I to the fourth assessment report of the Intergovernmental Panel on Climate Change Cambridge University Press Cambridge 2007 996 pp Iriti M Faoro F Chemical diversity and defence metabolism How plants cope with pathogens and ozone pollution International Journal of Molecular Sciences 2009 10 3371 3399 Ishida H Makino A Mae T Fragmentation of the large subunit of ribulose 1 5 bisphosphate carboxylase by reactive oxygen species occurs near Gly 329 Journal of Biological Chemistry 1999 274 5222 5226 Iyer NJ Tang Y Mahalingam R Physiological biochemical and molecular responses to a combination of drought and ozone in Medicago truncatula Plant Cell amp Environment 2013 36 706 720 Jarvis PG The interpretation of the variations in leaf water potential and stomatal conductance found in canopies in the field Philosophical transactions of the Royal Society of London B Biological Sciences 1976 273 593 610 Journal officiel des Communaut s europ ennes Directive 2002 3 CE du parlement europ en et du conseil relative l ozone dans l air ambiant 2002 L67 14 30 Kampfenkel K Van Montagu M Inz D Extraction and determinat
244. ndria in guard cells of Os treated plants could be related to the impairment of CO assimilation in response to ozone and subsequently the need to oxidize the excess of reducing power initially formed in the chloroplasts by photochemical reactions Scheibe et al 2005 Generally these conditions were suitable for an higher activity of the alternative oxidase a mitochondrial enzyme known as an antioxidant enzyme susceptible to dampen ROS generation and for which genes were more expressed under ozone conditions Ederli et al 2006 Vanlerberghe et al 2009 The increase of calcium content observed in response to ozone may be due to an alteration of the calcium efflux or an alteration of a channel among the many involved in calcium released from cellular compartments The initial activation of anion channel involved in stomatal closure is carried by an increase in cytosolic concentration Schroeder al 2001 In response to ozone if the cytosolic calcium content is maintained higher then it can affect anion efflux and inhibits stomatal opening The stomatal closure in response to an increased CO concentration is also slowed under ozone treatment Dumont et al 2013 The signaling leading to stomatal closure is mediated by B carbonic anhydrases BCA1 and BCA4 Hu et al 2010 Xue et al 2011 We showed that expression of the genes coding for 1 and BCA4 is strongly inhibited by ozone and are involved in signal
245. ne dinucleotide NADP nicotinamide adenine dinucleotide phosphate PEPC phosphoenolpyruvate carboxylase POD phytotoxic ozone dose ROS reactive oxygen species Rubisco ribulose 1 5 bisphosphate carboxylase oxygenase 36 Physiol Plant 148 2013 Laurence 1999 Bortier et al 2000 Di Baccio et al 2008 Renaut et al 2009 Street et al 2011 To predict the detrimental effect of on plants several indicators of critical levels have been proposed in the last 25 years First SUMOG sum of all hourly average concentrations over 0 06 ppm or sum of hourly concen trations above a threshold of 40 ppb O during daylight hours of the growing season were universally used Gothenburg Protocol 1999 However these indicators were based on atmospheric concentration and they were left aside in favor of flux based indices such as PODYy Phytotoxic Ozone Dose above a threshold of Y nmol m7 s This new index takes into account the real flux entering the leaf through stomata Gerosa et al 2009 and it is based on an empirical modeling of stomatal conductance UNECE 2004 In this concept the threshold takes into account the intrinsic detoxifying ability of the species in question which still remains poorly known Baumgarten et al 2009 As recently described two main Os detoxifying barriers should be considered and are highly variable depending on species genotype Dizengremel et al 2008 The first line
246. ng and non photosynthetic tissues Emes and Neuhaus 1997 Wakao et al 2008 An increase in its activity has been measured in plants submitted to various biotic and abiotic stresses Batz et al 1998 Nemoto and Sasakuma 2000 Valderrama et al 2006 particularly in response to O fumigation Dizengremel 2001 Fontaine et al 1999 Gaucher et al 2003 The regulation of non phosphorylating NADP GAPDH activity is not so well known but its implication in oxidative stress responses has been established Bustos et al 2008 Finally when studied individually most of these NADP dependent enzymes were found implied in cells capacity to increase the supply of cytosolic NADPH in response to oxidative stress However an integrated study to estimate the relative importance of each enzyme has not been carried out yet especially in response to high daily Os doses The main objective of our work was to study the reduc ing power NADPH regeneration capacity of poplar leaves under ozone treatment On the basis of the visible damage two poplar genotypes were selected Robusta defined as O3 sensitive and Carpaccio defined as O3 tolerant When we exposed the two genotypes to a high dose of 120 ppb O for 17 days they displayed similar Os influxes into leaves based on POD values So the differences in sensitivity could be attributed to a differential plasticity in leaf biochemical response In this context we compared the effects of tr
247. nificant differences between treatments T are indicated by p lt 0 05 lt 0 01 or lt 0 001 In contrast from the second week the opening speed was cut by half and the stabilization time was twice as long under treatment in Carpaccio and Cima Although Ci values increased along with increased CO the Ci Ca ratio remained stable data not shown In control air conditions Robusta displayed a significantly higher photosynthesis rate under low and high concentrations than Carpaccio and Cima around 1 3 times more Table 5 Under treatment with low or high concentrations all three genotypes had a lower photosynthesis rate than under ambient conditions The effect was more and more pronounced as fumiga tion time increased and appeared from the first week for Robusta and from the second week for Cima and Carpaccio with around 55 less photosynthesis the third week 3 6 Effects of ozone on the responses to variations in VPD In control air conditions Robusta was the slowest to close its stomata in response to increased VPD and Carpaccio was the fastest with almost a 10 fold difference between these extremes and a 6 fold difference for stabilization time Table 6 O3 had no clear effect on closing time or speed in Cima and Robusta but decreased Carpaccio closing speed significantly by almost one half and increased its stabilization time by almost 40 The effects of O4 on stomatal conductance
248. nmbiqnAT od reruns 7 ic6r06lq3 03 1eqrums c cp 1138 oj puns 160 193 SLSA 01 ews spuowo sap 150200 45 eseAp op amp uoprejooe euruoorg ome T v L ore D Tv euruo21uq O T 01 1 77 166 06 98 g L p L q8 Terus L 1602 HIS 5154 Jepus lt spuowo ay TSOLOO dS 95841 7 1 0118 7 OA esepopre tpojejo1 osv ope ouruoodt o e 1e rurs IT 08L867700 IWNXII W IT 68990 700 T 86TSTETOO 10420616200 107000 00 1218 CTOLAAd 009SSED 100 104 6 OTA POSMU IXAISI v IANS Id 00L8619500 0d lt 610 OT O 8d pysoussy 1xq1so 00 810D610 104 991 OT Tastmousn jxgiso 00 9 09 10 104 DVVOVOOLLIDVVOLIODIVOV IOLIV ILLOLLVODIDOIODlIOO DLLOLOIODVOODOLIDVIOL IOVVDDVDDDDV LLODDVD L VOVDLLOVOLLOVOIVOOVO VV LLLODIOOLOLOVODOV L LLOVODLLLLODOLVDOVDV VVVDIOVOLLOLLODIODVO 09S9EST000 ULdOd 0907755000 ULdOd O S OS6I00 ULdOd OSEVOSETOO ULdOd ITOLA Tan 6IVHL 7 O3ISOU JWI ESPAWT SC I DA e esejeudsoudouour 10 1 ouSoUT gZ6rSdldS 01 Terus aned ase 01 lt teseyeudsoudouo
249. nse towards a mechanistic risk assessment for forest trees New Phytol 174 7 9 27 Kirma M Araujo WL Fernie AR Galili G 2012 The multifaceted role of aspartate family amino acids in plant metabolism J Exp Bot 63 4995 5001 98 R sultats 28 Maeda H Dudareva N 2012 The Shikimate pathway and aromatic amino acid biosynthesis in plants Annu Rev Plant Biol 63 73 105 29 Noctor G Strohm M Jouanin L Kunert KJ Foyer CH et al 1996 Synthesis of glutathione in leaves of transgenic poplar overexpressing gamma glutamylcysteine synthetase Plant Physiol 112 1071 1078 30 Dghim AA Dumont J Hasenfratz Sauder MP Dizengremel P Le Thiec D et al 2013 Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity Physiol Plant 148 38 50 31 Kampfenkel K Van Montagu M Inz D 1995 Extraction and determination of ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165 167 32 Griffith OW 1980 Determination of glutathione and glutathione disulfide using glutathione reductase and 2 vinylpyridine Anal Biochem 106 207 212 33 R Development Core Team 2012 R A language and environment for statistical computing R Foundation for Statistical Computing Vienna Austria ISBN 3 900051 07 0 34 Pinheiro J Bates D DebRoy S Deepayan S R Development Core Team 2012 Linear and Nonlinear Mixed effects Models R package version 3 1
250. nster CL Clarke SG L Ascorbate biosynthesis in higher plants the role of VTC2 Trends in Plant Science 2008 13 567 573 Lombardozzi D Sparks JP Bonan G Levis S Ozone exposure causes a decoupling of conductance and photosynthesis implications for the Ball Berry stomatal conductance model Oecologia 2012 169 651 659 Lu Z Quifiones MA Zeiger E Temperature dependence of guard cell respiration and stomatal conductance co segregate in an population of Pima cotton Australian Journal of Plant Physiology 2000 27 5 457 462 Mao J Zhang YC Sang Y Li OH Yang HQ From The Cover A role for Arabidopsis cryptochromes and COPI in the regulation of stomatal opening Proceedings of the National Academy of Sciences of the United States of America 2005 102 12270 12275 Marenco A The troposheric ozone evolution during the XXth century Lettre du Programme International G osph re Biosph re Programme Mondial de Recherches sur le Climat PIGB PMRC 1994 n 1 10 Marten H Hyun T Gomi K Seo S Hedrich Roelfsema Silencing of NtMPK4 impairs CO induced stomatal closure activation of anion channels and cytosolic Ca signals in Nicotiana tabacum guard cells The Plant Journal 2008 55 698 708 Matyssek R Le Thiec D Low M Dizengremel P Nunn AJ Haberle KH Interactions between drought and O stress in forest trees Plant Biology Stuttg 2006 8 11 17 Mittler R Vanderauwera S Gollery M Van Breuseg
251. ntal au Mexique Mat riel et M thodes I MATERIEL VEGETAL GENERALITES SUR LE PEUPLIER L tude a t r alis e sur le peuplier arbre mod le depuis 2002 gr ce entre autre sa croissance rapide et son g nome de taille modeste d j s quenc Brunner et al 2004 Renaut et al 2009 Il fait partie de la famille des salicac es angiospermes dicotyl dones et est originaire de la zone temp r e de l h misph re nord Le genre Populus comprend une trentaine d esp ces ainsi que de nombreux hydrides naturels ou cr s par l homme class es en 6 sections botaniques Abaso Aigeiros Leucoides Populus Tacamahaca et Turanga sur la base de crit res cologiques et morphologiques Eckenwalder J E 1996 Dickmann et Kuzovkina 2008 Tableau 4 En France trois esp ces pures sont naturellement pr sentes le peuplier noir P nigra le peuplier tremble P tremula L et le peuplier blanc P alba Le peuplier tient une place importante pour la foresterie frangaise avec environ 200 000 ha de peupleraie Tableau 5 et une production d environ 1 400 000 m de grumes de peuplier Tableau 6 L importance de la populiculture s explique par la croissance rapide de l essence r colte en 15 20 ans et par la propri t polyvalente du bois permettant son emploi dans des d bouch s vari s Parmi ces utilisations on compte notamment les fili res suivantes boites d allumettes emballages
252. nthesis in response to ozone particularly in Carpaccio In addition ozone caused a remobilization of amino acids with a decrease of total amino acids pool an increase of cystein and putrescine especially in Carpaccio In addition the expression of gene encoding threonine aldolase was strongly induced only in the most resistant genotype T R sultats Carpaccio ASA level could partly explain the sensitivity to ozone for Robusta but not for Cima ASA level is not sufficient to account for ozone tolerance in poplar but that a sum of different factors is necessary including glutathione content 78 R sultats Introduction Tropospheric ozone is a powerful oxidizing agent which acts as phytotoxin 1 Its concentrations are still increasing and thus represent a significant threat to earth life with a decrease of vegetation growth reduced productivity and economic losses 2 3 In France the usual ozone concentrations are lower than 60 nmol mol but concentrations greater than 120 nmol mol are frequently recorded on several days each year 4 5 The current concentrations lead to economic losses due to a decline in forestry and agriculture productivity 6 7 8 Indeed in addition to a decrease of stomatal conductance and net CO assimilation by a direct effect of is observed on primary metabolism 9 10 This can reduce energy resources leading to slower growth and loss of biomas
253. nthesis pathways in relation with amino acid contents on three poplar genotypes Running title Detoxification and amino acids in ozone tolerance Jennifer Dumont Sarita Keski Saari Markku Kein nen David Cohen Nathalie Ningre Sari Kontunen Soppela Pierre Baldet Yves Gibon Pierre Dizengremel Marie No lle Vaultier Yves Jolivet Elina Oksanen amp Didier Le Thiec Abstract Ozone is an air pollutant causing oxidative stress by generation of reactive oxygen species ROS within leaf The capacity to detoxify ROS and repair ROS induced damages may contribute to define ozone tolerance Ascorbate and glutathione are known to be key players of detoxification Ozone effects on their biosynthesis and on amino acid metabolism were investigated in three euramerican poplar genotypes Populus deltoides x Populus nigra differing in ozone sensitivity Total ascorbate and glutathione contents were increased in response to ozone in all genotypes Reduced ascorbate ASA increased in the two least sensitive genotypes Carpaccio and Cima whereas the most sensitive genotype Robusta seemed unable to regenerate ASA from oxidized ascorbate DHA Increased ascorbate ASA DHA content correlated with the increase in gene expression on its biosynthetic pathway especially VTC2 Increased cysteine availability combined to increased expression of glutathione synthetase GSH1 and gamma glutamylcysteine synthetase GSH2 genes allow higher glutathione biosy
254. ntral roles of soluble redox couples Plant Cell Environ 29 409 425 Noctor G Queval G Gaklere B 2006 NAD P synthesis and pyridine nucleotide cycling in plants and their potential importance in stress conditions J Exp Bot 57 1603 1620 Noctor G Hager J Shengchun L 2011 Biosynthesis of NAD and its manipulation in plants Adv Bot Res 58 153 201 Pell EJ Eckardt N Enyedi AJ 1992 Timing of ozone stress and resulting status of Ribulose bisphosphate carboxylase oxygenase and associated net photosynthesis New Phytol 120 397 405 Pell EJ Sinn JP Brendley BW Samuelson L Vinten Johansen C Tien M Skillman J 1999 Differential response of four tree species to ozone induced acceleration of foliar senescence Plant Cell Environ 22 779 790 Pelloux J Jolivet Y Fontaine V Banvoy J Dizengremel P 2001 Changes in Rubisco and Rubisco activase gene expression and polypeptide content in Pinus halepensis M subjected to ozone and drought Plant Cell Environ 24 123 131 Pefiarrubia L Moreno J 1990 Increased susceptibility of ribulose 1 5 bisphosphate carboxylase oxygenase to proteolytic degradation caused by oxidative treatments Arch Biochem Biophys 281 319 323 Pitel JA Cheliak WM 1986 Effectiveness of protective agents for increasing activity of five enzymes from vegetative tissues of white spruce Can J Bot 64 39 44 Pl chl M Lyons T Ollerenshaw J Barnes JD 2000 Simulating ozone detoxification in the leaf apoplast th
255. ntrolled stomatal movements Nature Cell Biology 12 87 93 ICP Vegetation 2012 Harmens H Mills G eds Ozone pollution Impacts on carbon sequestration in Europe Bangor UK NERC Centre for Ecology amp Hydrology 88pp Jung J Y Kim Y W Kwak J M Hwang J U Young J Schroeder J I Hwang I amp Lee Y 2002 Phosphatidylinositol 3 and 4 phosphate are required for normal stomatal movements Plant Cell 14 2399 2412 Kivim enp M Sutinen S Medin E L Karlsson amp Selld n G 2001 Diurnal changes in microscopic structures of mesophyll cells of Norway spruce Picea abies L Karst and the effects of ozone and drought Annals of Botany 88 119 130 Kivim enp M Sutinen S Medin E L Karlsson P E amp Selld n G 2003 Cell structural changes in the needles of norway spruce exposed to long term ozone and drought Annals of Botany 92 779 793 71 R sultats Lawson T 2009 Guard cell photosynthesis and stomatal function New Phytologist 181 13 34 Lee Y Kim Y W Jeon B W Park K Y Suh S J Seo J Kwak J M Martinoia E Hwang I amp Lee Y 2007 Phosphatidylinositol 4 5 bisphosphate is important for stomatal opening The Plant Journal 52 803 816 Le Thiec D Rose C Garrec J P Laffray D Louguet P Galaup S amp Loosveldt P 1994 Alteration of element subjected to ozone fumigation and or water stress X ray microanalysis study Canadian Journal of Botany 72 86 92 M
