Plant Physiology Preview. Published on April 5, 2017, as DOI: /pp

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1 Plnt Physiology Preview. Published on April 5, 217, s DOI:1.114/pp Cytosolic nd chloroplstic DHARs cooperte in oxidtive stress-driven ctivtion of the slicylic cid pthwy Mrie-Sylvine Rhntniin 1, Shengchun Li 1,b, Gilles Chtel-Innocenti 1, Andrée Tuzet 2, Emmnuelle Isskidis-Bourguet 1, Amn Mhmdi 1c, Grhm Noctor 1 1 Institute of Plnt Sciences Pris-Scly (IPS2), UMR 9213/UMR143, Université Pris-Sud, CNRS, INRA, Université d'evry, Université Pris-Diderot, Sorbonne Pris-Cité, Bâtiment 63, 9145 Orsy, Frnce 2 Unité Mixte de Recherche ECOSYS / Pôle BIOCLIMATOLOGIE, INRA AgroPrisTech, Route de l Ferme, F-7885 Thivervl-Grignon, Frnce These uthors contributed eqully to this work b Present ddress : Hubei Collbortive Innovtion Center for Green Trnsformtion of Bio-Resources, College of Life Science, Hubei University, 4362 Wuhn, Chin. c Present ddress : Deprtment of Plnt Systems Biology, VIB, nd Deprtment of Plnt Biotechnology nd Bioinformtics, Ghent University, 952 Ghent, Belgium. Author for correspondence : grhm.noctor@u-psud.fr; FAX 33() Sttistics: Totl word count, excluding references: 6941 Abstrct: 198 words Introduction: 949 words Results: 2125 words Discussion: 218 words Mterils nd methods: 1247 words Min figures: 9 Supplementl Figures: 8 Supplementl Tbles: 6 Running title: Dehydroscorbte reductse in Arbidopsis 1 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved. Copyright 217 by the Americn Society of Plnt Biologists

2 Abstrct The complexity of plnt ntioxidtive systems gives rise to mny unresolved questions. One reltes to the functionl importnce of dehydroscorbte reductses (DHARs) in interctions between scorbte nd glutthione. To investigte this issue, we produced complete set of loss-of-function mutnts for the three nnotted Arbidopsis DHARs. The combined loss of DHAR1 nd DHAR3 expression decresed extrctble ctivity to very low levels but hd little effect on phenotype or scorbte nd glutthione pools in stndrd conditions. An nlysis of the subcellulr locliztion of the DHARs in Arbidopsis lines stbly trnsformed with GFP fusion proteins reveled tht DHAR1 nd DHAR2 re cytosolic while DHAR3 is chloroplstic, with no evidence for peroxisoml or mitochondril locliztions. When the muttions were introduced into n oxidtive stress genetic bckground (ct2), the dhr1 dhr2 combintion decresed glutthione oxidtion nd inhibited ct2-triggered induction of the slicylic cid pthwy. These effects were reversed in ct2 dhr1 dhr2 dhr3 complemented with ny of the three DHARs. The dt suggest tht (1) DHAR cn be decresed to negligible levels without mrked effects on scorbte pools; (2) the cytosolic isoforms re prticulrly importnt in coupling intrcellulr H 2 O 2 metbolism to glutthione oxidtion; (3) DHAR-dependent glutthione oxidtion influences redox-driven slicylic cid ccumultion One sentence summry Arbidopsis mutnts with negligible DHAR ctivity cn mintin wild-type scorbte sttus but decresed oxidtion of glutthione lters downstrem responses to intrcellulr H 2 O 2. Note: While GSH is often used s n bbrevition for (totl) glutthione, this mnuscript uses GSH specificlly to denote the reduced (thiol) form. Where the mening is intended to encompss both GSH nd glutthione disulfide (GSSG), glutthione is used. 2 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

3 Introduction Recent yers hve witnessed n ever-growing focus on the roles of rective oxygen species (ROS) in plnts, with molecules such s H 2 O 2 implicted in numerous developmentl processes nd environmentl responses (Dietz et l., 216). Key questions remin concerning exctly how the signling functions of these rective species re medited (Foyer nd Noctor, 216). When produced inside the cell, the ccumultion of ROS is limited by their rectivity with multiple ntioxidnt systems found in plnts. In the queous phse, such systems include ctlses, scorbte, glutthione, nd peroxiredoxins (Rouhier et l., 22; Tripthi et l., 29; Mhmdi et l., 21; Foyer nd Noctor, 211; Smirnoff, 211; Awd et l., 215). At lest some of the signling effects of ROS my involve secondry chnges in such ntioxidtive systems (Hn et l., 213). Ascorbte nd glutthione re often considered to work together in ROS removl. Following oxidtion, scorbte cn be regenerted through severl rections, only one of which, reduction of dehydroscorbte (DHA), is known to be strongly dependent on glutthione (Foyer nd Noctor, 211; Smirnoff, 211). When the scorbte-glutthione pthwy ws originlly formlized s chloroplst route for ROS processing, DHA reduction ws proposed to be non-enzymtic (Foyer nd Hlliwell, 1976). Although the chemicl rection between GSH nd DHA is rpid, proteins tht ctlyzed GSHdependent scorbte formtion from DHA hve been prtilly purified nd chrcterized from severl plnts, including spinch, rice, nd poplr (Foyer nd Hlliwell, 1977; Hossin nd Asd 1984; Kto et l., 1997; Shimok et l., 2; Lllement et l., 216). A recent survey of txonomiclly diverse species suggests tht plnts contin between two nd four genes encoding DHAR (Zhng et l., 215). In Arbidopsis, three genes tht belong to the glutthione S-trnsferse (GST) superfmily were nnotted to encode DHARs, nd ll three proteins were shown to hve DHAR ctivity in vitro (Dixon et l., 22; Dixon nd Edwrds, 21). Studies using tobcco lines overexpressing or underexpressing DHAR reveled roles for the enzyme in determining stomtl opening, plnt performnce, nd scorbte contents (Chen nd Gllie, 24, 26; Chen et l., 23). Other studies hve reported positive effects of overexpressing vrious DHARs (Kwon et l., 21, 23; Wng et l., 21; Yin et l., 21; Le Mrtret et l., 211; Chng et l., 216). Nevertheless, becuse plnts contin vrious proteins ble to ctlyze GSH-dependent DHA reduction nd becuse the rection cn occur non-enzymticlly t quite high rtes, questions remin surrounding the exct significnce of DHARs. Indeed, kinetic modelling of the chloroplst scorbte-glutthione pthwy suggested tht the chemicl rection could be lrgely predominnt (Polle, 21). For these resons, the physiologicl importnce of DHARs in oxidtive stress hs been 3 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