256. o reduction in internal concentrations Ci even if a direct effect of red light on guard cell chloroplasts is not excluded Shimazaki et al 2007 A light signal is transmitted from PHOT1 and PHOT to the plasma membrane H which is activated Protons efflux causes a hyperpolarisation of the membrane which drives the accumulation of by inward potassium channels a decrease in water potential and water uptake Schroeder et al 2001 Sugars produced by photosynthesis or resulting from degradation of starch can 66 R sultats also accumulate in guard cells to maintain stomatal opening Lawson 2009 In response to ozone we observed a decreased expression of PHOT1 and PHOT2 genes which can be linked to the slower opening to blue light Dumont et al 2013 In addition ROS have been shown to inhibit the blue light dependent H pumping and thus stomatal opening Shimazaki ef al 2004 The observed phosphorus content decreased in guard cell by ozone treatment could participate to the inhibition of H ATPase Phosphatidylinositol 4 5 bisphosphate PtdIns 4 5 P2 is an important signal molecule involved in various processes such as guard cell movements Jung et al 2002 Lee et al 2007 and especially in light induced stomatal opening We can hypothesize that if less phosphorus is present in the guard cell it will impact the concentration of PtdIns 4 5 P2 It has also been reported that ozone and ROS inhibit inward K channe
257. of defense is apoplastic ascorbate Smirnoff 1996 Pl chl et al 2000 When that barrier is overridden by Os or its ROS derivatives a second cytosolic line of defense is available within the cell Ascorbate glutathione flavonoids and phenolic compounds are the main actors of the second detoxifying barrier together with a set of ROS scavenging enzymes Castagna and Ranieri 2009 Symplastic ascorbate is regenerated by several cytosolic detoxifying reactions that are mainly connected to the Halliwell Asada Foyer cycle Foyer and Noctor 2011 which highly depends on the presence of cytosolic NADPH Fig 1 Thus its regeneration in the cytosol could be considered as a key factor for cells to face O3 generated oxidative stress Noctor 2006 Dizengremel et al 2009 The capacity for cytosolic NADPH regeneration tightly depends on enzymatic reactions linked to carbon metabolism In fact O3 exposure goes along with a reduction of the Calvin cycle turn over starting with a decrease in Rubisco activity Pell et al 1992 Dizengremel 2001 Bagard et al 2008 Dizengremel et al 2009 A simultaneous increase in PEPC activity has been observed in several C3 plants Landolt et al 1997 Dizengremel 2001 Dizengremel et al 2009 PEPC stimulation is linked to the anaplerotic replenish ment of citrate cycle intermediates but also to higher activity of two NADPH dependent cytosolic enzymes NADP malic enzyme NADP ME and NADP isocitrate dehydro
258. of ozone at 120 nmol during 13h was produced from pure with two ozone generators OZ500 Fischer Bonn Germany and CMG3 3 Innovatec II Rheinbach Germany and injected directly in the chambers 1 h after the beginning of the photoperiod until its end A set of analysers O341M Environment S A Paris France was used to monitor concentrations The flux was calculated and data are shown in Dumont ef al 2013 Gas exchange measurements h 1 1 The first fully expanded leaf was identified for gas exchange measurements 15 and 11 leaf from the top for Carpaccio Cima and Robusta respectively The stomatal 59 R sultats conductance gs of the abaxial lower and adaxial upper sides were measured with a leaf porometer SC 1 porometer Decagon Devices Inc Pullman Washington The measurements were carried out 3 times a week two hours after switching on the lighting in the morning Sampling for microscopy analysis At day 0 the first fully expanded leaf at the beginning of the experiment 15 11 and 11 leaf from the top for Carpaccio Cima and Robusta respectively of four trees of each genotype was sampled At day 11 the second fully expanded leaf at the beginning of the experiment of four and two other trees of each genotype submitted to ozone and control treatment respectively At day 18 the first fully expanded leaf at the beginning of the experiment of four trees of each genotype o
259. olyte concentrations in their cytosol Pandey et al 2007 Stomata react to changes in environmental parameters by opening or closing O3 has been shown to have different effects on stomatal movements it modifies opening and or closing speed but also opening and or closing rate in response to variations in light intensity Hoshika et al 2012 Paoletti 2004 Paoletti and Grulke 2010 VPD Grulke et al 2007 soil moisture Hayes et al 2012 CO2 concentration Onandia et al 2011 and ABA concentration Mills et al 2009 The purpose of this study was to characterize the impact of on the stomatal responses to four major environmental parameters ie blue light red light CO concentration and VPD on three Euramerican poplar genotypes The stomatal opening by light consists of two components the red light response driven by photosynthesis and the specific blue light response which is photosynthesis independent Shimazaki et al 2007 Wang et al 2011 We tested these components separately by studying blue light in darkness We focused on the speed and the amplitude of stomatal responses while studying the link with net assimi lation The regulation of stomatal conductance and thereby the regulation of stomatal uptake can be involved in the characteriza tion of sensitivity We hypothesized that the difference in sensitivity could also be linked to different stomatal behaviours in response to variations in envi
260. om the ANR and the Lorraine Region We thank the nursery of Gu m n Penfao for providing the cuttings References Ainsworth E A Yendrek C R Sitch S Collins W J Emberson L D 2012 The effects of tropospheric ozone on net primary productivity and implications for climate change Annual Review of Plant Biology 63 15 1 15 25 ASPA 2010 Bilan de qualit de l air en Alsace P riode 2000 2009 Avnery S Mauzerall D L Liu J Horowitz L W 2011 Global crop yield reductions due to surface ozone exposure 1 Year 2000 crop production losses and economic damage Atmospheric Environment 45 2284 2296 Bagard M Le Thiec D Delacote E Hazenfratz Sauder M P Banvoy J G rard J Dizengremel P Jolivet Y 2008 Ozone induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves Physiologia Plantarum 134 559 574 Betzelberger A M Gillespie McGrath J M Koester Nelson R L Ainsworth E A 2010 Effects of chronic elevated ozone concentration on antioxidant capacity photosynthesis and seed yield of 10 soybean cultivars Plant Cell amp Environment 33 1569 1581 Brosch M Merilo E Mayer Pechter P Puz rjova I Brader G Kangasjarvi J Kollist H 2010 Natural variation in ozone sensitivity among Arabidopsis thaliana accessions and its relation to stomatal conductance Plant Cell amp Environm
261. on 2008 155 453 463 Hopkins WG Physiologie v g tale Universit DB ed 2003 Hu H Boisson Dernier A Israelsson Nordstrom M Bohmer M Xue S Ries A Godoski J Kuhn JM Schroeder JI Carbonic anhydrases are upstream regulators of CO controlled stomatal movements in guard cells Nature Cell Biology 2010 12 87 93 sup pp 81 18 Hubbard KE Nishimura N Hitomi K Getzoff ED Schroeder JI Early abscisic acid signal transduction mechanisms newly discovered components and newly emerging questions Genes amp Development 2010 24 1695 1708 Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI Abscisic acid and signalling via calcium sensitivity priming in guard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus response analyses Annals of Botany 2011 1 13 Imai K Kobori K Effects of the interaction between ozone and carbon dioxide on gas exchange ascorbic acid content and visible leaf symptoms in rice leaves Photosynthetica 2008 46 387 394 International Co operative Programme Modelling amp Mapping ICP M amp M Modelling and Mapping Manual of the LRTAP Convention Chapter III Mapping Critical Levels for Vegetation 2004 114p International Co operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests ICP Forests Manual on methods and criteria for harmonized sampling 161 R f rences assessment monitoring a
262. on de la conductance stomatique et les processus de d toxication L objectif principal de cette th se tait de donner des pistes d am lioration de l estimation des seuils de risque l ozone pour la v g tation en approfondissant nos connaissances sur les effets de l ozone sur la r gulation stomatique et les processus de d toxication De plus l tude de trois g notypes de peuplier euram ricain caract ris s par une sensibilit plus ou moins forte l ozone avait pour but de d finir un ensemble de marqueurs de tol rance Les indicateurs de seuils de risque utilisant les flux d ozone sont bas s sur l utilisation de mod les de pr diction de conductance stomatique car les mesures de conductance stomatique en continu sur le terrain ne sont pas r alisables Deux grands types de mod les de conductance stomatique existent Parmi ceux ci l un des principaux est le mod le de Ball Berry Ball et al 1987 qui se base sur l assimilation nette de CO tandis que d autres tels que le mod le de Jarvis Jarvis 1976 repr sentent la conductance stomatique comme le produit de la conductance maximale relev e dans la litt rature par des fonctions r ductrices d crivant la r ponse de la conductance stomatique aux facteurs environnementaux et ph nologiques Il est admis qu en plus de dommages visuels 127 Conclusion et perspectives l ozone impacte les processus physiologiques entra nant une baisse diff ren
263. on of the United Nations 70 R sultats Felzer B S Cronin T Reilly J M Melillo J M amp Wang X 2007 Impacts of ozone on trees and crops Comptes Rendus Geoscience 339 784 798 Fischer R A 1968 Stomatal opening role of potassium uptake by guard cells Science 160 784 785 Goodstein D M Shu S Howson R et al 2012 Phytozome a comparative platform for green plant genomics Nucleic Acids Research 40 1178 1186 Grulke N E Neufeld H S Davison A W Roberts M amp Chappelka A H 2007 Stomatal behavior of ozone sensitive and insensitive coneflowers Rudbeckia laciniata var digitata in Great Smoky Mountains National Park New Phytologist 173 100 109 Hayes F Jones M L M Mills G amp Ashmore M 2007 Meta analysis of the relative sensitivity of semi natural vegetation species to ozone Environmental Pollution 146 754 762 Heath R L 1994 Possible mechanisms for the inhibition of photosynthesis by ozone Photosynthesis Research 39 439 452 Hetherington A M amp Brownlee C 2004 The generation of signals in plants Annual Review of Plant Biology 55 401 427 Hothorn T Bretz F amp Westfall P 2008 Simultaneous inference in general parametric models Biometrical Journal 50 346 363 Hu H Boisson Dernier A Israelsson Nordstr m M B hmer M Xue S Ries A Godoski J Kuhn J M amp Schroeder J I 2010 Carbonic anhydrases are upstream regulators in guard cells of CO2 co
264. on the lower side only in Carpaccio During stomatal opening the cytosolic concentration decreases because of efflux and storage in organelles especially the vacuole Genes encoding vacuolar antiporters are strongly down regulated which can lead to an impaired storage in the vacuole and higher cytosolic 67 R sultats concentrations In addition these genes are constitutively much less expressed in the sensitive genotype Robusta which is characterized by slower stomatal opening and closure even under ambient conditions Dumont ef al 2013 In response to ozone guard cells exhibit more mitochondria This increase could support the higher respiration in order to provide enough energy to meet the increased needs for detoxification and repair processes as observed previously in Norway spruce Kivim enp et al 2001 2004 They can also act as calcium buffer with a possible resting free concentration of 200 nM in mitochondria Stael et al 2012 and then be a key player in the spatiotemporal patterning of signals by rapidly removing Ca from the cytosol Hetherington and Brownlee 2004 Moreover it has recently been suggested that an influx of Ca in mitochondria can lead to a pulse of mitochondrial membrane potential to regulate respiratory bioenergetics and counteract ROS production in response to stress Schwarzlander et al 2012 At last the higher number of mitocho
265. osph rique et 2000 Marenco 1994 Vingarzan 2004 Synth se bibliographique Dans la troposph re les m me r actions ont lieu mais sont n anmoins limit es par le faible rayonnement UV dans la troposph re Un cycle de l ozone troposph rique d crit la formation naturelle limit e d ozone partir des pr curseurs primaires que sont les oxydes d azote en pr sence de compos s organiques volatils VOC et de lumi re Figure 4 Le cycle photochimique de l ozone dans la troposph re repose sur la photolyse du dioxyde d azote NO2 par le rayonnement des UV A qui produit du monoxyde d azote NO et un atome d oxyg ne 5 L atome d oxyg ne r agit ensuite avec du dioxyg ne pour former de l ozone 6 L ozone peut alors r agir avec NO pour reformer du NO 7 Les VOC peuvent n anmoins former des esp ces r actives radicaux comme HO ou qui vont substituer l ozone dans cette derni re r action 8 Les NO et les VOC produits par l activit humaine peuvent donc d s quilibrer ce cycle et entrainer une hausse des concentrations en ozone dans la troposphere 5 hv gt 6 O 0 M gt 03 M ou M est un partenaire de collision non affect par la r action 7 O2 8 NO t les concentrations ponctuelles deviennent parfois si importantes que l on parle de pics d ozone En effet l ensoleillement et la temp rature tant plus forts
266. osphoric acid 5 w v Homogenates were centrifuged for 10 min at 4 C with 14 000 g Ascorbate and dehydroascorbate were measured by the method of 31 scaled down for microplates To allow the use of a pipetting robot the trichloroacetic acid was replaced by metaphosphoric acid 5 w v Glutathione and glutathione disulfide were measured using glutathione and 2 vinylpyridine as described by 22 82 R sultats Real time quantitative RT PCR Total RNA was extracted from 100mg of fresh leaf powder using the RNeasy Plant Mini Kit Qiagen Courtaboeuf France according to the manufacturer s protocol Residual genomic DNA was removed by Dnase I Life Technologies Saint Aubin France treatment RNA quality was assessed using the automated gel electrophoresis Experion system Bio Rad Marnes la coquette France Quantification was performed by Quant iT riboGreen assay Life Technologies Saint Aubin France Reverse transcription was performed on 700 ng RNA using an iScript cDNA Synthesis Kit Bio Rad Marnes la coquette France The Alien QRT PCR Inhibitor Alert kit Agilent technologies Massy France was used to assess reverse transcription efficiency following the manufacturer s protocol Real time PCR was performed in a Mx3000P QPCR System Agilent technologies Waghaeusel Wiesental Germany according to the manufacturer s recommended cycling program 10 min 95 C 40 cycles of 5 s at 95 C and 20 s at 58 or 60 C depending on prim
267. owerWave HT USA Maximum rates were calculated automatically by the plate reader soft ware and then processed using Microsoft Excel Statistical analysis The data in this article are presented as means At each sampling date we performed a TYPE III two way analysis of variance ANOVA using R R Development 40 Core Team 2011 with genotype and treatment as variable factors The data were rearranged into a linear model and differences between control and O3 samples were considered significant if P lt 0 05 To compare the inter group means the ANOVA test was followed by a significant P lt 0 05 Tukey range test Linear regression was used to test the correlations between different enzymatic activity data and was followed by a two way ANOVA to compare r Using a linear logarithmic model in R no statistically significant chamber effect was found for any of the tested parameters throughout the whole fumigation period Results Visible injury and leaf fall The ozone fluxes cumulated via plant stomata in the absence of a threshold value POD were calculated for Carpaccio and Robusta They were averaged using the values obtained in the four O3 phytotronic chambers Throughout the whole fumigation period we observed a continuous increase in PODg which differed little between the two genotypes Fig 2 Even though ozone flux levels were similar the two genotypes showed differences in the occurrence and the aspec
268. oxication L importance du m tabolisme primaire en soutien aux m canismes de d toxication et de r paration a t soulign e et nous avons montr que les ph nom nes de tol rance ou de sensibilit l ozone ne peuvent tre appr hend s que par une tude approfondie avec un point de vue global sur tous ces facteurs Les conclusions de ces travaux n cessitent d tre confirm es en conditions naturelles toutes les exp riences ayant t conduites en conditions contr l es Il faudrait aussi v rifier leur pertinence au sein d autres esp ces et sur des arbres plus g s Dans notre tude l ozone seul des concentrations correspondant un stress chronique a t tudi pour nous permettre de comprendre finement les m canismes de r ponse ce stress mais il est important de noter que les pr dictions face au changement climatique indiquent entre autre une tendance une augmentation des temp ratures des concentrations en CO et de la fr quence des s cheresses ainsi que de leur intensit Diff rentes tudes se sont int ress es aux interactions entre l ozone et d autres stress comme le fort CO ou la s cheresse Matyssek et al 2006 Imai et Kobori 2008 Kontunen Soppela et al 2010 Lindroth 2010 Iyer et al 2013 Les interactions entre diff rents stress d pendent de l intensit de ces derniers de leur dur e mais aussi de l ajustement temporel des uns par rapport aux autres La chronologie peut avoir u