4 subject to some debte (Morell et l., 1997; Foyer nd Mullineux, 1998; Smirnoff, 211) nd the biologicl roles of the endogenous genes, nd the reltionships between them, remin to be elucidted. While DHA reduction is considered to be importnt to regenerte scorbte, nd therefore prevent its irreversible brekdown (Prsons nd Fry, 212), proteins tht cn oxidize GSH my be importnt in regulting redox signling pthwys (Hn et l., 213, 213b). Glutthione ccumultion, lrgely in the form of GSSG, hs long been known to occur in response to incresed intrcellulr H 2 O 2 (Smith et l., 1984; My nd Lever, 1993) but the extent to which such effects re DHA-dependent is not known (Rhntniin et l., 213). Other systems, such s GSTs or glutredoxin-peroxiredoxins, my couple ROS removl to GSH oxidtion (Rouhier et l., 22; Dixon et l., 29; Tripthi et l., 29). Although the scorbte-glutthione pthwy hs become ccepted s n importnt route for ROS metbolism in plnts, the extent to which the two ntioxidnts interct in vivo remins to be estblished (Noctor et l., 2; Foyer nd Noctor, 211). In Arbidopsis, three genes re nnotted to encode DHAR (Dixon et l., 22). While the encoded proteins my llow DHAR ctivity in both the chloroplst nd cytosol, some studies point to DHAR1 locliztion in the peroxisomes (Dixon et l., 22; Reumnn et l., 29; Greffen et l, 21; Tng nd Yng, 213). To dte, three studies hve ppered using single loss-of-function mutnts. The erliest reported substntil effect on the extrctble ctivity (bout 3% of wild-type) nd effects on ozone resistnce in dhr2 (Yoshid et l., 26). Very recently, much smller decrese ws reported in line lcking DHAR2 function, with most of the ctivity ssocited with DHAR1. Single mutnts for both genes were reported to show modified scorbte nd glutthione redox stte following photooxidtive stress (Noshi et l., 216). A similr study of DHAR3 lso reported decresed ctivity nd ltered responses to photooxidtive stress (Noshi et l., 216b). In this work, we imed to investigte the following distinct but relted questions. Wht re the rections tht re directly responsible for glutthione oxidtion during oxidtive stress? To wht extent re scorbte nd glutthione redox-coupled in optiml nd oxidtive stress conditions? How importnt re the different genes encoding Arbidopsis DHAR? To nswer these questions, we generted complete set of single, double, nd triple loss-of-function mutnts for the three Arbidopsis DHARs, nd nlyzed the locliztion of the three proteins in prllel. We lso introduced ll mutnt combintions into ctlse-deficient bckground (ct2) to produce double, triple nd qudruple mutnt lines. This pproch ws chosen becuse decresed ctlse ctivity forces H 2 O 2 metbolism through lterntive systems tht re dependent on cellulr reductnts 4 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

5 (Mhmdi et l., 21). Our nlysis of the dhr mutnt lines, grown in prllel in identicl conditions, shows tht DHAR ctivity cn be decresed to negligible levels without mrked effects on plnt growth or the scorbte pool. However, the three DHARs hve prtly redundnt roles in ensuring glutthione oxidtion when H 2 O 2 metbolism is enhnced, nd their combined bsence in these conditions impcts plnt phenotypes nd pthogenesis-relted (PR) signling. 5 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

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7 Results To nlyze the roles of the three DHAR genes, we obtined Arbidopsis T-DNA mutnts with insertions in the corresponding coding sequences. Homozygous lines were redily obtined for dhr muttions nd RT-PCR confirmed tht they were ll knockouts or severe knockdowns (Supplementl Fig. S1, left hlf). Loss of DHAR1 or DHAR3 function decresed extrctble lef ctivity by bout 5% while the effect of the dhr2 muttion ws much less mrked (Fig. 1A). From these lines, three double mutnts were produced. The dhr1 dhr2 double mutnt showed similr ctivity to dhr1 while the dhr1 dhr3 combintion decresed DHAR ctivity to 5% of the wild-type (Fig. 1A). Combining the dhr2 nd dhr3 muttions led to n increse in DHAR ctivity compred to the dhr3 single mutnt, pointing to compenstory effect tht ws presumbly linked to DHAR1 becuse triple mutnt, in which ll three genes were mutted, showed negligible ctivity (Fig. 1A). Functionl impct of dhr muttions in optiml nd oxidtive stress conditions When grown in stndrd conditions, none of the single, double, or triple mutnts showed ny difference in phenotype compred to Col- (Fig. 1B; Supplementl Fig. S2). Similrly, lef scorbte nd glutthione pools remined t wild-type levels nd reduction sttes (Fig. 1C,D; Supplementl Tble S1). As first test of the role of ech DHAR in stress responses, mutnts were grown in vitro in gr contining stressful concentrtions of cdmium, mnnitol, slt, or prqut, nd root growth ws mesured (Supplementl Fig. S3). Loss of DHAR function cused slight increse in cdmium sensitivity, which ws more evident t low concentrtions, nd which ws pprent even in the three single mutnts. Growth on mnnitol ws not substntilly ffected by the muttions but dhr1 dhr2, dhr1 dhr3, nd the triple mutnt showed somewht enhnced sensitivity to NCl (Supplementl Fig. S3). All three single muttions enhnced sensitivity to the oxidtive stress cused by prqut, lthough combintion of the muttions did not reinforce this effect (Supplementl Fig. S3). Tken together, the bove results suggest tht none of the three DHARs is required for plnt development but tht they my interct in complex mnner to determine stress resistnce. To explore this point further, the three muttions were introduced into ctlse-deficient bckground in which enhnced intrcellulr H 2 O 2 vilbility increses the demnd on reductnts such s scorbte nd glutthione (Mhmdi et l., 21). Thus, we produced double ct2 dhr1, ct2 dhr2, 7 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