269. p e OI gn 13 IT SYST MES DE DBTOXICATION tete tete Et PRAEERAT e te HERE RNA ei eee nere 13 1 Syst me de d fense antioxydatif esee eene eene rennen nennen 13 2 Voie d Halliwell sise i te e eme t icant tee itin 14 3 Autres enzymes et mol cules antioxydantes ses 15 4 Voie biosynthese de I ascorbate n tede eiie th Prati nee end 15 3 Voie de biosynth se du glutathion 16 OBJECTIFS DE RECHERCHE s scsccsccccscesscssscosscsoscessssescetccocsedsieionesbosecetosdgseseasissesssbessognedescousoebetestussienerecseses 17 I PROBEEMATIQUE env aee e baa o ee ende eds 18 I _ Neus de risque d lozonexzi oen ERE eei IE RT OE RES 18 2 Flux effectif d ozone mi ste a E 19 II OBIBGTIESu za ete t ERNST N 20 MATERIEL E T METHODES T 21 Le SNMIATERIEL VEGBTAT 5 aped ue deleta deridet 22 1 G n ralit s sur le peuplier sise 22 2 Geno Ypes Ll mete Bed tL LLL ex E A de AG nn Ld are 23 3 Conditions de Culture sh ese e a a 23 4 JIBr levementz men ttem ot beo Lo ag Re aD 23 IL nien enn 24 T Plan d exp rience indo a RUE deme Ss 24 2 Conditions environnementales ss 25 III SUIVI DE CROISSANCE BIOMASSES
270. pe were distributed in 8 phytotronic chambers with growth conditions identical to those in the growth chamber half of which were set for treatment 120 nmol mol for 13 h At the end of a 7 day long acclimation period the treatment started while control trees were exposed to charcoal filtered air for 18 days O3 was produced from pure with two ozone generators 0Z500 Fischer Bonn Germany and CMG3 3 Innovatec II Rheinbach Germany and injected directly in the chambers 1 h after the beginning of the photoperiod until its end A set of analysers O341M Environ ment S A Paris France was used to monitor concentrations 2 2 Plant growth and biomass Before fumigation five plants of each genotype were cut to measure initial biomass At the end of the experiment all the plants were harvested divided into stems leaves and roots and oven dried 60 C until constant weight was reached and then weighed Diameter at collar D and height H were recorded twice a week until the end of the experiment on each individual 2 3 Total chlorophyll content Total chlorophyll content was measured twice a week using a CCM 200 chlo rophyll meter Opti Sciences Hudson NH USA on the first fully expanded mature leaf at the beginning of the experiment 11th for Cima and Robusta and 15th for Carpaccio the age of leaves being the same between genotypes The values ob tained from the CCM Chlorophyll Content Index CCI were
271. perimental Botany 63 1525 1542 Torsethaugen G Pell amp Assmann S M 1999 Ozone inhibits guard cell K channels implicated in stomatal opening Proceedings of the National Academy of Sciences of the United States of America 96 13577 82 Untergasser A Cutcutache L Koressaar T Ye J Faircloth B C Remm M amp Rozen SG 2012 Primer3 new capabilities and interfaces Nucleic Acids Research 40 1 12 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A amp Speleman F 2002 Accurate normalization of real time quantitative RT PCR data by geometric averaging of multiple internal control genes Genome Biology 3 1 11 Vanlerberghe G C Cvetkovska M amp Wang J 2009 Is the maintenance of homeostatic mitochondrial signaling during stress a physiological role for alternative oxidase Physiologia Plantarum 137 392 406 Wittig V E Ainsworth E A Naidu S L Karnosky D F amp Long S P 2009 Quantifying the impact of current and future tropospheric ozone on tree biomass growth physiology and biochemistry a quantitative meta analysis Global Change Biology 15 396 424 73 R sultats Xue S Hu H Ries A Merilo E Kollist H amp Schroeder J I 2011 Central functions of bicarbonate in S type anion channel activation and OST protein kinase in CO signal transduction in guard cell The EMBO Journal 30 1645 1658 Zhang X Miao Y C An G Y Zhou Y Shangguan Z P Gao J F amp
272. piration is inhibited by ozone treatment 56 may explain the large decrease observed in our experiment in the expression of genes coding for enzymes involved in photorespiration such as glyoxylate aminotransferase Gly and Ser are two amino acids that are readily interconvertible As described by 57 there is a serine to glycine conversion by cytosolic Serine hydroxymethyltransferase SHMT connected with C1 metabolism and a glycine to serine 92 R sultats conversion by mitochondrial SHMT The expression of genes of mitochondrial SHMT GlyA3 and GlyA10 decreased in response to ozone whereas the expression of genes of cytosolic SHMT isoform was induced by ozone Ozone stimulates Cl metabolism via enhanced lignification in leaves 58 and thus could increase the production of Gly by cytosolic SHMT which could be used for glutathione production Gly can also be synthesised from Thr but this reaction has been poorly studied It has been suggested that Thr aldolase could contribute to maintaining Gly homeostasis in plants at times of transition in the use of the photorespiratory cycle 59 In response to ozone Thr aldolase could maintain Gly content and thus the observed increase of 7HA13 expression in Carpaccio could be helpful to tolerate ozone The stronger upregulation of GSH1 GSH2 and THA13 genes expression correlated with the increased concentration of reduced glutathione in the ozone tolerant genotype Carpaccio Our results sug
273. ponses to varying blue light red light leaf to air vapour pressure deficit VPD and CO were measured once a week on three plants of each genotype for each treatment on the leaf below the one used for Li 6200 measure ments the 12th for Cima and Robusta and the 16th for Carpaccio the age of leaves being the same between genotypes with three intercalibrated open gas exchange systems with 6400 40 Leaf Chamber Fluorometer Li 6400 LiCor Lincoln NE USA coupled with a custom built water trap Data were recorded automatically every 30 s For each test only one environmental parameter was changed as summarized in Table 1 Leaf temperature was 25 C for all conditions Blue light and VPD responses were studied in complete darkness in order to bypass the effects of photosynthesis and in case of VPD response to allow the control of leaf temperature Red light response was studied in presence of blue light and CO response was studied at saturating light conditions in order to have a complete aperture of stomata We used 400 pmol for red light blue light and CO responses in order to keep the CO concentration used in phytotronic chambers To study VPD responses we used 100 CO to open stomata even in the dark For calculating closing or opening speeds we used as shown in Fig 1 e Stomatal conductance at the initial state 2 0 means of 10 measurements after reaching a steady state before changing environment
274. posed to ambient or ozone treatment at 2 4 11 15 and 17 days Mean SE GIDH16 AMBIENT OZONE 12 10 c 5 5 808 a o Bos A T 504 z 0 0 T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA GME13 AMBIENT OZONE o o o o 1 Normalized expression o 1 wh T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA LGalDH9 AMBIENT L4 o Normalized expression o a 1 OZONE 15 um 0 0 T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA sampling dates day 2 4 11 15 17 sampling dates day 2 4 11 15 17 sampling dates day GME5 AMBIENT nbi T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA OZONE sampling dates day 2 4 11 15 17 o Normalized expression o o o 1 o h LGalDH1 AMBIENT OZONE 5 sampling dates 270 day 2 a 2 2 L 4 o 8 11 c 15 E 505 17 0 0 T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA VTC25 AMBIENT OZONE 1 2 g 1 07 9 sampling dates 2 day 508 2 4 D 8 0 6 11 g 15 3 17 04 dica til m M T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA VTC46 AMBIENT OZONE Normalized expression o o o o 1 gt o 1 0 0 T T T T T CARPACCIO CIMA ROBUSTA CARPACCIO CIMA ROBUSTA VTC4
275. quelle l air est renouvel circuit ouvert Figure 38 Les param tres environnementaux sont contr l s temp rature VPD CO lumi re Les concentrations en H20 et CO le rayonnement quantique la temp rature de bloc de l air et de la feuille le d bit d air et la pression sont mesur s de mani re simultan e l aide de toute une s rie de capteurs majoritairement group s au niveau de la chambre de mesure un IRGA analyseur de gaz infrarouge pour les concentrations d H2O et de un capteur Quantum sensor Li 190 pour la lumi re ext rieure un capteur GaAsP pour la lumi re l int rieur de la chambre une thermistance pour la temp rature de bloc et d air un thermocouple pour la temp rature de feuille un capteur de flux pour le d bit d air dans la console et un capteur de pression dans 29 Uy Co Wo Figure 39 Param tres mesur s utilis s dans le calcul de la transpiration et de la conductance stomatique transpiration nette a assimilation u et flux nets entrant et sortant w et w concentrations en eau entrante et sortante c et c concentration en entrante et sortante Figure 40 Mesures r alis es l aide de l appareil de mesure d changes gazeux Licor 6400 Mat riel et M thodes la console La transpiration et la conductance stomatique sont ensuite calcul es selon 8 et 9 en se basant sur des mesures faites sur un circuit de r f rence air similaire l
276. r des exp riences ant rieures pour leurs r ponses contrast es l ozone n croses chute de feuilles conductance stomatique activit PEPC croissance Carpaccio apparait comme le g notype le plus tol rant suivi de Cima puis de Robusta qui est le g notype le plus sensible Ce choix permet une meilleure compr hension des diff rents m canismes utilis s en r ponse l ozone et d approcher les diff rences intra sp cifiques 3 CONDITIONS DE CULTURE Pour chaque exp rience de fumigation le mat riel v g tal a t cultiv dans les m mes conditions Les boutures de peuplier issues de la p pini re foresti re de Gu m n Penfao ont t plant es dans des pots de 5 L remplis de terreau N P K 14 16 18 1 2 kg m Gramoflor SP1 Universel 6 semaines avant le d but de la fumigation d ozone Figure 28 Au fond des pots 1 2 cm de billes d argile de diam tre 8 16 permet d am liorer le drainage de l eau De plus 10 mg de nutricote T 100 Nutricote T 100 N P K MgO 13 13 13 2 Fertil Boulogne Billancourt France ont t ajout s pour fertiliser le milieu ce qui correspond une dose finale de 20 mg pot Enfin pour tamponner le pH 1 mg l de calcaire magn sium a t utilis Dans la chambre de culture la temp rature et l humidit relative ont t maintenues 23 C 75 le jour et 20 C 85 la nuit avec une photop riode de 14 h 8 h 22 h Les arbres ont t irrigu s manuellement tous les jours Avant
277. r expression 65 a b PC2 14 9 PC4 8 1 6 p Edi T i 5 L 4 E i 0 x 2 Ne N 3 oA A ix 2 O oO o 6 20 lp oA A sA 9 oA oH is oA at kA 40 E 20 sA 2 3 X N d QA 4 N 20 sA P A 6 unc ci 8 7 6 5 4 3 2 0 1 2 3 4 6 7 8 PCI 26 5 5 erp 999 4 N 3 Low be 2 Upp Low N Upp e Upp A Upp LOW a 1 Upp Low ao Low Upp Low Upp Los 0 BUR Upp PP sLow Upp AL Y a indi Pow 2 X N oUpp F sLow Low 3 S aLow Low P 4 B e 5 5 4 3 2 1 0 1 2 4 5 10 1 0 35 c TOY 0 30 0 25 6 aNHX113 SLACI CLCa6 TPK11 0 20 AKT2 Neue MIS ALMT610 0 15 610 KAT36 2 010 E aCAXI aCLCa s NHX114 1 2 0 05 aGLR25 A A aioe AHA1115 4O0ST2 CAI 0 05 ALMT68 aGLUR32 GORK12 0 10 ech 2 18 GLUR3 0 15 0 20 29 0 25 0 30 025 020 015 010 0 05 0 00 0 05 010 0 15 020 26 5 4 aGLUR3 0 35 KAT36 4087218 110 0 25 29 1ALMT6 0 20 114 id aGLR25 JALMT68 2 aTPCI
278. raires Fy 7 cumul s de l atmosph re travers les stomates au del ou 28 LI 6400XT Console With 6400 01 CO Injector System Liquid CO2 man L Figure 38 Sch ma de fonctionnement de l appareil Li 6400 Mat riel et M thodes d un seuil de Y nmol m s Comme recommand par Fagerli et al 2011 nous avons utilis 2 _ 1 un seuil de 1 nmol m s 4 AOT40 Y max 05 40 0 At i 5 PODy max F 0 At i 6 CUO FaeAt i 7 Fst 03 X gs avec n le nombre d heures At Ih et Y 1 nmol m s Comme les conditions environnementales dans les phytotrons taient stables nous avons appliqu pour nos calculs de flux une conductance stomatique constante au cours de la journ e De plus les valeurs de conductance stomatique des jours sans mesure ont t valu es partir des jours adjacents Bagard et al 2008 M me si nous ne prenons pas en compte une ventuelle baisse de conductance au cours de la photop riode nous appliquons le m me principe pour tous les arbres 3 ASSIMILATION NETTE DE CO ET CONDUCTANCE STOMATIQUE A LA VAPEUR D EAU LI 6400 PRINCIPE DE L APPAREIL L appareil Li 6400 est un syst me ouvert portable de mesure de photosynth se permettant de r aliser des mesures d changes gazeux pas de distinction des faces La feuille est plac e dans une chambre dont les conditions lumineuses intensit et qualit sont contr l es et dans la
279. ramicrotome mounted on Formwar copper grids stained with uranyl acetate and lead citrate 4 Examination of sections was performed with a transmission electron microscope TEM PHILIPS CM12 operating at 80kv 60 R sultats Stomatal density and size of stomata As stomata are located on both sides of Populus leaf the three freeze dried discs of each sample were divided in two pieces to study separately the two sides of each sample They were observed under a controlled pressure scanning electron microscope SEM with the use of a backscattered secondary electron detector BSD at accelerating voltage of 15kV and magnification of 250x for stomatal density and 800x for size measurements after being evaporated by carbon Three and one digital images of each side of each disc of each sample at 250x and 800x were captured The total number of stomata within an image was determined by manual counting using the convention of excluding stomata overlapping the margins of the image and was converted to the number of stomata per mm of abaxial and adaxial leaf surface Stomata length and width were also measured on 6 stomata by image at 800x X ray microanalysis The material to be analysed was prepared according to the method used in Le Thiec et al 1994 Leaves were examined under a SEM model 1450VP Leo Cambridge UK at 15 kV equipped with a dispersive energy microanalysis system EDX silicon drift detector of 80mm Oxford Instrumen
280. re chaque analyse V rifier que toutes les analyses aient r ussies V rifier qu il reste suffisamment de place sur le disque dur Vider la bouteille poubelle Nettoyer la surface dans le bouclier ionique avec 50 50 Purger les canaux Nettoyer avec 98 d ac tonitrile et 0 1 de formique acide pendant 15 min 0 4 mL min puis avec du meOH pendant au moins 30 min jusqu ce que la colonne semble propre v rifier sur le PDA Direct control Start data zero data Les lignes doivent tre stables Nettoyer le spectrom tre de masse avec 50 50 MeOH H20 0 2 mL min pendant 10 20 min Laisser le spectrom tre de masse allumer 5 min de plus avec un flux nul Laisser la phase soluble se stabiliser avant de lancer une nouvelle analyse environ 10 min et v rifier de nouveau sur le PDA la stabilisation V rifier que le Corona donne une ligne de base inf rieure 0 5 pA La pression de d part doit toujours rester la m me d pend de la colonne Laisser les appareils en veille apr s les analyses chaque jour 143 Annexes S il n y a pas d analyses pr vues pendant une journ e ne pas oublier de vider la bouteille poubelle apr s le nettoyage teindre le spectrom tre de masse teindre les lampes du PDA Dans le cas o aucune analyse n est pr vue pendant plus d une journ e teindre le Corona Quand toutes les analyses sont finies purger les canaux utilis s C et D avec de l eau Millipore
281. re performed on MeV 35 36 Results Sensitivity to ozone The sensitivity of Cima was mainly characterised by total biomass losses Table 1 whereas Robusta was the most sensitive based on foliar injuries with the largest necrosis Figure 1 and the highest percentage of leaf fall Table 1 Carpaccio appeared to be the most resistant genotype displaying only brown stippling on the leaves located at the bottom of the trees with the lowest percentage of fallen leaves and biomass losses twice smaller than Cima Effects of ozone on ascorbate concentrations In ambient condition the concentration of total ascorbate was significantly higher in Carpaccio than in Cima and Robusta Table 2 Ozone treatment had a strong effect on ascorbate availability The concentration of ASA increased significantly in response to ozone in particular at the last sampling date in Carpaccio with a 2 8 fold increase and in Cima with 85 Table 2 Concentration of total ascorbate and total glutathione in ambient condition in three poplar genotypes Genotypes Constitutive total ascorbate Constitutive total glutathione concentration umol 071 concentration nmol GSH mg Carpaccio 9 95 0 17a 0 47 0 01a Cima 7 68 0 15c 0 36 0 01b Robusta 8 62 0 28b 0 35 0 015 Concentration of total ascorbate and total glutathione in ambient condition in three genotypes Carpaccio Cima and Robusta Mean of all the sampling dates 2 4 11 15 and