8 nd ct2 dhr3 mutnts, nd from these lines produced three triple nd one qudruple mutnt (Supplementl Fig. S1, right). Anlysis of extrctble lef DHAR in ct2 reveled tht the ctivity ws incresed in response to the oxidtive stress cused by ctlse deficiency (Fig. 2A, top). Anlysis of double ct2 dhr mutnts suggested tht DHAR1 ws responsible for most of the DHAR ctivity in the ct2 bckground, nd tht the reminder ws lrgely due to DHAR3. Activities in ct2 nd ct2 dhr2 were similr (Fig. 2A, top), suggesting tht, s in the Col- bckground, DHAR2 mkes minor contribution to the extrctble lef ctivity. Consistent with this, no ctivity ws detected in ct2 dhr1 dhr3 or the qudruple ct2 dhr1 dhr2 dhr3 mutnt (Fig. 2A, top). As previously reported, the ct2 mutnt showed only bout 2% of wild-type lef ctlse ctivity (Quevl et l., 29) nd this ws not ffected by ny of the combintions of dhr muttions (Fig. 2, bottom). The ctivities of two other enzymes of the scorbte-glutthione pthwy, glutthione reductse (GR) nd scorbte peroxidse (APX), were incresed in ct2 but the presence of the dhr muttions hd little effect on either (Fig. 2A, middle pnels). The dhr muttions did not ffect rosette mss in the ct2 bckground (Fig. 2B, bottom) but two mutnt combintions significntly impcted the ct2-triggered phenotype (Fig. 2B, top). While ct2 nd most of the derived ct2 dhr lines showed clerly visible hypersensitive response (HR)-like lesions on specific leves, these were much less pprent in ct2 dhr1 dhr2 nd ct2 dhr1 dhr2 dhr3 (Fig.2B, top). Geneticlly or phrmcologiclly induced ctlse deficiency cuses ccumultion of glutthione (Smith et l., 1984; My nd Lever, 1993; Willekens et l., 1997; Mhmdi et l., 21; Quevl et l., 211). Accordingly, lef glutthione ws bout 2-fold higher in ct2 thn in Col-, with the difference being lmost entirely due to GSSG (Fig. 3, bottom). As result, the glutthione reduction stte, which ws bove 9% in Col-, ws only 64% in ct2 (Supplementl Tble S2). Glutthione ws significntly less oxidized in the triple ct2 dhr1 dhr2 nd qudruple ct2 dhr1 dhr2 dhr3 mutnts (Supplementl Tble S2), nd this ws ccompnied by decresed totl glutthione in these two lines (Fig. 3, bottom). All other ct2 dhr lines showed similr glutthione sttus to ct2. As previously reported, scorbte is less ffected in ct2 thn is glutthione (Mhmdi et l., 21, 21b; Hn et l., 213). No difference in totl scorbte contents ws observed in ny of the mutnts reltive to Col- (Fig. 3, top). However, severl lines in the ct2 bckground showed slightly but significntly more oxidized scorbte pool s result of the presence of dditionl muttions in dhr (Supplementl Tble S2). 8 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

9 21 Subcellulr locliztion of the three DHARs Fusion proteins were constructed for ll three DHARs with the GFP tg fused C- or N-terminlly (DHAR-GFP or GFP-DHAR, respectively). The six constructs were trnsformed into the qudruple ct2 dhr1 dhr2 dhr3 nd the triple dhr1 dhr2 dhr3 mutnt bckgrounds, nd T1 plnts were nlyzed. Despite repeted ttempts, we were not ble to regenerte plnts expressing GFP-DHAR1 in either the ct2 dhr1 dhr2 dhr3 or dhr1 dhr2 dhr3 bckgrounds. DHAR1-GFP showed cytosolic locliztion in the leves of the qudruple mutnt bckground (Fig. 4). A similr locliztion ws lso observed for DHAR1-GFP in the roots of this line (Supplementl Fig. S4) nd in the lef cells of the dhr1 dhr2 dhr3 triple mutnt bckground (Supplementl Fig. S5). Fluorescence ptterning did not overlp with the positive control for peroxisoml locliztion (Supplementl Fig. S6) DHAR2-GFP nd GFP-DHAR2 fusions showed similr fluorescence ptterns to the negtive GFP control, confirming cytosolic locliztion in the leves of both genetic bckgrounds (Fig. 4; Supplementl Fig. S5). Both DHAR2 fusions lso gve cytosolic signl in the roots (dt not shown). In contrst to DHAR1 nd DHAR2, DHAR3-GFP ws ssocited with the chloroplst. The DHAR3-GFP signl showed close overlp with chlorophyll utofluorescence in the leves of both the complemented qudruple nd triple mutnts (Fig. 4; Supplementl Fig. S5). To estblish whether DHAR3 might lso be trgeted to the mitochondri, we compred GFP fluorescence with mitotrcker signls nd with positive control for dul chloroplst-mitochondrion trgeting (PDF1B-GFP) in roots. Mitotrcker fluorescence reveled no overlp with the DHAR3-GFP signl (Supplementl Fig. S4). Wheres PDF1B-GFP fluorescence ptterns were observed in both the lrger plstids nd the smller punctte mitochondri, DHAR3-GFP signls were only observed in plstids (Supplementl Fig. S4). When the GFP ws fused to the DHAR3 N terminus (GFP-DHAR3), no ssocition with the chloroplst ws observed; insted diffuse pttern of stining ppered (Fig. 4; Supplementl Fig. S5), consistent with trgeting of the ntive DHAR3 to the chloroplst through n N terminl signl peptide sequence. In conclusion, the dt suggest tht DHAR1 nd DHAR2 re cytosolic while DHAR3 is loclized in plstids. This distribution ws not ffected by the presence of the ct2 muttion. Functionl complementtion of the ct2 dhr1 dhr2 dhr3 qudruple mutnt To further evlute the importnce of the three DHARs in oxidtive stress responses, we nlyzed the bility of DHAR-GFP fusion proteins for ech of the three DHARS to complement extrctble ctivity, glutthione contents, nd phenotypes in T2 individuls of the ct2 dhr1 dhr2 dhr3 mutnt. For ech DHAR, two independent lines were obtined tht showed 3:1 segregtion for 9 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

10 ntibiotic resistnce in T2 (Supplementl Tble S3). In resistnt DHAR1-GFP nd DHAR3-GFP plnts, DHAR ctivity ws restored to bout 5% of the vlue in ct2 (Fig. 5). No increse in extrctble DHAR ctivity ws detected in ntibiotic-resistnt individuls from the qudruple mutnt complemented with DHAR2-GFP (Fig. 5). Complementtion of ct2 dhr1 dhr2 dhr3 plnts with DHAR1 or DHAR3 cused significnt increses in GSSG (Fig. 6,B). As result, the glutthione sttus of DHAR1-GFP+ nd DHAR3-GFP+ plnts ws more similr to the single ct2 mutnt thn to the prent qudruple mutnt line (Fig. 6, B). No effect on scorbte ws observed (Fig. 6,A). Despite the lck of detectble increse in extrctble ctivity in DHAR2-GFP+ complemented lines, introduction of this protein lso incresed glutthione contents nd decresed the glutthione reduction stte (Fig. 6D). However, the effect ws somewht different from tht produced by introduction of DHAR1 or DHAR3. Complementtion with DHAR2 cused glutthione to increse bove ct2 levels nd to remin more reduced thn in ct2, lbeit less reduced thn in the uncomplemented qudruple mutnt line (Fig.6D). Agin, no effect on scorbte ws observed (Fig. 6C). Complementtion with DHAR1, DHAR2 or DHAR3 restored the ct2 lesion phenotype in the ct2 dhr1 dhr2 dhr3 qudruple mutnt. Two experiments with different btches of T2 seed reveled 3:1 phenotype segregtion (Supplementl Tble S4). In first step to nlyze this effect more closely, lesions were quntified using imging softwre. Quntifiction in Col- nd the eight ct2 dhr lossof-function lines showed tht ll lines crrying the ct2 muttion presented lesions on bout 15% of the totl rosette surfce, except the two lines in which DHAR1 nd DHAR2 functions were both lost (ct2 dhr1 dhr2 nd ct2 dhr1 dhr2 dhr3; Fig. 7A). The ct2-dependent lesions could be fully restored by complementtion of the qudruple mutnt with ny of the three DHARs (Fig. 7B). The role of the different DHARs in oxidtive stress linked to ctlse deficiency ws further tested using 3-mino-1,2,4-trizole (3-AT). Like the ct2 muttion, ctlse inhibition by this herbicide engges GSH-dependent processes, which become pprent s ccumultion of GSSG (My nd Lever, 1993). Sprying with 3-AT cused bleching phenotype within two dys in the wild-type (Supplementl Fig. S7). The ppernce of this phenotype ws inhibited in dhr1 nd, more clerly, in dhr1 dhr2 nd dhr1 dhr3 double mutnts, s well s the triple dhr1 dhr2 dhr3 mutnt line (Fig. 7C; Supplementl Fig. S7). Similr to effects observed in the ct2 bckground (Fig. 3), 3-ATinduced ccumultion of GSSG ws significntly decresed in dhr1 dhr2 nd, especilly, in dhr1 dhr2 dhr3 (Fig. 7D). Consequently, glutthione remined significntly more reduced in these lines thn in the wild-type treted with 3-AT (Supplementl Tble S5). 1 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

11 Roles of DHARs in oxidtive stress-triggered ctivtion of the slicylic cid pthwy nd cell deth To explore the processes underlying the phenotypic effects described bove, we nlyzed the effect of the three dhr muttions on trnscripts of key gene involved in SA synthesis (ICS1) nd two SAdependent pthogenesis-relted genes (PR1, PR2). All three genes were mrkedly induced in response to oxidtive stress in ct2 but less so in the qudruple ct2 dhr1 dhr2 dhr3 line (Fig. 8). In the Col- bckground, the triple loss of DHAR function lso decresed bsl levels of PR1 nd PR2 trnscripts lthough ICS1 trnscripts were slightly incresed (Fig. 8). In second pproch, we profiled the lesion phenotype of the leves from Col- nd ll eight lines contining ct2 nd dhr muttions in combintion. In the ct2 single mutnt, cell deth ws pprent on the oldest leves, both s whitish lesions nd trypn blue-positive stining (Fig. 9A). Similr phenotypes to ct2 were observed in ct2 dhr1, ct2 dhr2, ct2 dhr3, ct2 dhr1 dhr3, nd ct2 dhr2 dhr3 (Fig. 9A). By contrst, lesions were restricted to one or two of the very oldest leves in ct2 dhr1 dhr2 nd the qudruple mutnt (Fig. 9A). Hence, cler phenotypic difference ws pprent between these two lines nd the others crrying the ct2 muttion, prticulrly on leves four nd five (Fig. 9A). Mesurements of glutthione specificlly in these leves confirmed the effect of the combined dhr1 nd dhr2 muttions on ct2-triggered effects on glutthione (Fig. 9B). Anlysis of slicylic cid (SA) in the sme leves reveled tht while ll other ct2-crrying genotypes ccumulted SA to round 2 µg.g -1 FW in leves four nd five, SA remined t close to wild-type levels in ct2 dhr1 dhr2 nd in the qudruple mutnt (Fig. 9C). To ssess the significnce of this effect, the qudruple mutnt ws selected for n nlysis of resistnce to virulent bcteri. While bcteril growth ws substntilly lower in ct2 thn in the wild-type, the simultneous presence of the three dhr muttions cused prtil loss of this induced resistnce (Supplementl Fig. S8). Although no difference in bcteril growth ws observed between Col- nd dhr1 dhr2 dhr3, SA ccumulted somewht less in response to infection in the triple mutnt (Supplementl Fig. S8). Similrly, the combined loss of the three DHAR functions decresed SA ccumultion following infection of the qudruple mutnt compred to ct2 (Supplementl Fig. S8). 11 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