282. re plot of and PC2 A PC3 and PC2 C and the corresponding PCA loading plots B and D of the Amino acids and polyamines concentrations in leaves of three poplar genotypes Carpaccio orange circles Cima green squares and Robusta blue triangles submitted to ambient A dotted line or Ozone treatment O solid line at five sampling dates 1 2 3 4 and 5 corresponding to 2 4 11 15 and 17 days respectively x fold concentration change Putrescine PUT E Tryptophan TRP E _ Cysteine CYS Phenylalanine Isoleucine_ ILE Serine SER Asparagine ASN Threonine THR Aspartic acid ASP Glutamic acid GLU Proline PRO _ 11 VAL Glutamine GLN E um um E Glycine GLY i H See Spermidine SPE Methionine MET CE ysine Lys LI IN ornithine ORN Figure 8 Ozone effects on amino acid concentrations in poplar leaf Heat map representing the effects of ozone on the Amino acids and polyamines concentrations in leaves of three poplar genotypes Carpaccio orange border Cima green border and Robusta blue border at five sampling dates 1 2 3 4 and 5 corresponding to 2 4 11 15 and 17 days respectively Compounds are clustered by Pearson correlation R sultats Figure 7D Most of the amino acids and polyamines concentrations decreased with time in response to ozone except for Put Trp Cys Phe Ile His Leu and Tyr Figure
283. rease for Carpaccio and Cima respectively 0 001 ns ns ns 0 001 0 00621 ns 3 5 Effects of ozone on the responses to variations in CO In control air conditions Robusta closed its stomata significantly more slowly than the other two genotypes under an increased CO2 concentration 3 3 and 1 7 times more slowly than Carpaccio and Cima respectively leading to a significant lengthening of the time needed for stomatal conductance to stabilize Table 5 The treat ment had no effect on closing speed or stabilization time in Robusta Genotype effect Carpaccio Cima Cima Robusta Robusta Carpaccio Robusta 90 J Dumont et al Environmental Pollution 173 2013 85 96 Ambient 8 Ozone 1 10 4 A lt 1 00 1 Ts E 0 90 1 3 T 0 80 070 8 e 0 60 1 8 0 50 7 O 040 0 30 1 10 1 00 0 90 0 80 7 0 70 4 0 60 4 0 50 4 0 40 p 0 30 dperpepererpeOrp96rp6rP TUTUP Cima Chlorophylls g m 1 10 1 00 4 080 0701 2 B 0 60 E 0504 5 0404 V 0 30 0 5 10 15 20 Time day Ambient Ozone D 10 7 A umol m s N 10 4 T A umol m s 10 4 A umol m 7 87 Time day Fig 4 Effects of O3 treatment Ozone JAmbient on chlorophyll content g m 2 A B and C and net CO assimilation m s D E and F in the leaves of Carpaccio A Cima B and E and Robusta C and Sig
284. rences in stomatal responses to Robusta was the shortest genotype and displayed the most important decrease in leaf production and the highest percentage of fallen leaves It seemed more sensitive to Os with more visible leaf injuries data not shown It had the highest stomatal conductance and the highest chlorophyll content but it also showed slower stomatal reactions than the other two geno types in the control treatment Its sensitivity may be linked to that stomatal conductance characteristic in accordance with the study of Brosch et al 2010 which concluded about a major role of stomatal conductance in sensitivity Indeed Robusta Two adjustment models were used a linear regression lin solid line with y 6 6588x 13 964 2 0817x 10 446 and y 3 0586x 17438 as equations respectively for Carpaccio Cima and Robusta for chlorophyll content and y 4 5184x 11 267 3 0024x 1 1513 and 1 6972x 4 7525 for net assimilation and a polynomial regression of order 2 pol dotted line with 0 0283x 6 9386x 14 511 0 0588x 1 0742x 13 742 and y 0 0803x 4 5982x 23145 as equations respectively for Carpaccio Cima and Robusta for chlorophyll content and 0 4423x 8 4054x 6 2965 0 2457x 0 4456x 5 7112 and y 0 0404x 2 4066x 6 5356 for CO net assimilation experienced a higher flux thus a higher POD and as suggested by Paoletti
285. rer le gel marqu en m langeant 65 uL gel filtr 1 uL de marqueur Attention vortexer puis ranger tout de suite l obscurit Mettre 1 5 uL d chantillons en barrettes D naturer les chantillons en mettant 2 min 70 C puis conserver 4 C Laver l lectrode en mettant 800 uL de solution de lavage dans la puce de lavage et en l ins rant dans la machine pendant 2 min ensuite vider et ranger la puce de lavage Laver l lectrode 2 fois en mettant 800 uL d eau dans la puce de rin age et l ins rant dans la machine pendant 2 min ensuite vider et ranger la puce de ringage Mettre 9 uL de gel marqu dans le trou gs orange d une nouvelle puce Attention ne pas faire de bulle et ne pas toucher le fond Initialiser la puce dans la machine en choisissant la pression et le temps indiqu s en fonction du type de puce 149 Annexes Mettre 9 uL de gel marqu dans le trou gs jaune de la puce initialis e Attention ne pas faire de bulle et ne pas toucher le fond Mettre 9 uL de gel NON marqu dans le trou G Attention ne pas faire de bulle et ne pas toucher le fond Mettre 5 uL de loading buffer dans les puits 1 12 et le puit L Mettre 1 uL d chantillon dans les puits 1 12 Mettre 1 uL de ladder dans le puit L Mettre la plaque vortexer Lire la plaque A la fin ne pas oublier de refaire la proc dure de nettoyage de l lectrode VII
286. ression de genes et principalement ceux impliqu s dans la photosynth se le m tabolisme du sucrose et la synth se d osmolytes Gupta ef al 2005 Les sucres solubles sont des signaux distincts et n agissent pas tous de la m me mani re Le sucrose et le glucose en plus de leur r le osmoprotecteur servent de substrat la respiration cellulaire Le glucose peut alimenter les hexokinases mitochondriales et chloroplastiques afin de r duire la production de ROS dans ces compartiments cellulaires Valluru et Van den Ende 2011 Le fructose quand lui pourrait tre li par l interm diaire de la production d erythrose 4 Phosphate la synth se de m tabolites secondaires comme la lignine ou les compos s ph noliques dont la synth se est induite par l ozone Hilal et 2004 Le raffinose et le galactinol pourraient agir comme d toxiquants lorsqu ils sont accumul s lors d un stress notamment au sein des chloroplastes Nishizawa et al 2008 Valluru et Van den Ende 2011 Or le g notype le plus r sistant Carpaccio se caract rise par des concentrations constitutives en sucre notamment glucose fructose mannose et galactose plus importantes que les deux autres g notypes La disponibilit en sucres solubles plus importante peut s av rer un facteur de r sistance au stress ozone gr ce aux caract ristiques antioxydantes de certains sucres mais aussi pour pouvoir r pondre plus facilement aux besoins n cessaires la
287. riainen M Karjalainen R 1994 Plant defence systems induced by ozone Plant Cell amp Environment 17 783 794 Karlsson P E Braun S Broadmeadow M Elvira S Emberson L Gimeno B S Le Thiec D Novak K Oksanen E Schaub M Uddling J Wilkinson M 2007 Risk assessments for forest trees the performance of the ozone flux versus the AOT concepts Environmental Pollution 146 608 616 Lee S C Luan S 2012 ABA signal transduction at the crossroad of biotic and abiotic stress responses Plant Cell amp Environment 35 53 60 Le Thiec D Rose C Garrec J P Laffray D Louguet P Galaup S Loosveldt P 1994 Alteration of element content in guard cells of Norway spruce Picea abies subjected to ozone fumigation and or water stress X ray microanalysis study Canadian Journal of Botany 72 86 92 Massman W J 1998 A review of the molecular diffusivities of H20 CO CH4 CO 50 NH3 N20 NO and NO in air and near STP Atmospheric Envi ronment 32 1111 1127 Matyssek R Wieser G Ceulemans R Rennenberg H Pretzsch H Haberer K Low M Nunn AJ Werner H Wipfler P OfSwald W Nikolova P Hanke D E Kraigher H Tausz M Bahnweg G Kitao M Dieler J Sandermann H Herbinger K Grebenc T Blumenr ther M Deckmyn G Grams T E E Heerdt C Leuchner M Fabian P H berle K H 2010 Enhanced ozone strongly reduces carbon
288. rmalized expression o o P4 Figure 6 Ozone effects on gene expression of gamma glutamylcysteine synthase glutathione synthase and threonine aldolase Effects of ozone on the expression of putative genes mean SE of gamma glutamylcysteine synthase A glutathione synthase B and threonine aldolase C on three poplar genotypes Carpaccio orange border Cima green border and Robusta blue border at five sampling dates 2 4 11 15 and 17 days PC1 40 11 amp N N o o o k 0 1 2 3 4 5 6 7 PC2 17 0 32 0 30 0 28 0 26 PC1 40 o o o o o o o o o o zb v EAS e N A e o N gt 0 04 0 02 0 00 TARY aG ASP aGLY LU ORN AVAL LEU aspesLYS 5 aHIS 0 20 0 10 0 00 0 10 0 20 0 30 PC2 17 0 40 0 50 0 5 0 4 0 3 PC3 11 0 1 0 2 0 3 GLY HA M T ORN SPE LEU CYS TRP aGLN aSER 5 4G ASP LU AVAL LYS HIS PC2 17 0 00 0 10 0 20 0 30 PC2 17 0 40 0 50 Figure 7 Representation of genotype and ozone effects on amino acid concentrations in poplar leaf Principal Component Analysis sco
289. rom the 11th fumigation day NADP G6PDH and NADP ME profiles made it possible to differentiate between the two genotypes with a higher activity in Carpaccio than in Robusta At the same time Carpaccio was able to maintain high levels of NADPH in the cells while NADPH levels decreased in Robusta O3 treated leaves All these results support the hypothesis that the capacity for cells to regenerate the reducing power especially the cytosolic NADPH pool contributes to improve tolerance to high ozone exposure Received 13 June 2012 40 10 1111 1399 3054 2012 01686 Introduction Ozone O3 is a harmful phytotoxic stressor for plants Fuhrer et al 1997 Matyssek and Innes 1999 Ashmore 2005 Karnosky et al 2007 Wittig et al 2009 It causes serious damage including visible injury and growth decline that can lead sensitive species to death In trees its harmful effect can vary with growth conditions age structure or the presence of other stressors Vollenweider et al 2003 Matyssek et al 2004 Karnosky et al 2005 UNECE 2005 McLaughlin et al 2007 Poplar has been demonstrated to be among the most sensitive tree species but its sensitivity varies depending on clones genotypes Karnosky et al 1996 Yun and Abbreviations Chl Chlorophyll G6PDH glucose 6 phosphate dehydrogenase GAPDH glyceraldehyde 3 phosphate dehydrogenase ICDH NADP isocitrate dehydrogenase ME malic enzyme ML mature leaves NAD nicotinamide adeni
290. ronmental parameters under treatment In this context we also monitored the global impact of on biomass growth chlorophyll content photosynthesis and stomatal conductance by gas exchange measurements All our results were linked to POD to determine if other parameters than biomass could be modelled as a linear regression in relation with POD or if other types of adjustments are necessary 2 Material and methods 2 1 Plant culture and exposure conditions Cuttings of three Euramerican poplar genotypes Populus deltoides x Populus nigra Carpaccio Cima and Robusta with similar stomatal densities were selected from a previous study on the basis of contrasting responses to O i e visible injury growth number offallen leaves data not shown and planted in 5 L pots filled with loam N P K 14 16 18 1 2 kg m 3 Gramoflor SPI Universel fertilized by adding 20 mg of slow release nutritive granules Nutricot T 100 N P K MgO 13 13 13 2 Fertil Boulogne Billancourt France One mg 17 of magnesian limestone was added toregulate the pH The experiment was first conducted for 3 weeks on Carpaccio and Cima and was later duplicated on Robusta with half of the chambers The plants were grown in growth chambers for 5 weeks at 75 85 relative humidity day night with a 14 h light period 250 umol m s PPFD at mid leaf height from 08 h00 to 22 h00 and a temperature of 23 20 C Twenty nine individuals of each genoty
291. roplastes et les mitochondries Les observations ont t produites 80 kV sur un MET PHILIPS 12 39 RESULTATS 40 R sultats I REGULATION STOMATIQUE 1 EFFETS DE L OZONE SUR LA REPONSE STOMATIQUE AUX PARAMETRES ENVIRONNEMENTAUX LUMIERE BLEUE LUMIERE ROUGE CO ET DEFICIT DE PRESSION DE VAPEUR D EAU DANS TROIS GENOTYPES DE POPULUS DELTOIDES X NIGRA Les stomates r gulent les changes gazeux entre la plante et l atmosph re et plus particuli rement la transpiration et l entr e de CO La conductance stomatique varie en fonction des conditions environnementales les stomates tant ouverts quant les conditions sont les plus favorables La lumi re bleue et rouge induit une ouverture stomatique tandis qu une augmentation de VPD et de entraine une fermeture stomatique La vitesse de r action des stomates permet dans un environnement en constante variation de saisir les meilleures opportunit s pour limiter l vapotranspiration tout en assimilant un maximum de carbone De plus la r gulation de la conductance stomatique joue un r le important pour limiter les flux entrants d ozone et ainsi le stress oxydatif induit par celui ci Cependant il a t montr que la r ponse des stomates aux facteurs environnementaux est alt r e sous l effet de l ozone avec des vitesses ralenties Paoletti 2005 Paoletti and Grulke 2010 L objet de l tude pr sent e ci apr s tait de caract riser pr cis ment
292. rough the direct reaction with ascorbate Planta 210 454 467 Queval G Noctor G 2007 A plate reader method for the measurement of NAD NADP glutathione and ascorbate in tissue extracts Application to redox profiling during Arabidopsis rosette development Anal Biochem 363 58 69 R Development Core Team 2011 R A language and environment for statistical computing R Foundation for Statistical Computing Vienna Austria ISBN 3 900051 07 0 URL http www R project org Reich 1983 Effects of low concentration of on net photosynthesis dark respiration and chlorophyll 49 contents in aging hybrid poplar leaves Plant Physiol 73 291 296 Renaut J Bohler S Hausman JF Hoffmann L Sergeant K Ashan N Jolivet Y Dizengremel P 2009 The impact of atmospheric composition on plants a case study of ozone and poplar Mass Spectrom Rev 28 495 516 Ribas Pe uelas J Elvira S Gimeno BS 2005 Contrasting effects of ozone under different water supplies in two Mediterranean tree species Atmos Environ 39 685 693 Smeets K Cuypers A Lambrechts A Semane B Hoet P Van Laere A Vangronsveld J 2005 Induction of oxidative stress and antioxidative mechanisms in Phaseolus vulgaris after Cd application Plant Physiol Biochem 43 437 444 Smirnoff N 1996 The function and metabolism of ascorbic acid in plants Ann Bot 78 661 669 Street NR Tallis MJ Tucker J Brosch M Kangasjarvi J Broadmeadow M Taylor G 2011
293. rs les stomates absorption e niveau d antioxydants en r ponse la pression oxydative effets biologiques L AF4Y et le PODy prennent en compte la r gulation des flux par la conductance stomatique en se basant sur le mod le multiplicatif de Jarvis Or ce mod le ne prend pas en compte l impact direct de l ozone sur ses diff rentes fonctions Afin de calculer finement les flux effectifs d ozone il convient donc d am liorer le calcul de la conductance stomatique pour prendre en compte l impact direct de l ozone sur la r gulation des mouvements stomatiques aux diff rents param tres environnementaux et de d finir l intensit de d toxication cellulaire 19 Objectifs de recherche II OBJECTIFS Cette th se a pour but d indiquer des pistes pour am liorer les seuils de risque l ozone Mon travail comporte deux volets compl mentaires visant d une part am liorer l aspect d toxication inclure dans les seuils de risques l ozone et d autre part prendre en compte dans les calculs de conductance stomatique l impact de l ozone sur la r gulation stomatique L utilisation de trois g notypes de peupliers nous aide comprendre les diff rences intra sp cifiques et cibler des processus ou m tabolites cl s impliqu s dans la sensibilit l ozone Un suivi de traits cophysiologiques tels que le diam tre la hauteur les biomasses le nombre de feuilles tomb es la conductance stomatique nous permet de rel
294. rting from malate the anapleurotic pathway of the Krebs cycle can also use NADP ME which is also likely to increase the pool of cytosolic NADPH NADP ME 46 activation has been shown in several C3 and C4 plants in response to several abiotic stresses known to modify cell redox homeostasis Valderrama et al 2006 Maier et al 2011 especially in response to Dizengremel et al 2009 Guidi et al 2009 In addition by supplying NADPH and pyruvate NADP ME participates in fatty acid biosynthesis Edwards and Andreo 1992 and also in defense related lignin and flavonoid formation in stress conditions Casati et al 1999 In our work the stimulation of NADP ME activity occurred in the two poplar genotypes but the effect was more important in Carpaccio the tolerant genotype Furthermore in that genotype the correlation between PEPC activity and NADP ME activity appeared stronger These results argue in favor of an important role for NADP ME in providing cytosolic NADPH in response to ozone treatment and perhaps in conferring Carpaccio higher tolerance to ozone Considering that the same relation Physiol Plant 148 2013 was not found in other species Guidi et al 2009 that last conclusion will have to be supported by other results In case of a stress the stimulation of NADP G6PDH activity is supposed to supply the cytosol with NADPH However its involvement in the pentose phosphate pathway also implies providing higher amo