12 39 12 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

13 Discussion The present study ws conducted with the im of resolving severl outstnding questions relted to the importnce of genes encoding DHAR nd the functioning of the scorbte-glutthione pthwy in H 2 O 2 processing. From prllel nlysis of complete set of Arbidopsis mutnts for the three DHAR-encoding genes, together with trnsformed lines complemented with ech DHAR, we re led to the following conclusions. Almost ll of the extrctble DHAR ctivity in Arbidopsis leves is ttributble to cytosolic DHAR1 nd chloroplstic DHAR3 In ddition to the chloroplst nd cytosol, DHAR ctivity hs been reported in purified pe peroxisomes nd mitochondri (Jiménez et l., 1997). Trgeting experiments in Arbidopsis hve estblished tht these orgnelles contin APX, MDHAR, nd GR, in some cses s result of dultrgeting with the chloroplst or cytosol (Chew et l., 23; Kty nd Reumnn, 21). Although DHAR1 does not contin known peroxisoml trgeting sequence, it ws ssigned locliztion in this orgnelle on the bsis of proteomics nlyses nd experiments with yellow fluorescent protein (YFP) fused to the DHAR1 C terminus (Reumnn et l., 29). For unknown resons, we were unble to recover GFP-DHAR1 plnts. However, we could not find ny evidence tht the protein is found in orgnelles from nlysis of lines trnsformed with constructs with GFP fused to the DHAR1 C terminus. Our dt suggest tht DHAR1 is cytosolic protein, in greement with nother study using YFP (Grefen et l., 21). If so, two of the Arbidopsis DHARs would be cytosolic, s DHAR2 ws clerly locted in this comprtment, whether the GFP ws fused N- or C-terminlly. A pe gene encoding GR ws the first to be shown to encode trgeting sequences directing dul locliztion in the chloroplst nd mitochondri (Creissen et l., 1995). This observtion ws confirmed in Arbidopsis, where APX, MDHAR, nd DHAR ctivities were lso detected in mitochondri (Chew et l., 23). In the present study, DHAR3-GFP signls were clerly ssocited with the chloroplst but not the mitochondri. Our study therefore suggests tht the lef DHAR ctivity in Arbidopsis is distributed pproximtely eqully between the cytosol nd chloroplst, linked minly to the ctivities of DHAR1 nd DHAR3, respectively. A minor contribution to the cytosolic ctivity would come from DHAR2. This is consistent with two recent reports on single dhr mutnts (Noshi et l., 216,b) but in contrst to n erlier nlysis of dhr2 (Yoshid et l., 26). 13 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

14 The subcellulr distribution we report for the Arbidopsis DHARs is consistent with erlier predictions (Dixon et l., 22), s well s informtion vilble for the three poplr genes (Tng nd Yng, 213). Locliztion of DHARs ws recently compred in Ze mys, Pice bies, nd Selginell moellendorffii (Zhng et l., 215). All three species contined one gene encoding chloroplstic DHAR nd either one or two genes encoding cytosolic isoforms, with fourth gene in mize coding for protein loclized in the vcuole (Zhng et l., 215). Interestingly, both of the DHARs we found to be cytosolic were identified in proteomics study of proteins ssociting with the plsm membrne, long with severl other GSTs nd peroxidses (Mrmgne et l., 27). Proteins other thn the three DHARs, such s certin glutredoxins or GSTs (Chew et l., 23; Dixon et l., 29), my be responsible for DHAR ctivities previously reported to be ssocited with the peroxisomes nd mitochondri. However, very low residul ctivities were detected in both the dhr1 dhr2 dhr3 triple nd ct2 dhr1 dhr2 dhr3 qudruple mutnts (Figs 1 & 2), indicting tht other proteins probbly hve reltively low cpcities. Arbidopsis cn mintin lef scorbte pools nd redox sttes when DHAR ctivity is negligible, even in conditions of oxidtive stress In greement with our previous nlyses, lef scorbte ws highly reduced in Arbidopsis Col- (bout 9%). A high scorbte reduction stte is predicted if most of the pool is found in comprtments tht contin non-limiting regenerting systems dependent on more powerful reductnts, such s ferredoxin, NAD(P)H, nd glutthione (Foyer nd Noctor, 211, 216). Previous studies in tobcco hve implicted DHAR s mjor plyer determining scorbte redox stte nd contents. A DHAR-silenced tobcco line with decrese of bout 7% in lef ctivity hd more oxidized scorbte pool while scorbte ws more reduced in strong overexpressors (Chen et l., 23; Chen nd Gllie, 24, 26). The Arbidopsis genotypes reported here show decreses in DHAR ctivity from 1% to 1% s result of gene-specific loss of function. In strk contrst to the studies in tobcco, we observed no mrked effects on scorbte contents or redox stte. Significntly enhnced oxidtion, reltive to ct2, ws observed in some of the ct2 dhr lines but effects were slight, with scorbte remining bout 8% reduced (Supplementl Tble S2). We cnnot discount role for DHARs in mintining scorbte in other conditions. For exmple, chnges in scorbte redox stte were reported in dhr1 nd dhr2 single mutnts subjected to high light (Noshi et l., 216, 216b). However, these effects were reltively slight. Our nlysis, in which ll combintions of the three dhr muttions were exmined, provide little evidence for mjor role for DHARs in mintining scorbte pools in Arbidopsis grown in moderte light with or without intrcellulr oxidtive stress. The bility to mintin lrgely reduced lef scorbte pool when DHAR ctivity is 14 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

15 negligible my be explined by the presence of other routes tht cn either reduce DHA, or else void its formtion by reducing MDHA. Such mechnisms notbly include non-enzymtic reduction of DHA by GSH nd reduction of MDHA by chloroplst ferredoxin or by NAD(P)H-dependent reductses (Polle, 21; Smirnoff, 211; Gest et l., 213; Johnston et l., 215; Noctor, 215). DHARs cooperte in glutthione oxidtion triggered by oxidtive stress Although complete loss of DHAR function in dhr1 dhr2 dhr3 did not gretly ffect scorbte, it clerly impcted the responses to oxidtive stress triggered by ctlse deficiency. Accumultion of GSSG is the clerest nd most redily quntifible biochemicl mrker of ltered redox stte cused by decresed cpcity for removl of intrcellulr H 2 O 2 (Willekens et l., 1997; Quevl et l., 29, 211). By comprison, reproducible increses in H 2 O 2 itself re much more difficult to detect in ct2 (Mhmdi et l., 21b, Noctor et l., 215). This suggests tht, when ctlse cpcity is decresed, lterntive H 2 O 2 -reducing rections re redily elicited, dmpening increses in H 2 O 2 nd insted leding to oxidtion of key redox buffers such s glutthione. Exctly how this occurs remined uncler: DHARs re only one of severl clsses of GSH-dependent enzymes tht could potentilly be involved in H 2 O 2 processing, nd genes encoding severl of these enzymes cn be up-regulted longside GSSG ccumultion (Mhmdi et l., 21b; Rhntniin et l., 213). Given the differentil oxidtion of glutthione compred to scorbte, one simple interprettion would be tht GSH is coupled to H 2 O 2 metbolism vi pthwys tht re scorbte-independent, such s GSHdependent peroxidses. Our dt provide in plnt evidence ginst this interprettion, becuse they show tht t lest one DHAR must be functionl in order to observe significnt oxidtion of glutthione when H 2 O 2 vilbility is incresed. If ll three DHARs re knocked out, GSSG ccumultion, whether induced phrmcologiclly or geneticlly, is lmost completely inhibited (Figs 6-7). Of the three double loss-of-function combintions, the clerest effect on glutthione sttus ws observed for dhr1 dhr2. This underscores the potentil importnce of the cytosolic isoforms in coupling scorbte nd glutthione pools, nd is consistent with previous studies demonstrting the importnce of cytosolic APX1 or GR1 in ct2-triggered responses (Mhmdi et l., 21b; Vnderuwer et l., 211) The importnce of the cytosol in this context my be relted to the peroxisoml loction of the H 2 O 2 tht becomes vilble when ctlse is deficient, lthough decresed glutthione oxidtion ws lso reported for single dhr1 nd dhr2 mutnts fter exposure to high light (Noshi et l., 216). DHAR3 my be more significnt in chloroplstic processing, lthough our dt provide little evidence tht this isoform plys specific role in 15 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

16 response to prqut-induced oxidtive stress, which lrgely origintes in this orgnelle (Supplementl Fig. S3). Intriguingly, lthough glutthione ws only mrginlly more oxidized in ct2 dhr1 dhr2 thn ct2 dhr1 dhr2 dhr3, the chloroplstic DHAR3 ws just s effective s the cytosolic DHAR1 in restoring ct2 glutthione sttus (Fig. 6). Differences between ct2 dhr1 dhr2 dhr3 DHAR3+, in which glutthione sttus ws quite similr to ct2 (Fig.6) nd ct2 dhr1 dhr2, in which GSSG ccumultion ws somewht lower (Fig. 3), my reflect differences in DHAR3 expression levels. In ny cse, the bility of chloroplstic DHAR3 to complement the qudruple mutnt rises questions bout the potentil importnce of redox exchnge cross the inner envelope membrne. Chloroplsts hve long been known to be competent in GSSG nd DHA uptke (Anderson et l., 1983) nd, more recently, chloroplst envelope glutthione nd scorbte trnsporters hve been identified in Arbidopsis (Mughn et l., 21; Miyji et l., 214). While our dt provide in plnt evidence tht DHARs re importnt in coupling scorbte nd glutthione pools during oxidtive stress, they lso identify importnt questions for further study. The first reltes to why scorbte stys lrgely reduced even when glutthione is mrkedly oxidized, nd why DHAR function cn ffect glutthione but not scorbte. Severl fctors my explin these observtions. As discussed bove, GSH-independent mechnisms exist for regenerting scorbte, notbly from MDHA, implying tht reltively minor prt of scorbte regenertion is linked to glutthione oxidtion (Polle, 21). Another importnt fctor reltes to differences in comprtmenttion of the two ntioxidnts. In mny cell comprtments, glutthione nd scorbte re highly reduced under most conditions (Meyer et l., 27; Noctor et l., 216), with DHA nd GSSG ccumulting t specific loctions. Much of the GSSG ccumulted during oxidtive stress is found in the vcuole, thus llowing it to escpe reduction by GR (Quevl et l., 211). We re currently investigting the processes responsible for this distribution, which my be significnt in t lest two respects. First, it could be crucil to llow GSSG generted in comprtments such s the cytosol to be useful s stble tissue mrker of oxidtive stress. Second, it my contribute to homeostsis of the NADP(H)-glutthione redox couples, nd therefore influence cellulr redox signling. The second question reltes to the exct biochemicl function of DHAR2. We found little genetic evidence tht this protein is ble to ctlyze GSH-dependent conversion of DHA to scorbte. Extrctble DHAR ssys reveled little effect of either the dhr2 muttion or of complementtion with DHAR2. This is surprising becuse the recombinnt DHAR2 hs been shown to be competent in GSH-dependent reduction of DHA, lbeit with lower specific ctivities compred to DHAR3 nd, prticulrly, the other cytosolic isoform, DHAR1 (Dixon et l., 22). Our study suggests tht DHAR2 16 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