295. s 11 12 13 Ozone enters the leaves through stomata and is degraded in the apoplast generating reactive oxygen species ROS 14 These chemically reactive molecules can destroy or modify several cellular components like proteins lipids or nucleic acids 15 but in parallel they have a key role in cell signalling 16 17 18 A balance between ROS production and detoxification must be maintained by the two ways of plant defence against Os i e regulation of stomatal conductance and detoxification of ROS generated during degradation 14 19 20 Differences in sensitivity to ozone between species or genotypes may be related to one of these two processes 21 22 23 A constitutive detoxification system is already operative at the entrance of O in the leaf The pool of reduced ascorbate ASA in the apoplast is the first line of defence to avoid ROS to reach the plasmalemma where it would initiate an oxidative burst and induce a wide range of metabolic changes 14 The Foyer Halliwell Asada pathway ensures the reduction of 79 R sultats ascorbate by a sequence of redox reactions involving glutathione and NAD P H 18 The antioxidant power is not only limited to ascorbate and glutathione the detoxification process being a complex network of different types of metabolites and enzymes such as polyphenols carotenoids tocopherols superoxide dismutase and catalase 17 24 In combination with the regen
296. s diff rences de sensibilit l ozone sise 107 4 Changements induits par l ozone dans les m tabolites primaires de trois g notypes de peuplier 124 CONCLUSION ET PERSPECTIVES asar oreore soa S eod ek vanno seca eu ra eS euenit aene UR Up ex 126 dU M lt es 2548 133 I PROTOCOLE DU DOSAGE DE L ASCORBATE ET DU GLUTATHION FORMES OXYDEES ET REDUITES EN MICROPLAQUE teres en IER RINT e e RUE YE te et eto 134 5 EXIFICIOI SEINS ix ere LAT M xc X mE E E 134 6 iDosage dscorb le sente oe AR 135 Zn JDosageglutathion d D 137 II PROTOCOLE D EXTRACTION ET D ANALYSE PAR HPLC DES CAROTENO DES eee 139 PROTOCOLE D ANALYSE DES SUCRES COMPOSES PHENOLIQUES ET ACIDES AMINES eee 140 1 140 2 Analyse d s sucres eiit de b ets 142 3 Analyse des compos s ph noliques ss 143 4 Analyse des Acides amines ase eerte i e nete 144 IV PROTOCOLE D EXTRACTION DES ARNS AVEC LE KIT RNEASY PLANT MINT 147 V PROTOCOLE DE TRAITEMENT DNASE I 148 VI PROTOCOLE DE VERIFICATION DE LA QUALITE DES ARNS A L EXPERION ss 149 VII PROTOCOLE DE DOSAGE DES ARNS EN 22 200 150
297. s que nous ayons tudi s se caract rise par une chute de feuilles pr coce et importante D apr s nos r sultats sa sensibilit peut s expliquer par diff rents facteurs Au niveau stomatique il se distingue par une conductance stomatique plus lev e et surtout par des vitesses de r actions tr s faibles La lenteur de ses r ponses ne lui permet pas de tirer partie des conditions favorables au cours d une journ e et entraine probablement des pertes en eau plus importantes et une entr e d ozone plus forte De plus il n arrive pas maintenir un pool de NADPH n cessaire au processus de r g n ration des antioxydants dont l ascorbate laissant s accumuler la forme oxyd e Carpaccio l inverse est le g notype le plus r sistant que nous ayons tudi Sa tol rance se traduit par la combinaison de nombreux facteurs de r sistance tant au niveau stomatique qu au niveau m tabolique Il se caract rise par des vitesses de r ponse des stomates rapides lui permettant de saisir toutes les opportunit s pour perdre le moins d eau possible laisser entrer peu d ozone et assimiler suffisamment de carbone A ceci s ajoute de bonnes capacit s de d toxication avec notamment des concentrations constitutives en ascorbate et en glutathion importantes Il arrive maintenir un pool de NADPH suffisant pour r g n rer ses antioxydants Il dispose aussi constitutivement de concentrations en sucres plus importantes et remobilise fortement ses acid
298. s would make it possible to assess their role in ozone tolerance mechanisms Acknowledgements This work is part of the Vulnoz project funded by the following French institutions Agence Nationale de la Recherche Minist re de l Enseignement Sup rieur et de la Recherche The authors thank St phane Martin Fabien Spicher and Jacques Banvoy for technical support Pierre Montpied for advice about statistical analyses and Annie Buchwalter for English proof reading References Abat JK Saigal PT Deswal R 2008 S nitrosylation another biological switch like phosphorylation Physiol Mol Biol Plants 14 119 130 Ashmore MR 2005 Assessing the future global impacts of ozone on vegetation Plant Cell Environ 28 949 964 Bagard M Le Thiec D Delac te E Hasenfratz Sauder MP Banvoy J G rard J Dizengremel P Jolivet Y 2008 Ozone induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves Physiol Plant 134 559 574 Batz O Logemann E Reinold S Hahlbrock K 1998 Extensive reprogramming of primary and secondary metabolism by fungal elicitor or infection in parsley cells Chem Biol 379 1127 1135 Baumgarten M Huber C B ker P Emberson L Dietrich HP Nunn AJ Heerdt C Beudert B Matyssek R 2009 Are Bavarian Forests southern Germany at risk from ground level ozone Assessment using exposure and flux based ozone indices Environ Pollut 157 2091 2107 Bohler S B
299. s correspondants identifi s chez Arabidopsis sont encadr s Synth se bibliographique 15 gs gsw 1 51 16 Isw Imax X fonen x f gnt x max fmin Chew x fvpa x Lava 3 REGULATION DES MOUVEMENTS STOMATIQUES La r gulation des mouvements stomatiques est un l ment d terminant pour les capacit s d adaptation des plantes lors de changements de facteurs environnementaux En effet la conductance stomatique r gule l entr e de et les pertes en eau au niveau des feuilles Les variations de facteurs environnementaux entrainent la fermeture ou l ouverture des stomates afin d optimiser les changes gazeux en fonction des conditions environnementales L ouverture des stomates est provoqu e par la force motrice li e l augmentation du volume des cellules de garde et la pression de turgescence caus es par une entr e d eau dans l apoplasme Ces flux d eau accompagnent l accumulation d ions K provoqu e par une hyperpolarisation de la membrane suite l activation d une pompe proton Fischer 1968 Outlaw and Manchester 1979 L ouverture stomatique est ensuite maintenue par l accumulation de sucres La fermeture des stomates r sulte d un processus inverse impliquant en plus le calcium Ca En effet plusieurs facteurs comme l acide abscissique ABA ou une hausse de concentration en CO peuvent stimuler l absorption de ce qui entra ne une d polarisation de la membrane puis une sortie d ions et malate et
300. sejod oj WIS 1 8S8S0EZ00 INXBosI HOXNSdd VDVOVVDVVVVODIODODIV 0080070071204 0 1 080 OTTIHSTIV 99 lt lt 4 8cc901 JO Jo Sooquo 099059000 ULdOd 09 piopfiaand p4o81un998 DYHO osequss POLOIA DT 224 jxqiso IODLVODODIVVOVVIOLOL 8 juopuodop urxopoug oj IIS 17908806000 INXIoW C INTO ODLVVDVOVVVDDDDOLV LL 0078009001204 9LVDOD 06ETE8TIV 00zTSO8TIV Jo goz 8 10 09 9AT1 101do201 8 090 7SZ000 ULdOd ordonouor c e ouueqo 8 21120000 8 VDVVOVOVODDVODLLVVOV Josmooid 103d9991 oj IIS 166106200 VIOLVVOVODOLVOVVOLOD 0000 70700 H10d TEANTO LLLVOODVOLOLDIVOOLOL 8010 lt 000 JNXBou 0805157100 ULdOd VVVVOVOLLOVDOVVOODDOL 0077S IDF T0 ANTO losrisserv Jo 7101 Sopouio 09 3runqns ouueuo UOT 8 8 JO oj 9796 T OLSOISS000 ULdOd 10309091 oyeurejnia juerd 11108000 couosno VOOLLV IODVVODVVODVVO 1ejrurs 10dooo1 D 00590 700 WNXH9A S EWTO d IOLVODIOVVDVDLVILODDO 00LC0TOSO00 1nod STATO TATO loszeessiv Jo TOT Sojoyuo 00 626201 Md 01 SUNIDGD 5 O ouueqo pue sisdopiqv4y 0696782 9 y T 01 00
301. shington DC EPA 600 R 05 004aF cF 2006 166 R f rences Vahisalu T Puz rjova I Brosch M Valk E Lepiku M Lindgren O Moldau H Pechter P Wang Y S Salojarvi J et al Ozone triggered rapid stomatal response involves production of reactive oxygen species and is controlled by SLACI and OSTI The Plant Journal 2010 62 3 442 453 Valluru R Van den Ende W Myo inositol and beyond Emerging networks under stress Plant Science 2011 181 387 400 Vingarzan R A review of surface ozone background levels and trends Atmospheric Environment 2004 38 3431 3442 Vlachokostas C Nastis SA Achillas C Kalogeropoulos K Krmiris I Moussiopoulos N Chourdakis E Banias G Limperi N Economic damages of ozone air pollution to crops using combined air quality and GIS modelling Atmospheric Environment 2010 44 3352 3361 Vollenweider P Ottiger M G nthardt Goerg M S Validation of leaf ozone symptoms in natural vegetation using microscopical methods Environmental Pollution 2003 124 101 118 Wang FF Lian HL Kang CY Yang HQ Phytochrome B is involved in mediating red light induced stomatal opening in Arabidopsis thaliana Molecumar Plant 2010 3 246 259 Wang Y Noguchi K Terashima I Distinct light responses of the adaxial and abaxial stomata in intact leaves of Helianthus annuus L Plant Cell amp Environment 2008 31 1307 1316 Wang Y Noguchi K Terashima I Photosynthesis dependent and independ
302. side by 6 in Carpaccio and 8 in Cima No ultrastructural damage in guard cells was visible in response to ozone Fig 2 Ozone has no effects on the number of chloroplasts or starch grains but it increases strongly the number of mitochondria in guard cells of both sides of leaf Fig 2b c Analyses of the element content in guard cells In ambient condition the calcium chlorine potassium and phosphorus contents are 30 15 20 and up to 25 higher respectively in the guard cells of the lower side than of the upper side of leaf in all the genotypes The calcium content is around 25 lower in Cima than in Carpaccio and Robusta on both sides Fig 3a The chlorine content in Robusta is twice as important as in Carpaccio and Cima Fig 3b The potassium content is slightly lower in Carpaccio than in Cima and Robusta on both sides Fig 3c The phosphorus content in guard cell of the lower side of the leaf is more than 10 higher in Cima than in the other two genotypes whereas on the upper side there is no difference From 11 days of treatment ozone increases strongly the calcium content of guard cells of the upper side of leaf in the three genotypes especially in Carpaccio Fig 3e In contrast on the lower side the calcium content is only increased in Carpaccio and is even decreased by 10 in Robusta In response to ozone from 11 days of treatment while in Carpaccio the chlorine content in guard cells on both sides of the leaf is grea
303. side in three poplar genotypes more or less sensitive to ozone We attempt to precise which step of stomatal movement was disturbed to explain the sluggish behaviour of stomata induced by ozone In order to see if a particular ion flux was modified and at what channels it could be linked we combine element content X ray microanalyses and gene expression analyses on microdissected stomata of adaxial and abaxial sides Finally a possible change in the ultrastructure of guard cells was checked by microscopy techniques MATERIALS AND METHODS Plant culture and exposure conditions The plant material was grown as described in Dumont et al 2013 Briefly cuttings of three Euramerican poplar genotypes Populus deltoides x Populus nigra Carpaccio Cima and Robusta selected from a previous study on the basis of contrasting responses to i e visible injury growth number of fallen leaves data not shown were planted and grown in a growth chamber for 5 weeks Twenty nine plants of each genotype were transferred for an acclimated week in eight fumigation chambers at 75 85 relative humidity day night with a 14h light period 250 umol m s at mid leaf height from 08 00 to 22 00 and a temperature of 23 20 C day night The experiment was first conducted for 3 weeks on Carpaccio and Cima and was later duplicated on Robusta with half of chambers Four fumigation chambers were used for each treatment charcoal filtered air or fumigation
304. signaling and involved in ozone tolerance via stomatal movement in Tobacco Plant amp Cell Physiology 2005 46 1902 1914 Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Miltros T Dirks W Hellsten U Putnam N et al Phytozome a comparative platform for green plant genomics Nucleic Acids Research 2012 40 1178 1186 Griffith OW Determination of glutathione and glutathione disulfide using glutathione reductase and 2 vinylpyridine Analytical Biochemistry 1980 106 207 212 Grulke NE Neufeld HS Davison AW Roberts M Chappelka AH Stomatal behavior of ozone sensitive and insensitive coneflowers Rudbeckia laciniata var digitata in Great Smoky Mountains National Park New Phytologist 2007 173 100 109 160 R f rences H iki E Freiwald V Julkunen Tiitto Beuker E Holopainen T Oksanen E Differences in leaf characteristics between ozone sensitive and ozone tolerant hybrid aspen Populus tremula x Populus tremuloides clones Tree Physiology 2009 29 53 66 Han R M Zhang J P Skibsted LH Reaction dynamics of flavonoids and carotenoids as antioxidants Molecules 2012 17 2140 2160 Hashimoto M Negi J Young J Israelsson M Schroeder JI Iba K Arabidopsis HT1 kinase controls stomatal movements in response to CO2 Nature Cell Biology 2006 8 391 397 Heath RL Modification of the biochemical pathways of plants induced by ozone What are the varied routes to change Environmental Polluti
305. silyl trifluoro ac tamide pendant 1h 37 C Les chantillons sont ensuite transf r s dans des inserts pour proc der l analyse Les sucres les alcools de sucres les polyols et les acides ph noliques ont pu tre analys s par chromatographie en phase gazeuse Agilent Technologies 6890N Network GC system coupl e un spectrom tre de masse Agilent 5973 Network d tecteur s lectif de masse GC MS avec une injection d bit divis 33 Figure 44 Principe de la technique de RT qPCR m thode au fluorochrome SYBR Green Dans une premi re tape les ARN totaux trait discontinu rouge sont r trotranscrits en ADN compl mentaire ADNc trait continu noir par une transcriptase inverse en pr sence d une amorce sp cifique antisens et d oligos dT Dans une seconde tape un petit fragment 100 200 nucl otides de l ADNc trait continu rouge est amplifi par PCR l aide d une paire d amorces sens et antisens sp cifiques du g ne tudi en pr sence de SYBR Green toiles grises Ce produit devient fortement fluorescent toiles roses lorsqu il s intercale dans un ADN double brin chaque cycle de PCR le morceau d ADN est amplifi et l intensit de fluorescence est lue et enregistr e Cela permet de quantifier le nombre de mol cules d ADN double brin et par comparaison avec une gamme talon d en d duire la quantification absolue du nombre de mol cules ADNc et donc pr sent dans l
306. similation This reduction is almost only due to the ozone effect on the leaf upper side Indeed stomatal conductance on the lower side is weakly impacted or even not at all in Cima A deposition effect of ozone from top to bottom is not excluded despite the air renewal in growth chamber and could result in higher ozone flux on the leaf upper side than on the lower side In addition the upper side is exposed to a more intense light which may generate additional ROS production Apel and Hirt 2004 The lower stomatal conductance in response to ozone may be due to an amplified ROS production caused by higher ozone flux and light intensity on the upper side Ozone leads to sluggish stomatal responses to variation of environmental parameters Paoletti 2005 Paoletti and Grulke 2010 Dumont ef al 2013 This effect is not caused by ultrastructural damage but is more likely due to a perturbation of the ion fluxes in guard cells In our study we observed a lower stomatal conductance under ozone resulting from a smaller and or lower opening in the morning since the measurements have been carried out two hours after the beginning of the light phase This is coherent with the slower opening in response to increase of blue light and the lower amplitude of opening in response to increased red light shown in response to ozone Dumont al 2013 Phototropins function as blue light receptors in guard cells whereas stomatal response to red light is linked t
307. sink strength of adult beech Fagus sylvatica resume from the free air fumigation study at Kranzberg Forest Environmental Pollu tion 158 2527 2532 McAinsh MR Evans N H Montgomery L T North K A 2002 Calcium signalling in stomatal responses to pollutants New Phytologist 153 441 447 MEDDTL 2010 Bilan de la qualit de l air en France en 2010 Mills G Hayes F Wilkinson S Davies W J 2009 Chronic exposure to increasing background ozone impairs stomatal functioning in grassland species Global Change Biology 15 1522 1533 Onandia G Olsson A K Barth S King J S Uddling J 2011 Exposure to moderate concentrations of tropospheric ozone impairs tree stomatal response to carbon dioxide Environmental Pollution 159 2350 2354 Pandey S Zhang W Assmann S M 2007 Roles of ion channels and transporters in guard cell signal transduction FEBS Letters 581 2325 2336 Paoletti 2004 Ozone slows stomatal response to light and leaf wounding in a Mediterranean evergreen broadleaf Arbutus Unedo Environmental Pollution 134 439 445 Paoletti E Grulke N E 2010 Ozone exposure and stomatal sluggishness in different plant physiognomic classes Environmental Pollution 158 2664 2671 Pei Z M Murata Y Benning G Thomine S Klusener B Allen G Grill E Schroeder J I 2000 Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells
308. sociation curve was generated in order to check amplification product specificity The reaction mixture contained 10uL Brillant III Ultrafast SYBR green QPCR Master mix 0 3uL ROX Agilent Technologies 250 nM of gene specific primers and 2 2 uL cDNA diluted at 5 ng uL in each 20 uL reaction No template control NTC reactions were prepared for each gene The Ct values were determined with the same threshold and PCR efficiency was estimated using a standard curve for each gene and each genotype Efficiency values were taken into account in all subsequent calculations Gene expressions were analysed using GeneNorm program Vandesompele et al 2002 Mestdagh 62 a b 0 7 0 7 0 6 0 6 0 5 0 5 0 4 a dod t 0 4 0 3 0 3 0 2 0 2 0 1 0 1 0 0 10 20 d e 0 4 0 4 a 5 0 3 0 3 2 0 2 0 2 E 0 1 0 1 on 0 0 g h 0 4 0 4 0 3 0 3 0 1 0 1 0 0 0 10 20 10 20 4 1 0 10 20 10 Time day 10 20 Fig 1 Effects of ozone control in white and ozone in black on stomatal conductance gs of leaf a b c of the upper side only d e f and the lower side only g h i of Carpaccio a d g circles Cima b e h squares and Robusta c f i triangles Significant differences between the two treatments are indicated by p 0 05 p 0 01 or p 0 001 R sultats et al 2009 allowing the identification of five reference
309. society and the environment FAO IPC Rome Italy 2008 135p Dizengremel P Effects of ozone on the carbon metabolism of forest trees Plant physiology and biochemistry 2001 39 729 742 Dizengremel P Le Thiec D Bagard M Jolivet Y Ozone risk assessment for plants Central role of metabolism dependent changes in reducing power Environmental Pollution 2008 156 11 15 Dizengremel P Le Thiec D Hasenfratz Sauder MP Vaultier MN Bagard M Jolivet Y Metabolic dependent changes in plant cell redox power after ozone exposure Plant Biology 2009 11 35 42 Dumont J Spicher F Montpied P Dizengremel P Jolivet Y Le Thiec D Effects of ozone on stomatal responses to environmental parameters blue light red light CO and vapour pressure deficit in three Populus deltoides x Populus nigra genotypes Environmental Pollution 2013 173 85 96 Eckenwalder J E Systematics and evolution of Populus In biology of Populus and its implications for management and conservation Part I Chapter 1 Edit par Stettler R F Bradshaw H D Jr Heilman P E et Hinckley T M NRC Research Press National Research Council of Canada Ottawa Canada 1996 pp 7 32 Einig W Lauxmann U Hauch B Hampp R Landolt W Maurer S Matyssek R Ozone induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves New Phytologist 1997 137 673 680 European Environment Agency Spatial assessmen
310. su su su su su o opoedi1e ejsnqoy su su su su su su su su su su su su su su su 9 ejsnqoy euir su su su su su su su su su su su su su su su t9 eurr oroedae5 juaunean x adAjous9 su 00 0200 su su su su su su su su su su su su opDoedie ejsnqoy su su su su su su su su su su su su su su su ejsnqoy eun su su su su su su su su su su su su 166000 su su ewp edep 3239 adAjouay 100 0 gt 100 0 gt su 100 0 gt su su su su su 100 0 gt su su su su ejsnqoy 100 0 gt 619200 su 100 0 gt su su su su su su su su su su su eun 82000 su su 100 0 su su su EHZOO O su su LS00 0 su su su su omoedie PJJ 990 FIDL 261 6701 T 6 81 060 FLOS 060 7989 6804866 92 LFZ 1196 000 00 100 COO 200 600 00 F 1270 00 F 8CO v0 O 6CO 91020 STO F LLLI 200 8607 660 0400 260 496 8 0 9201 0601 7 0 LFT LFLZ 6007800 COOFILO 000 270 200 T ZOO 620 9007 LEO qusiquiy ejsnqoy GG0 F EVO 860 FLLOL 680 8251 897 V90TF6LG CLOTFEGL IL T IC 8797 8710 1007 00 000 600 900 S0O T 020 100 T 9 O 100 F970 91020 6071 0261 LEUT 6091 97 FOVSI ILOFIL8 960 67 6071 887 EL FC CC FLE OL 00 SUO 500 600 00 0 0 00 FLEO 500 SEO 200 620 JUaIquiy Pew c9 0T C09 980 L88 88170071 8707086 66 EGOTFEVL ILFCE 871 8 FSZ 100 600 100 100 2000 900 00 F 910 00 O O 0 0 T 91020 SCI 7 9261 680 FCOVI CUL T 8261 7607928 1COTS8SL PLOFLGL 97
311. sultats Leuning 1990 Leuning 1995 mais il est important de souligner qu un tel mod le n cessite d tre coupl un mod le appropri de photosynth se comme le mod le de Farquhar et al 1980 Le calcul de la conductance stomatique l ozone gs consiste appliquer un facteur de diffusion au calcul de la conductance stomatique la vapeur d eau gsw 15 Le mod le d Emberson Jarvis 16 utilise une conductance stomatique maximale et des fonctions r ductrices d pendantes de facteurs environnementaux ph nologie lumi re temp rature d ficit de pression de vapeur d eau VPD potentiel hydrique du sol swp Mais celui ci ne tient pas compte de l impact temporel de l ozone sur la conductance stomatique il faut donc r ussir inclure une fonction ozone fOs dans le mod le comme l ont fait Danielsson et al 2003 sur la pomme de terre L ozone ayant des r percussions sur la r ponse des stomates aux facteurs environnementaux il convient d tudier son impact sur les diff rents facteurs entrant dans le calcul afin de d finir la fonction ozone et la place ad quate o l int grer R Type QUAC1 Anion H AHA1 AHA11 y X gt LD D Vacuole AKT1 AKT2 3 KAT1 KAT2 KC1 Figure 15 Canaux et transporteurs ioniques impliqu s dans les mouvements stomatiques A gauche les prot ines de transport activ es lors de la fermeture et droite lors de l ouverture des stomates Les gene
312. t Cell Environ 35 454 484 43 Linster CL Clarke SG 2008 L Ascorbate biosynthesis in higher plants the role of VTC2 Trends Plant Sci 13 567 573 44 Wolucka BA Van Montagu M 2007 The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants An opinion Phytochemistry 68 2602 2613 45 Gilbert L Alhagdow M Nunes Nesi A Quemener B Guillon F et al 2009 GDP D mannose 3 5 epimerase GME plays a key role at the intersection of ascorbate and non cellulosic cell wall biosynthesis in tomato Plant J 60 499 508 100 R sultats 46 Wolucka BA Van Montagu M 2003 GDP mannose 3 5 epimerase forms GDP L gulose a putative intermediate for the de novo biosynthesis of vitamin C in plants J Biol Chem 278 47483 47490 47 Torabinejad J Donahue JL Gunesekera BN Allen Daniels MJ Gillaspy GE 2009 VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants Plant Physiol 150 951 961 48 Gatzek S Wheeler GL Smirnoff N 2002 Antisense suppression of L galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L galactose synthesis Plant J 30 541 553 49 Ishikawa T Dowdle J Smirnoff N 2006 Progress in manipulating ascorbic acid biosynthesis and accumulation in plants Physiol Plant 126 343 355 50 Alhagdow M Mounet F Gilbert L Nunes Nesi A Garcia V et al 2007 Silencing of
313. t a unique indicator of resistance is very unlikely but it is rather the sum of several factors that increases the resistance 94 Y ROS VTC2 L galactose Phenols P pathway flavonoids Antioxydant IROS AS DHA vues GSSG GSH amino acids NADP NADPH Glutamate Oxidative stress Lignin carbon metabolism Glycine v glutamylcysteine modified decrease TCA cycle m of photosynthesis cystine Threonine Aspartate family Alanine amino acids Glutamate Figure 10 Scheme of the factors partly explaining ozone sensitivity among poplar genotypes Synthesis of parameters that may be involved in the difference in ozone sensitivity between the three genotypes Colour frames Carpaccio in orange Cima in green and Robusta in blue represent an increase of concentration or gene expression italics the thickness showing the intensity of the increase Only parameters that varied differently among genotypes are colour framed No decrease has been shown in our study to be involved in the differences of sensitivity between the genotypes R sultats Acknowledgements The authors are thankful to St phane Martin Fabien Spicher and Patricia Ballias for technical assistance We thank Olivier Forestier and his team from the State Forest nursery of Gu men Penfao for providing the cuttings This work was supported by the COST action FP0903 STSM the IFR110 EFABA t
314. t induire des pertes en eau et une diminution de l assimilation L ozone parvenant entrer dans la feuille va cr er un stress oxydatif par la formation de radicaux libres oxyg n s ROS dans l apoplaste Un processus de d toxication combin un processus de r paration doit permettre de lutter contre les effets d l t res de ces ROS Une des principales voies de d toxication est la voie d Halliwell Asada Foyer dont l ascorbate et le glutathion sont les acteurs majeurs De plus l ascorbate tant pr sent dans l apoplaste c est le premier pouvoir s opposer aux effets induits par les ROS Cette tude vise mettre en vidence l effet de l ozone sur les concentrations en ascorbate et en glutathion sous leurs diff rentes formes oxyd e et r duite Afin de ne pas tre d bord par les ROS le pouvoir r ducteur dont l ascorbate et le glutathion sont deux acteurs majeurs doit tre maintenu gr ce aux processus de r g n ration et de synth se de novo Nous avons tudi l expression des g nes impliqu s dans la voie de biosynth se de l ascorbate et du glutathion en compl ment de l analyse des teneurs en acides amin s utilis s dans ces voies Les conclusions suivantes ont pu tre tir es ozone engendre une augmentation des concentrations en ascorbate Chez le g notype sensible cette augmentation n est due qu une hausse de la forme oxyd e tandis que chez les autres g notypes c est une hausse de la forme r
315. t of PMIO and ozone concentrations in Europe 2005 EEA Technical report 2009 n 1 159 R f rences Fischer RA Stomatal opening role of potassium uptake by guard cells Science 1968 160 784 785 Fiscus EL Booker FL Burkey KO Crop responses to ozone uptake modes of action carbon assimilation and partitioning Plant Cell amp Environment 2005 28 997 1011 Foyer CH Noctor G Redox homeostasis and antioxidant signaling A metabolic interface between stress perception and physiological responses 2005 17 1866 1875 Fuhrer J Skarby L Ashmore MR Critical levels for ozone effects on vegetation in Europe Environmental Pollution 1997 97 91 106 Fuhrer J Ozone risk for crops and pastures in present and future climates Naturwissenschaften 2009 96 173 194 Galano A Vargas R Martinez A Carotenoids can act as antioxidants by oxidizing the superoxide radical anion Physical Chemistry Chemical Physics 2010 12 193 200 Galant A Preuss ML Cameron JC Jez JM Plant glutathione biosynthesis diversity in biochemical regulation and reaction products Frontiers in Plant Science 2011 2 1 7 Gerlier D Plumet S Herschke F Dynamique de l ARNome du virus de la rougeole Virologie 2007 11 231 245 Gomi K Ogawa D Katou S Kamada H Nakajima N Saji H Soyano T Sasabe M Machida Y Mitsuhara I et al A mitogen activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid
316. t of leaf injuries due to treatment 120ppb For Robusta damage rapidly spread onto the first fully expanded leaves located at the bottom of the trees and resulted in severe large mottle necrosis on large surfaces Fig 3D The last fully expanded leaves ML of this genotype displayed the same type of injury but to a lesser extent Fig 3B with brown flecks near the edges in some cases For Carpaccio the first symptoms also occurred on the first fully expanded leaves and started with chlorotic flecks on the total leaf areas resulting in moderate internal brownish stippling Fig 3C At the end of the treatment some ML also exhibited rare small brown stippling on their upper surface Fig Due to W Carpaccio 15 A Robusta n 510 4 5 4 1 1 1 1 1 1 1 1 1 1 1 1 1 5 10 15 Fig 2 Time course of POD values during fumigation Carpaccio and Robusta genotypes Values represent means of n 20 se and represent the average POD values obtained in four chambers Physiol Plant 148 2013 Fig 3 Comparison of ozone induced leaf injury observed on the last fully expanded leaves A B and the first fully expanded leaves C D of Carpaccio A C and Robusta B D genotypes Both genotypes were exposed to a dose of 120 ppb for 17 days Table 1 Percentages of fallen leaves during ozone treatment calculated in relation to total numbers
317. tagna A Ranieri A 2009 Detoxification and repair process of ozone injury From Os uptake to gene expression adjustment Environ Pollut 157 1461 1469 Chai MF Wei PC Chen QJ An R Chen J Yang S Wang XC 2006 NADK3 a novel cytoplasmic source of NADPH is required under conditions of oxidative stress and modulates abscisic acid responses in Arabidopsis Plant J 147 665 674 Di Baccio D Castagna A Paoletti E Sebastiani L Ranieri A 2008 Could the differences in O3 sensitivity between two poplar clones be related to a difference in antioxidant defense and secondary metabolic response to influx Tree Physiol 28 1761 1772 Dizengremel P 2001 Effects of ozone on the carbon metabolism of forest trees Plant Physiol Biochem 39 729 742 Dizengremel P Le Thiec D Bagard M Jolivet Y 2008 Ozone risk assessment for plants central role of metabolism dependent changes in reducing power Environ Pollut 156 11 15 Dizengremel P Le Thiec D Hasenfratz Sauder MP Vaultier MN Bagard M Jolivet Y 2009 Metabolic dependent changes in plant cell redox power after ozone exposure Plant Biol 11 35 42 Doubnerov V Ryslava H 2011 What can enzymes of C4 photosynthesis do for C3 plants under stress Plant Sci 180 575 583 Edwards GE Andreo CS 1992 NADP malic enzyme from plants Phytochem 31 1845 1857 Emes MJ Neuhaus HE 1997 Metabolism and transport in nonphotosynthetic plastids J Exp Bot 48 1995 2005 Fares S Loreto F
318. tal Pollution 173 2013 85 96 1000 gt 100 0 gt 096100 VSO 6001 L8 0 F CHET SUL CE6 6661 VEO tV F EG 9L adAjouas pue UO1Jeriea 200 Jaye UONepruisse 1 5 su su su su su su su su su 9ET F SESI 65676 1831 EVEL OL EL 861 88 TL 107 88 81 T su su su VLOvVO O 279000 su su su su YEO 0061 Scc 1692 F 6 6961 F ETSI 281 06 91 IV su su su su su su 10007 10007 1000 gt 160 029 F 1971 SEO F 079 TOT T VL SI S 0 F 99 S L6 0 lt 71 2 5 7 su su su su su su 700 0 100 0 gt 166000 vel lt 01 9 16 71 6 648 991 SEIL TTI 6176 821 071 jown Ly uonerreA TO 55 _s su su su 100707 su su su su su OST EVOL PTET 68 8 9S7 F 89 1 ESS T 86LE TTI 81 SVL 1221 T su su su 100707 su 000 su su su EVLI 9106 8S C868 2811 F OLLT OTS9 SLT C9CC 691 F ETLI 1 t9 opoedie ejsnqoy EQ ejsnqoy eum to eurr opoedae5 x adAjouo oroedae ejsnqoy ejsnqoy eur PUII 0ID2ed1e 32399 adfjoua9 ejsnqoy onDedie 323g 3usunea1 91020 quaiquiy eJsnqoy 91020 juemquiv Pew 91020 quaiquiy oroedie5 sumjQueuneal adAjouay 0 0 gt d JuedyIUsIS
319. talase est une enzyme qui dismute le peroxyde d hydrog ne en eau Les peroxydases sont une famille d enzymes catalysant la d gradation du peroxyde d hydrog ne avec comme second substrat un compos r ducteur L ascorbate peroxydase et la glutathion peroxydase en font partie 4 VOIE DE BIOSYNTHESE DE L ASCORBATE L ascorbate est un l ment important pour la r gulation des radicaux libres oxyg n s dans plusieurs compartiments cellulaires travers la voie d Halliwell Asada La voie de synth se principale de l ascorbate utilise le galactose Figure 25 cependant d autres voies 15 Apoplasme MDHAR NADP ASA NADPH 3 DHA ou GLR 6556 H 0 Y NADP 7 T j 4 NADP SS gt Chloroplaste Mitochondrie A NAD P H Figure 24 Sch ma des syst mes de d toxication des ROS dans les diff rents organites des cellules v g tales Abr viations APX Ascorbate peroxydase ASA Ascorbate CAT Catalase DHA D hydroascorbate DHAR D hydroascorbate r ductase EIM Espace inter membranaire F Flavono de F Radical Flavono de GLR Glutaredoxine GPX Glutathion peroxydase GR Glutathion r ductase GSH Glutathion r duit GSSG Glutathion oxyd GuPx Guaiacol Peroxydase MDHA Monod hydroascorbate MDHAR Monod hydroascorbate r ductase NAD P Nicotinamide ad nine dinucl otide phosphate oxyd NAD P H Nicotinamide ad nine dinucl otide phosphate r duit P
320. te de la conductance stomatique et de l assimilation de CO Wittig et al 2007 Ainsi il a t observ un d couplage entre les deux Lombardozzi et al 2012 Comme nous l avons aussi montr Dghim ef al 2012 l ozone impacte directement la photosynth se en plus de la conductance stomatique en induisant une modification de l activit des enzymes carboxylantes Rubisco et PEPC et en modifiant chez les esp ces les plus sensibles la disponibilit en NADPH ce qui pourrait ralentir la r g n ration du RibuloseBisPhosphate La relation troite liant l assimilation et la conductance stomatique n est donc plus valide en pr sence d ozone et rend les mod les de pr diction de la conductance stomatique utilisant cette relation tels que le mod le de Ball Berry inadapt s De la m me mani re nous avons montr que l ozone modifie la r ponse des stomates aux variations de diff rents facteurs environnementaux Dumont ef al 2013 L encore les fonctions sur lesquelles se fondent les mod les multiplicatifs de pr diction de conductance stomatique ne sont plus valides lors d un stress ozone et rendent les mod les incertains Cependant ces mod les peuvent tre am lior s pour prendre en compte les effets directs de l ozone sur la photosynth se et sur la r ponse stomatique aux variations de param tres environnementaux Pleijel et al 2002 Danielsson et al 2003 Lombardozzi et al 2012 Nous avons montr que l utilisation