17 is ctive in GSH oxidtion in plnt, lthough we note tht complementtion of the ct2 dhr1 dhr2 dhr3 mutnt with this protein produced different glutthione signture from tht observed when the mutnt ws complemented with DHAR1 or DHAR3. Further work will be required to elucidte this question, lthough we tenttively suggest tht DHA is not the only physiologicl cceptor substrte for DHAR2-dependent oxidtion of GSH. This might explin why DHAR2 cn contribute to glutthione oxidtion in vivo even though the ssocited DHAR ctivity, mesured in vitro, ppers to be negligible. Compromised DHAR ctivity uncouples oxidtive stress from ctivtion of the slicylic cid pthwy Loss of CAT2 function induces SA ccumultion in the bsence of pthogens nd ssocited pthogenesis-relted responses, including resistnce to bcteri (Chouch et l., 21, 212). Induction of these responses is influenced by glutthione sttus (Mhmdi et l., 21b; Hn et l., 213). Here, we show tht when glutthione remins close to wild-type sttus (in ct2 dhr1 dhr2 or the qudruple mutnt), much of the SA response is bolished (Figs 8 nd 9). Thus, coopertion between the DHARs, nd prticulrly the cytosolic forms, is required to couple oxidtive stress to the ctivtion of downstrem signling. The importnce of the cytosolic isoforms could be linked to their prtil recruitment to the plsm membrne (Mrmgne et l., 27), possibly to set pproprite conditions for redox signling t this loction. However, the close correltion between glutthione nd SA contents (Fig. 9) suggests tht the sttus of this key cellulr thiol/disulfide compound my lso be n importnt fctor. If so, this would dd to dt showing tht SA-relted signling is compromised in mutnts for chloroplst-cytosol glutthione exchnge (Mughn et l., 21) nd in ct2 lines in which glutthione synthesis is prtilly blocked (Hn et l., 213). It lso provides further evidence tht the ctivtion of H 2 O 2 -triggered signling inside the cell depends on secondry modultion of ntioxidnt sttus, nd notbly tht of glutthione. In conclusion, our study found no evidence for n essentil role for ny single DHAR but provided direct in plnt evidence tht coupling between scorbte nd glutthione is functionlly importnt in H 2 O 2 metbolism. DHARs seem to be dispensble for mintennce of lef scorbte, even in stressful conditions. Rther, one function of these proteins my be to djust intrcellulr glutthione sttus in oxidtive stress conditions to ensure pproprite ctivtion of signling through downstrem pthwys such s those involving SA. If so, these enzymes might more ppropritely be nmed glutthione dehydrogenses, rther thn dehydroscorbte reductses. 17 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

18 Mterils nd methods Plnt mterils nd mutnt chrcteriztion All the Arbidopsis mutnts used in this study were in the Columbi genetic bckground, nd ll seeds were obtined from the Nottinghm Arbidopsis Stock Centre ( Lines crrying T-DNA insertions in the DHAR1 (At1g1957), DHAR2 (At1g7527) nd DHAR3 (At5g1671) genes were identified using insertion mutnt informtion obtined from the SIGnAL Web site ( While T-DNA lines for DHAR1 nd DHAR2 were obtined from the SALK collection (SALK_5238 nd SALK_2689 respectively), the T-DNA line for DHAR3 ws from the SAIL collection (SAIL_435_A9). The ct2 mutnt ws ct2-1 (Quevl et l., 29). From these lines, the following resources were produced by crossing: dhr1 dhr2, dhr1 dhr3, dhr2 dhr3, dhr1 dhr2 dhr3, ct2 dhr1, ct2 dhr2, ct2 dhr3, ct2 dhr1 dhr2, ct2 dhr1 dhr3, ct2 dhr2 dhr3 nd ct2 dhr1 dhr2 dhr3. After verifiction of heterozygotes in F1 plnts by PCR, double, triple nd qudruple homozygotes were identified similrly in the F2 genertion nd llowed to produce F3 seeds, which were used for experiments. Expression nlyses were performed by semiquntittive RT-PCR. Totl RNA ws extrcted with TRIzol regent (Invitrogen) following the mnufcturer s instructions. Reverse trnscription nd first-strnd cdna synthesis were performed using the SuperScript III First-Strnd Synthesis System (Invitrogen). ACTIN2 trnscripts were mesured s loding control. Primer sequences re listed in Supplementry Tble S6. Plsmid constructs nd plnt trnsformtion Full-length cdna clones of Arbidopsis DHARs were obtined from the RIKEN BioResource Center Arbidopsis full-length cdna collection ( The cdna clones encoding DHAR1 (RAFL19-3-N19), DHAR2 (RAFL9-83-E23), nd DHAR3 (RAFL19-84-K2) were moved s SlI-NotI frgments into the vector pentr2b (Invitrogen) nd sequenced, using primer pirs listed in Supplementry Tble S6. An LR rection ws then used to trnsfer cdnas from the resulting entry vectors to the pproprite gtewy binry destintion vectors fusion ccording to the mnufcturer s instructions (Invitrogen). The pb7wgf2 vector ws used for N-terminl GFP fusion nd the ph7fwg2 for C-terminl GFP fusion (Krimi et l., 22). In these constructs, the expression of DHAR1, DHAR2 or DHAR3 ws driven by the 35S promoter of the Culiflower mosic virus. Purified plsmids were nlyzed nd sequenced to confirm successful fusion constructs. Agrobcterium tumefciens strin GV311 pmp9 (Koncz nd Schell, 1986) ws trnsformed with confirmed binry vector constructs (Höfgen nd Willmitzer, 1988) nd used to trnsform developing florl tissues of 4-week-old 18 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