321. th tase y ECS Noctor 2006 16 OBJECTIFS DE RECHERCHE 17 Tableau 3 Principaux indicateurs d valuation de l impact de l ozone sur la v g tation Mesure Param tres Unit O3 Danger AOT40 ou SUM60 ppb h Ozone atmosph rique 7 Entr e 4 gj POD ou CUO ou Exposition mmol m Conductance stomatique Q flux Flux effectif valuation du flux D toxication mmol m risque Conductance stomatique et indicateur de la d toxication Objectifs de recherche I PROBLEMATIQUE 1 SEUILS DE RISQUE A L OZONE Les effets n fastes de l ozone sur la sant l environnement et l conomie agricole et foresti re repr sentent une pr occupation importante pour l Union Europ enne qui exige une am lioration des seuils de risque l ozone pour la v g tation En effet aujourd hui l impact de l ozone est valu l aide de plusieurs indicateurs Tableau 3 Ces indicateurs varient en fonction des param tres de mesure utilis s et en fonction de leur pr cision Un bon indicateur doit tre le plus pr cis possible tout en restant le plus simple d utilisation d o certains compromis L AOT40 1 mesur en ppb a t d velopp et adopt au niveau europ en dans les ann es 90 s Fuhrer et al 1997 Il correspond l exposition cumul e au del d une concentration limite de 40 ppb chaque jour clairements gt 50 pendant la p riode de v g tation Il permet actue
322. the two genotypes Fig 6C D In Robusta NADP G6PDH activity constantly increased and reached a maximum at the end of the fumigation period day 17 Fig 6D In 42 Carpaccio the activity reached a maximum on day 11 of the treatment Fig 6C On days 11 and 15 a significant genotype effect was detected for O3 treated trees with higher activity values for Carpaccio Table 2 NADP ME activity increased under treatment and reached Physiol Plant 148 2013 P Carpaccio control B Robusta control 4 Carpaccio ozone Robusta ozone 4 a O 3 L KKK n KKK k k Bl 2 Auk 4 E 4 _ Tq C ger o amp 5r M a o H iP 1 ES KEE 1 KK E 1r pee a da a a aca dE aaa acaba 6 12 18 6 12 18 Time d Fig 5 Effect of treatment on PEPC activity A B and on Rubisco C D activity nkat protein leaf protein extracts of Carpaccio and Robusta B D genotypes n 4 st Significant differences between the Os treated and control leaf extracts are indicated by P lt 0 05 P lt 0 01 and P lt 0 001 a maximum value on day 15 for the two genotypes Fig 6E F For this enzyme a genotype effect was observed for O3 treated trees on days 11 15 and 17 with higher activity values for Carpaccio Table 2 Similar to NADP ME and NADP G6PDH NADP ICDH activi
323. the fumigation period On day 15 NADH NAD ratios in O3 treated leaves dropped by approximately 43 A Carpaccio control Carpaccio ozone NADP GAPDH nkat mg prot B Robusta control A Robusta ozone j xxx NADP G6PDH nkat mg prot as KKK T nkat mg prot SE G KKK KKK D NADP ICDH nkat mg prot __ a a a a a dE 6 4 a H 4 18 6 12 18 Time d Fig 6 Effect of treatment on NADP GAPDH activity A B on NADP G6PDH activity C D on NADP ME activity E F and on NADP ICDH G H activity nkat protein in leaf protein extracts of Carpaccio A C E G and Robusta B D F genotypes n 4 se Significant differences between the O3 treated and control leaf extracts are indicated by P lt 0 05 P lt 0 01 and P lt 0 001 50 6096 for the two genotypes with a concomitant increase of the NAD H pool Table 3 For Carpaccio the NADPH NADP ratio increased while it decreased by 50 for Robusta On day 15 the NADP H pool tended to decrease for the two genotypes and the total amounts of all the pyridine nucleotide forms reduced as well as oxidized were fairly similar for either control or treatment for the two genotypes Discussion In this study high daily exposure 120 ppb in
324. tion losses and economic damage under two scenarios of O pollution Atmospheric Environment 45 22977 2309 Castagna A amp Ranieri A 2009 Detoxification and repair process of ozone injury From uptake to gene expression adjustment Environmental Pollution 157 1461 1469 Dizengremel P 2001 Effects of ozone on the carbon metabolism of forest trees Plant Physiology and Biochemistry 39 729 742 Dizengremel P Le Thiec D Bagard M amp Jolivet Y 2008 Ozone risk assessment for plants central role of metabolism dependent changes in reducing power Environmental Pollution 156 11 15 Dodd A N Kudla J amp Sanders D 2010 The language of calcium signaling Annual Review of Plant Biology 61 593 620 Dumont J Spicher F Montpied P Dizengremel P Jolivet Y amp Le Thiec D 2013 Effects of ozone on stomatal responses to environmental parameters blue light red light and vapour pressure deficit in three Populus deltoides x Populus nigra genotypes Environmental Pollution 173 85 96 Ederli L Morettini R Borgogni A Wasternack C Miersch O Reale L Ferranti F Tosti N amp Pasqualini S 2006 Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone treated tobacco plants Plant Physiology 142 595 608 FAO 2006 Global Forest Resources Assessment 2005 Progress towards sustainable forest management In FAO Forestry Paper Rome Food and Agricultural Organisati
325. tique de chaque g notype pour chaque traitement sur chaque face de la feuille Les observations ont t faites avec les r glages suivants tension d acc l ration des lectrons EHT 15 kV Intensit de Sonde ISonde 1 nA distance focale WD 12 mm grandissement 250X La taille des 38 MT a l Figure 45 A Syst me de microdissection laser Palm Microbeam Zeiss B Principe de fonctionnement du syst me de microdissection laser C Morceaux d piderme restant sur la lame apr s microdissection D Stomates propuls s dans le tube Eppendorf faisceau incident lectrons r trodiffus s lectrons secondaires RX chantillon lectrons diffus s faisceau transmis Figure 46 Sch ma des interactions entre le faisceau d lectrons et la mati re Mat riel et M thodes stomates a t mesur e dans les m me conditions mais un grandissement de 800X Chaque demi disque a donn lieu 3 r p titions sur 3 champs diff rents pour l observation de la densit stomatique et un champ permettant d observer 6 stomates pour les mesures de taille 3 MICROANALYSE X DES ELEMENTS MINERAUX MICROSCOPIE ELECTRONIQUE A BALAYAGE La microanalyse des rayons X a permis de doser les concentrations min rales l int rieur des cellules de garde Les analyses ont t r alis es sur les m mes chantillons que ceux ayant servis pour l observation au MEB avec 2 champs de 5 stomates par
326. tly reduced in Robusta the opposite effect is 64 Fig 2 General view of guard cells submitted to a control and b ozone treatments Proliferation of mitochondria was typical in ozone treatment c Mitochondria m vacuoles v cell wall cw and starch grain st inside chloroplasts can also be seen Bars 1 um 12 D 0 25 0 20 0 15 0 10 0 05 c Element contents dry weight in guard cells d 05 0 00 Relative of element contents dry weight guard cells e f g h 50 0 40 0 30 0 20 0 10 0 0 0 10 0 20 0 40 0 20 0 0 0 20 0 40 0 60 0 80 0 50 0 40 0 30 0 20 0 10 0 0 0 10 0 20 0 0 0 10 0 20 0 30 0 40 0 50 0 60 0 11 18 Time day Fig 3 Element contents Ca a and e Cl b and f K c and g P d and h in guard cells of the upper pastel colour and lower sides bright colour of leaves of three poplar genotypes Carpaccio orange Cima green and Robusta blue before fumigation a b c d and effect of ozone relative to control 6 after 11 and 18 days of treatment e f g h Significant differences between genotypes sides of leaves or treatments are indicated by p lt 0 05 p 0 01 or p 0 001 17 401554 8 700777 ORinf ORsup Fig 4 Heat map representing the effects of ozone on t
327. tre les broyages refroidir les chantillons dans de l azote liquide et m langer les chantillons Faire un blanc avec seulement du solvant et le m lange de standards internes chaque jour 140 Annexes Ajouter 650 uL de 0 005 de BHT dans 80 de MeOH D gazer le solvant chaque jour apr s usage sous atmosph re azot e pendant 5 min et stocker 20 C Ajouter 200 uL de m lange de standards internes Pour faire le m lange 2 mL de 1 mg mL d acide benzoique d5 dans 1 1 2 mL 1 mg mL D glucose C13 dans 1 1 MeOH H5O 1 mL de biochanin A dans 100 de MeOH 600 uL de 2 8 mg mL glycerol d8 dans 1 1 MeOH H 0 et 1 mL de 0 1 mg mL d alanine d3 dans 1 1 Secouer 15 sec avec le TissueLyser Ajouter de nouveau 650 uL de 0 005 de BHT dans 80 de MeOH et m langer pendant 15 sec avec le TissueLyser D gazer le solvant chaque jour apr s usage sous atmosph re azot e pendant 5 min et stocker 20 C Extraire 15 min 4 C 1400 rpm Centrifuger pendant 2 min 10 C 12000 rpm environ 13500 g Pr lever le surnageant et le garder sur la glace Ajouter 2 fois 650 uL de 0 005 de BHT dans 100 de MeOH V rifier que l chantillon est bien m langer dans le solvant et m langer 15 sec avec le TissueLyser Faire une seconde extraction de 5 min 4 C 1400 rpm Centrifuger 12000 rpm environ 13500 g pendant 2 min a 10 C Combiner les surnageants
328. trifug 4 C pendant 10 min 14000 g L ascorbate et le d hydroascorbate ont t mesur s l aide de la m thode d crite par Kampfenkel et al 1995 adapt e l utilisation d un robot de pipetage par le remplacement de l acide trichlorac tique par de l acide m taphosphorique 5 La m thode est bas e sur la r duction du en par et la r action ult rieure des produits Fe II avec le 2 2 bipyridyl pour former un complexe color dont on suit l apparition 550 nm Pour doser l ascorbate total un traitement pr alable avec l agent r ducteur Dithiothreitol DTT est n cessaire afin de r duire l ascorbate Pour le dosage du glutathion nous avons suivi 405 nm la r duction glutathion r ductase d pendante du 5 5 dithiobis 2 nitro benzoic acid avec ou sans pr traitement avec du 2 vinylpyridine VPD qui vient neutraliser la forme r duite du glutathion pour ne doser que la forme oxyd e selon la m thode d crite par Griffith 1980 3 CAROTENOIDES Toutes les tapes de l extraction l analyse des chantillons ont t r alis es en maintenant les chantillons 4 C l obscurit les carot noides tant photosensibles Protocole d taill en annexe 2 Les carot noides et les chlorophylles ont t extraits partir de 10 mg de poudre de feuille lyophilis e dans 1 mL de m thanol puis 1 mL d hexane contenant chacun 0 01 de butylhydroxytolu ne BHT Les surnag
329. tropines la lumi re bleue Mao et al 2005 La lumi re rouge active la photosynth se et stimule l accumulation de sucrose dans les cellules de garde en l absence d apport d ions K alors que la lumi re bleue stimule l entr e 11 Rapid Ca independent pathway PCR SLAC1 ee channel ERORE JannE gt il High AT I 2 7 mm gt nee gt Mesophyll cell lt gt and malate release A 4 Na a ABCBI4 Guard cell malate re uptake wall malate Stomatal closure Figure 18 Mod le simplifi illustrant les fonctions de g nes r cemment identifi s et de m canismes impliqu s dans le contr le des mouvements stomatiques en r ponse au CO Kim et al 2010 Figure 19 Nouveau mod le montrant la s quence d v nements induits par le entra nant l activation des canaux anioniques de type S et la fermeture des stomates Xue et al 2011 Synth se bibliographique de K et de la synth se de malate et l hydrolyse de l amidon Zeiger et al 2002 De plus les cryptochromes CRY1 et CRY2 agissent en plus des phototropines lors de la r ponse des stomates la lumi re bleue et en parall le des phytochromes lors de la r ponse la lumi re rouge Boccalandro et al 2012 Les cryptochromes interviendraient dans la r ponse la lumi re bleue et la lumi re rouge en r duisant les niveaux d ABA D une mani re g n rale il sembl
330. ts Point and Identify Module was used to target guard cell across the freeze dried surface at magnification 1000x X ray microanalyses of element content were performed on 10 guard cells of each leaf side of each disc of each sample Guard cells microdissection After 18 days from the beginning of the fumigation the first fully expanded leaf at the 11 and 11 leaf from the top for Carpaccio Cima and beginning of the experiment 15 Robusta respectively of three trees of each genotype of each treatment was sampled The leaves were cut in half by removing the midrib and were immediately flash frozen in liquid nitrogen They were freeze dried at 40 C for 40 h at a pressure of 10 Pa in a FreeZone Plus freeze dryer Labconco Kansas City Missouri and then the temperature was turned back progressively to 20 C within 10 h Leaves were stored under vacuum in a desiccator at room temperature to avoid rehydration Pieces of lower and upper epidermis were isolated by scraping gently the epidermis with a razor blade above a glass side The pieces were fixed by evaporation of few drops of ethanol Laser microdissection and pressure catapulting of guard 61 R sultats cells were carried out using the PALM MicroBeam system Carl Zeiss MicroImaging GmbH Jena Germany For each sample 500 stomatal complexes were cut and catapulted in 0 2 ml tubes with adhesive cap The room temperature was kept at 19 C and air humidity decreased to 45 to
331. ty was stimulated in O3 treated leaves of the two genotypes Fig 6G H The activity reached a peak on day 15 for the two genotypes However at that sampling date NADP ICDH activity was lower in Carpaccio Table 2 Regarding the relationship between NADP dependent enzyme activities versus PEPC activity an approximate linear correlation prevailed except for NADP GAPDH Fig 7A D The correlation for NADP ICDH versus PEPC was high r gt 0 9 and no difference between the two genotypes was found Fig 7D On the other hand the genotype dependent linear relation differed for NADP ME and NADP G6PDH versus PEPC P values of 0 0003 and 0 0076 respectively For those two NADP dependent enzymes the slopes of the linear regression were steeper for Carpaccio Fig 7A C than for Robusta A linear relation was also found between NADP ICDH versus NADP ME activity in the two genotypes Fig 7E with a higher slope for Carpaccio However the positive slope of this linear relation was below 1 showing a higher increase of NADP ME activity in O3 treated leaves of the two genotypes as compared to ICDH activity Leaf pyridine contents In control conditions the contents in oxidized or reduced pyridine nucleotide forms did not change in the leaves Physiol Plant 148 2013 of either poplar genotype Fig 8 However changes were noticed in response to treatment Concerning NADPH its contents decreased in Robusta from the start of
332. uaunea x sdAjous obedie e snqoy euy o199ed 1e9 adAjouay ejsnqoy onDedIe payja 91020 ejsnqoy 91020 JU3Iquiy 91020 quaiquiy oroedie5 sumjQueuneal 5 adAjoua9 S0 0 gt d queoyiusis pa1eprsuoo are uonoe1ojur ad joueS x pue PJ w oum oy paads Surso eyeuo s _s z A 25012 ejeuuojs 10 ua s 1 SYIM 61021 Jaye 0 1JUOD JOU 10 EQ JO our 021 5 ejsnqoy pue ew sadAjouas 1e dod 22113 ay Jo AS F sueaur our 202 Jouni 0021 03 007 W014 20 e 03 sesuodsa1 oSuetpxo 528 rureu AG 94 S IEL J Dumont et al Environmental Pollution 173 2013 85 96 95 middle of the day and higher net CO assimilation Fig 7A undu lated area When light decreases Robusta may lose more water but its carbon gain may be limited by low light In the presence of Os but before any effects of on stomatal conductance the uptake of Robusta may be higher at midday leading to higher sensitivity Since 2011 the threshold for PODy calculations for trees has been 1 nmol m 2571 so that a linear relation with biomass loss can normally be obtained In our study we show that the use of POD to get a linear relation with physiological parameters is not always sufficient Differences of sensitiv
333. ueline Nicolas Agn s Dominique Adeline Pascal Pascale Erwin Rosine Sandrine Mireille Lysiane Laurence Marie Paule Damien Bernard Caroline Fran ois Jean Marie Daniel Bernard St phane Patrick Claude Rapha l Damien Nathalie Carole Dorine LISTE DES FIGURES ET DES TABLEAUX FIGURES Figure 1 Structure de la mol cule d ozone Figure 2 R partition de l ozone atmosph rique selon l altitude Document mis par la NASA Figure 3 Cycle de g n ration destruction de l ozone ou cycle de Chapman Yip 2000 Figure 4 Cycles de l ozone stratosph rique et troposph rique U S EPA 2006 Figure 5 Evolution des concentrations europ ennes en ozone ppb mesur es entre 1870 et 2000 Marenco 1994 Figure 6 Concentrations annuelles pass es actuelles et pr dites en ozone troposph rique Vingarzan 2004 Figure 7 Cartes des valeurs d AOT40 indicateur de seuil de risque l ozone pour la v g tation en 2005 pour les cultures et pour les for ts EEA 2009 Figure 8 Exemples de sympt mes foliaires sur des feuilles de peupliers soumis un traitement l ozone Photos F Spicher A D coloration feuille de droite Par rapport une feuille t moin feuille de gauche B et C n croses plus ou moins importantes entre les nervures de feuilles de peuplier et D marbrures sur des aiguilles de pin d Alep Photo V Calatayud Figure 9 Variation en pourcentage de la biomasse totale biomasse folia
334. ui O ISOUI 03 1e rurs toAneynd oseyeydsoydouow p 10 0 150 03 1e rurs ossocspiv octeeSeiv Jo Jo 2 So oq10 09 Z 9 UILUEJIA 1e ruis IT OTOLEETOO T SOTETETOO JWXIoW 1 6S8L0ETO0O IWNXII W T TE6TOETOO JNXII W 76129100 INXII W 00 9S19010 104 0001109910 104 ETHOIA OT IOSIMOUOD jxgiso 0061009001204 lt 0 0000 001919110109 10 100 900 1218 VCOLA7d 002 800 I90DVVILLLOLLLOVOLOLO L DVOLVVODVODLLLLODVO L VVOVVOLVOIVODLIOLDDIO VVOLVOVVODDIOVDVDVD L ILVVOLODIOLLLODIVODDV IODLLVVOOLLOLOVOVDIO VVIODDOLLOLIVVODIVOOV LLLODDVODVODVVDOD LLV L DVVVOVLIVOVIDVODIVOOO VILVODIOLLLO JIOJIOV IOVO O Z10S9100 ULdOd OTTIOS9T00 ULdOd 0091059000 ULdOd OrrrISS000 ULdOd Ot680STIOO ULdOd TOITOLA 9IPOLA 9YOLA STOLA TITOLA Table S2 ANOVA the main effects and significant interactions of genotype treatment and sampling date on the concentrations of ascorbate total reduced form oxidized form and redox ratio DF Degrees of Freedom Metabolite name DF F value p value Total ascorbate ASA DHA Genotype 2 40 310 lt 0 0001 Treatment 1 44 752 lt 0 0001 Time 4 4 315 0 003 Genotype x time 4 9 977 lt 0 0001 Reduced ascorbate ASA Genotype 2 10 3582 0 0001 Treatment 1 8 1068 0 0056 Time 4 5 4790 0 0006 Genotype x time 8 4 1325 0 0004 Treatment x time 4 6 2252 0 0002 Genotyp
335. ujours aid e de bon c ur et qu avec toi mes journ es auront t bien plus joyeuses gr ce ta spontan it ta bonne humeur et ton rire communicatif surtout quand tu as te remercie beaucoup Ir ne te dois une sp ciale d dicace car sans toi et ton esprit de g n rosit il n y aurait pas eu de manuscrit En tout cas toujours directe tu n en restes pas moins quelqu un d essentiel pour l quipe car tu es avant tout quelqu un d humain toujours l pour veiller sur les autres J ai pu d couvrir le monde de la microscopie en autre gr ce vous Lu et Jo lle Vous m aurez appris toujours avec patience Vous aurez t mes guides vers l infiniment petit J ai aussi pu faire mes premiers dans l univers de et des statistiques Cierre merci de m avoir fait profiter de tes connaissances et de tes conseils tes c t s on en apprend toujours Franck tu auras t mon sauveur tu auras su pr server mes donn es et ce malgr mon karma quelque peut capricieux ce n tait pas gagn alors merci et f licitations Je n oublierai certes pas Cyndie un petit rayon de soleil Ca fait du bien en Lorraine mais malheureusement ca dure jamais assez longtemps ce fut un plaisir de travailler avec toi St phane merci pour ton aide dans gestion des chambres phytotroniques D sol e de t avoir autant emb t soir et week end compris Ce n tait pas toujours facile mais on aura r ussi Aur lie tu auras t un
336. unettes de protection Les d chets seront limin s dans les diff rents containers correspondants aux produits 152 Annexes Produits pr parer Pr paration du tampon phosphate 0 2 M pH 7 2 pour 100 mL A 4 290 0 2 M B Na gt HPO 02M 3 12 g 100 mL 2 84 g 100 mL M langer 28 ml de A et 72 ml de B Pr paration du fixateur Concentration finale Volume 10 ml Volume 50 ml Glutarald hyde 25 2 5 1 5 Tampon phosphate 0 2 M pH 7 2 0 1 M 5 25 Eau distill e 4 20 Faire le m lange des diff rents produits et mettre dans la glace Pr paration de la r sine EPON DER 736 8 mL EMbed 812 2mL NMA 8 9 mL DMP 30 0 28 mL M langer les diff rents produits dans l ordre avec un barreau aimant en les ajoutant les uns apr s les autres Conserver le m lange 4 C Sortir la r sine temp rature ambiante avant de l utiliser voir la mettre quelques minutes l tuve 50 C Ne pas agiter trop fortement il faut viter les bulles 153 Annexes Une faible quantit de fixateur est d pos e sur la zone pr lever l aide d une lame de scalpel le mat riel est d coup et plong dans un tube contenant du fixateur 4 C Temps de la fixation 4 heures 8 C un l ger passage sous vide permet une meilleure p n tration du fixateur Lavage dans le tampon phosphate 0 1 M pH 7 2 plusieurs bains de 15 min ou la nuit
337. unts of sugar intermediates for lignin biosynthesis which is believed to play a defensive role in case of O3 exposure Caban et al 2004 Our work showed that its activity was also stimulated in the two genotypes in response to fumigation but with higher values in Carpaccio The high correlation between the increase in NADP G6PDH and PEPC activity levels supports the hypothesis that in case of O3 induced oxidative stress several pathways all likely to provide the cytosol with NADPH were activated simultaneously Conversely NADP GAPDH also known to provide cytosolic NADPH displayed much lower changes in their activity levels in response to treatment NADPH is considered as one of the most vital factors in cell growth maintenance and detoxification The large necroses and the early abscissions observed on Robusta leaves could result from lower NADPH availability In Robusta a drop in NADPH content was evidenced especially at the end of O3 exposure while Carpaccio was able to maintain the same pool of reduced pyridine nucleotides as the controls all along the treatment These results are in agreement with the fact that Carpaccio is more able to regenerate NADPH by increasing certain cytosolic NADP dependent dehydrogenase activities in case of stress In good accordance with what is commonly observed at the cell level NADPH NADP ratio remained higher than the NADH NAD ratio for which the reduced form NADH can rea
338. ures r alis es l aide de l appareil de mesure d changes gazeux Licor 6400 Figure 41 Sch ma du dispositif de mesure avec 3 appareils Li 6400 intercalibr s Figure 42 Exemple de r ponses de la conductance stomatique A une augmentation de lumi re bleue et B une augmentation de VPD Figure 43 Plateau robotis de ph notypage enzymatique haut d bit Figure 44 Principe de la technique de RT qPCR m thode au fluorochrome SYBR Green Gerlier et al 2007 Figure 45 A Syst me de microdissection laser Palm Microbeam Zeiss B Principe de fonctionnement du syst me de microdissection laser C Morceaux d piderme restant sur la lame apr s microdissection D Stomates propuls s dans le tube Eppendorf Figure 46 Sch ma des interactions entre le faisceau d lectrons et la mati re Figure 47 Analyse en composantes principales bas e sur les concentrations des carot noides des chlorophylles et des terp nols Figure 48 Concentration en carot noides A en chlorophylles B et en terp nols C dans trois g notypes de peuplier Carpaccio Cima et Robusta apr s 2 4 11 15 et 17 jours de traitement ozone ou control Figure 49 Analyse en composantes principales bas e sur les concentrations des compos s ph noliques chez 3 g notypes de peuplier euram ricain Carpaccio en orange Cima en vert et Robusta en bleu soumis ou non un traitement ozone Figure 50 Analyse en composantes principales bas e sur les
339. uve d eau Remettre les plaques sur le robot dans le bon ordre Il va ajouter 10 uL de NEM sur les plaques DTT et 10 uL d eau sur les autres Laisser incuber 1 min temp rature ambiante Ajouter sous hotte 80 uL par puits de r actif A B Attention ne pas trop exposer la lumi re 135 Annexes Laisser incuber 40 min 37 C Lire sur lecteur de plaque 550 nm Les plaques avec le DTT permettent de doser l ascorbate le d hydroascorbate Les plaques sans DTT ne dosent que la forme r duite de l ascorbate Solution pr parer 0 4 M Tampon phosphate pH 7 4 12H50 35 8 g 250 mL d eau di sodium hydrogen orthophosphate 12 hydrate BDH cat No 102 485B b NaH PO 5 52 g 100 mL d eau sodium dihydrogen orthophosphate 1 hydrate BDH cat no 102454R Ajouter b a pour tre pH7 4 se conserve 1 mois 4 C utiliser temp rature ambiante Acide m taphosphorique 5 5 g dans 100 mL d eau se conserve 1 mois 4 C 5 mM DTT agent r ducteur dissoudre 8 mg DTT dithiothreitol dans 10 mL de 0 4 M tampon phosphate pH7 4 Peut tre stock 20 C par aliquots de 1 2 mL ou par 15 mL 0 5 NEM N ethylmaleimide retire le DTT exc s 50 mg NEM dans 10 mL d eau utiliser sous hotte Peut tre stock 20 C par aliquots de 0 6 mL ou par 15 mL R actif color H3PO4 FeCl 18 5 mL d acide orthophosphorique 85 31 5 mL
340. vuo P Peltonen P Kangasj rvi J Auvinen P Paulin L M Oksanen E Vapaavuori E Differential gene expression in senescing leaves of two silver birch genotypes in response to elevated CO2 and tropospheric ozone Plant Cell amp Environment 2010 33 1016 1028 Langebartels C Wohlgemuth H Kschieschan S Gr n S Sandermann H Oxidative burst and cell death in ozone exposed plants Plant Physiology and Biochemistry 2002 40 567 575 Larkin MA Blackshields G Brown NP Chenna R McGettigan PA McWilliam H Valentin F Wallace IM Wilm A Lopez R et al Clustal W and Clustal X version 2 0 Bioinformatics 2007 23 2947 2948 Lawson T Guard cell photosynthesis and stomatal function New Phytologist 2009 181 13 34 Lawson T Oxborough K Morison JI Baker NR Responses of photosynthetic electron transport in stomatal guard cells and mesophyll cells in intact leaves to light CO and humidity Plant Physiology 2002 128 52 62 Leuning R Modelling stomatal behaviour and photosynthesis of Eucalyptus grandis Australian Journal of Plant Physiology 1990 17 159 175 Leuning R A critical appraisal of a combined stomatal photosynthesis model for C3 plants Plant Cell amp Environment 1995 18 339 355 163 R f rences Lindroth RL Impacts of elevated atmospheric CO2 and O3 on forests phytochemistry trophic interactions and ecosystem dynamics Journal of Chemical Ecology 2010 36 2 21 Li
341. was unable to regenerate it efficiently Ascorbate is regenerated by monodehydroascorbate reductase MDHAR and dehydroascorbate reductase DHAR at the expense of NAD P H or GSH 18 Ozone leads to an increase of respiration which can 89 Total amino acids AMBIENT OZONE 200000 150000 sampling dates day 2 4 2 100000 11 50000 CARPACCIO CIMA ROBUSTA CARPACCIOCIMA ROBUSTA Figure 9 Ozone effect on the total amino acid concentration Total amino acids content in leaves of three poplar genotypes Carpaccio Cima and Robusta exposed to ambient or ozone treatment at 2 4 11 15 and 17 days Mean SE umol g DW with alanine d4 as the internal standard R sultats participate in the supply of reducing power when the photosynthesis is decreased 30 As shown by 30 in ozone treated leaves Carpaccio sustains high levels of NAD P H while in Robusta NAD P H levels decreased The inability of Robusta to regenerate its ASA pool in response to ozone was not due to a decrease of GSH It may be linked to the decrease of NAD P H an alteration of NAD P H turnover 30 or an insufficient activity of MDHAR and DHAR The tolerance of Carpaccio to ozone might be related to its capacity to regenerate ASA but must be combined with other factors of tolerance GSH is the reducing co factor for several enzymes involved in ROS detoxification thus playing a central role as an antioxidant but also as a component of cell sign
342. were not linked to variations in Ci values which remained stable data not shown In control air conditions there was no significant genotype effect on photosynthesis Table 6 From the second week whether with high or low VPD increased respiration in the three geno types 352 50 and 80 increase under low VPD 65 90 and 50 increase under high VPD for Carpaccio Cima and Robusta respectively Such increases were only significant for Robusta 4 Discussion 4 1 Effects of ozone on net CO assimilation In our experiments we observed a decrease of stomatal conductance which reduced ozone uptake directly followed by an important decrease in net assimilation As highlighted by Wittig et al 2009 net assimilation reduction can have important effects on plant biomass that are in accordance with the slight decrease in total biomass we observed but it can also result to important ecosystem effects with a decrease of the carbon sink strength In dark conditions respiration was more important under O3 treatment and in the presence of low light when photosynthesis prevailed on respiration for the control plants respiration remained more important than photosynthesis for the plants under treatment When light intensity was sufficient to have higher photosynthesis than respiration for the plants under O3 treatment net CO assimilation was still much lower than in the J Dumont et al Environmental Amb
343. x ANHXI13 Pao m CAX3 gt S 0 05 z TPKI 4 20872 GLUR32 0 00 4GORKI2 hee ALMT610 0 05 1115 4KATI 2 39 16 1 5 1GORK4 0 15 0 20 aNHXI ATPKII 0 25 4CLCa 0 30 0 20 0 15 0 10 0 05 0 00 0 05 0 10 0 15 0 20 0 25 0 30 0 35 0 40 Fig 5 Principal Component Analysis score plot of and PC2 a PC3 and b and the corresponding PCA loading plots c and d of the normalized gene expression in guard cells of the upper upp or lower low sides of leaves of three poplar genotypes Carpaccio orange circles Cima green squares and Robusta blue triangles submitted to ambient A or ozone treatment O after 18 days PC3 10 1 R sultats of CLCa than Cima Fig 5d Cima is mostly characterized by higher expression of TPK11 and lower expression of GLUR3 OST218 and PHOT29 than Carpaccio The differentiation between the two sides is due to some genes preferentially expressed on the lower side of leaves such as AHAII QUACI or CLCa6 except in Robusta GLUR32 is also more expressed on the lower side but only in ambient conditions whereas SLAC and OST2 have higher expression in ozone conditions only on the lower side DISCUSSION Whatever the genotype in response to ozone the total stomatal conductance is lower thus limiting ozone uptake and risk of oxidative stress but also carbon as
344. y but in the evening as stomata may close more slowly the O3 flux may be more important Finally net CO assimilation may be limited by the lower stomatal conductance in the morning and during the day and in the evening the limitation may come from lower light so that the higher conductance has no positive effect 5 Conclusions O3 impacts stomatal conductance by decreasing it but in this study we report specific effects of on poplar responses to four different environmental parameters Our results show that the effect of O3 should be taken into account in the stomatal conduc tance models used for risk assessment and not only as related to phenology The responses of stomatal conductance to variations in blue light red light CO and VPD are directly altered by Os Further studies are needed to characterize these impacts and improve stomatal conductance models like the DO3SE model Moreover to explain how modifies the response to those parameters we need to improve our knowledge of the impact of on the different signalling steps and of the ion channels involved in the stomatal movements due to variations in environmental parameters Acknowledgements The authors are thankful to Cyril Bur St phane Martin Franck Radnai and Aur lie Heinis for technical assistance The research was supported by the French National Research Agency ANR project VMCS Vulnoz and Jennifer Dumont was supported by a PhD grant fr
345. ynthesis 43 This is consistent with our results as the expression of VTC25 the most responsive gene involved in the L galactose pathway correlated with ascorbate accumulation in response to ozone Although we observed an increased expression of VTC416 VTC4162 VTC46 and LGalDHI it has been suggested that a gene expression increase of L Galactose 1 P phosphatase which is involved in both L galactose and animal like pathways 47 is not always correlated with change of activity and that L Galactose dehydrogenase LGalDH exerts little control on ascorbate biosynthesis 43 48 L Galactono 1 4 lactone dehydrogenase GLDH catalyzes the last step of ascorbate biosynthesis by the L galactose pathway and the salvage pathway Although the transcript level of GLDH generally follows the same trend as ascorbate content the effect of increase in GLDH gene expression can vary between species and even between organs and is not always correlated with ascorbate content 49 50 51 In our experiment GLDH16 expression was barely increased in response to ozone and thus cannot explain the increase in ascorbate In conclusion our results suggest that in poplar under ozone stress increased ascorbate levels are associated to a transcriptional induction of ascorbate biosynthesis mainly by genes encoding VTC2 9 R sultats Glutathione de novo synthesis in response to ozone may be linked to increase in GSH1 and GSH2 gen
346. ypes ii The expression of 1115 16 and 0572 is quite strongly repressed in guard cells of both side of leaf in almost all the genotypes iii The expression of 087218 CLCa TPK11 CHL NHX114 CLCa6 GORK and SLACI is globally enhanced especially for 087218 in guard cells of both side of leaf in almost all genotypes iv The expression of genes such as AKT2 GLR25 CA4 KATI 2 PHOT2 QUACI PHOTI and GORK4 is repressed in guard cells of both side of leaf in almost all genotypes As illustrated by the PCA the variance in gene expression in guard cells was explained by 27 15 10 and 8 by four components PC1 PC2 PC3 and respectively Fig 5 PCI shows the ozone effect by separating ambient and ozone samples PC2 isolates the sensitive genotype Robusta from Cima and Carpaccio Fig 5a PC3 separates samples from the two sides of leaf only in Carpaccio and Cima even if the separation is clearer in Carpaccio than in Cima Fig 5b differentiates Cima from Carpaccio The separation is mainly due to genes shown in Fig 4 to be regulated under ozone treatment such as and 057218 and Fig Sc Robusta is principally characterized by a higher expression of PHOT29 and 4 and a lower expression of CHL TPKI CAXI6 and CAX3 than Carpaccio and Cima Carpaccio is essentially typified by higher expression of NHX110 and KAT36 under ambient conditions and a lowe

Rôle de la régulation stomatique et de la capacité de détoxication

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R&ocirc;le de la r&eacute;gulation stomatique et de la capacit&eacute; de d&eacute;toxication

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Rôle de la régulation stomatique et de la capacité de détoxication