19 Arbidopsis triple dhr1 dhr2 dhr3 nd qudruple ct2 dhr1 dhr2 dhr3 mutnts plnts using the florl dip method (Clough nd Bent, 1998). Subcellulr locliztion nd genetic complementtion 1 dy-old seedlings of the triple dhr1 dhr2 dhr3 nd qudruple ct2 dhr1 dhr2 dhr3 mutnts were stbly trnsformed with the DHAR1-GFP or GFP-DHAR1, the DHAR2-GFP or the GFP-DHAR2 nd the DHAR3-GFP or the GFP-DHAR3. Trnsformed T1 seeds were germinted on gr nd identified bsed on their resistnce to hygromycin or were screened by using Leic (Deerfield, IL) dissecting microscope equipped with mercury lmp nd epifluorescence filter set (Krimi et l., 22). Resistnt nd PCR-positive trnsgenic plnts were trnsferred to growth chmber nd mintined up to the T2 genertion, when segregtion nlysis ws performed on complemented ct2 dhr1 dhr2 dhr3 lines. Two independent lines were selected nd used for nlysis of enzyme ctivities, glutthione, nd scorbte. To determine subcellulr locliztion, lef sections from T1 trnsformed plnts were mounted onto slide in wter nd imging ws performed with Leic SP8 scnning confocl microscope using 63x PLAN APO oil immersion objective (Numericl Aperture, 1.4). GFP ws excited with 488 nm lser nd fluorescence ws detected in 493 nm to 53 nm detection window. As chloroplst mrker, chlorophyll utofluorescence ws excited t 633 nm nd detected t nm. As peroxisome nd dul chloroplst-mitochondrion mrkers, lines expressing GFP-MFP2 (A5 line; Cutler et l., 2) or PDF1B-GFP (Giglione et l., 2) were used. To check for possible mitochondril locliztion, trnsformnts were stined with the mitochondrion-selective probe MitoTrcker Red (fluorescent dye; CMTMRos; Invitrogen) following the instruction mnul. Fluorescence ws excited with 578 nm lser nd detected in 582 nm to 69 nm detection window. Both detection chnnels were recorded using hybrid detectors (Hmmtsu) with sequentil cquisition to void ny crosstlk. Plnt growth nd stress tretments For plnts grown on soil, seeds were first incubted for 2 dys t 4 C then trnsferred to controlled-environment growth chmber in 16 h photoperiod nd n irrdince of 2 μmol. m 2 s 1 t lef level, 2 C/18 C, 65 % humidity, nd given nutrient solution twice per week. Unless otherwise stted, plnts were smples fter three weeks growth. Smples were rpidly frozen in liquid nitrogen nd stored t 8 C un l nlysis. All dt re mens ± SE of t lest three biologicl replictes obtined from different plnts, nd experiments were repeted t lest twice. 19 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

20 For in vitro stress tretments, seeds were sown on commercilly vilble.5 MS medium (Bslt slt mixture M221; Duchef) solidified with.8% gr (Phyto-Agr HP696; Klys) nd grown in 16 h light, 8 h night, 2 C growth chmber without sugr. Plnts were grown verticlly for 1 dys on medi supplemented with, 1, or 5 μm CdCl 2 ; 5 or 1 mm NCl ; 1 or 2 mm mnnitol nd.1 or.1 µm prqut. For 3-AT tretment, seeds were sown on soil s described bove. After 3 weeks, plnt leves were spryed once with 2 mm 3-AT, then lef smples were collected fter 48 h nd immeditely frozen with liquid nitrogen for further nlysis. To ssess resistnce to virulent bcterium, plnts grown in soil s bove were inoculted with Pseudomons syringe pv. tomto (Pto) strin DC3 s in Chouch et l. (212) using bcteril titer of 1 6 colony forming units (cfu) ml -1. Smples were tken for counting immeditely fter infiltrtion ( h) or 48 h lter. Qudruplicte biologicl smples, ech of two lef discs, were used. Lesion quntifiction, trypn blue stining, nd pthogen tests Percentge lesion re in ct2, or bleching induced by 3-AT tretment, ws quntified using IQmterils softwre. For trypn blue stining, rosette leves were infiltrted three times under vcuum with lctophenol trypn solution (2.5 mg/ml trypn blue, 25 % lctic cid, 23 % wter sturted phenol, 25 % glycerol, nd wter). Smples were then wshed with distilled wter nd heted over boiling wter. After cooling, smples were clered with chlorl hydrte solution (8 g of chlorl hydrte dissolved in 2 ml of glycerol (5 %) nd 1 ml of wter). The solution ws replced severl times. Smples were mounted in chlorl hydrte solution nd observed with mcroscope (Nikon AZ1 Multizoom). Trnscript quntifiction Totl RNA ws extrcted with TRIzol (Invitrogen) following the mnufcturer s instructions. RNA qulity ws determined by gel electrophoresis nd concentrtion estimted using nnodrop spectrophotometer t 26 nm. Reverse trnscription nd first-strnd cdna synthesis were performed using the SuperScript III First-Strnd Synthesis System (Invitrogen). qpcr ws performed ccording to Quevl et l. (29). Primer sequences re listed in Supplementry Tble S6. 2 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

21 Enzyme ssys nd metbolite nlysis Extrctble enzyme ctivities were mesured ccording to protocols detiled in Noctor et l. (216). Briefly, ctlse ws mesured by the removl of H 2 O 2 monitored t 24 nm, DHAR s GSHdependent formtion of scorbte from DHA t 265 nm, APX s H 2 O 2 -dependent scorbte oxidtion t 29 nm, nd GR s GSSG-dependent NADPH oxidtion t 34 nm. Oxidized nd reduced forms of glutthione nd scorbte were mesured by plte-reder ssy s described by Quevl nd Noctor (27). Totl SA ws mesured ccording to the protocol of Chouch et l. (21). 21 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

22 Acknowledgments We re grteful to the Slk Institute Genomic Anlysis Lbortory for providing the sequenceindexed Arbidopsis T-DNA insertion mutnts, to the Nottinghm Arbidopsis Stock Centre (UK) for supply of seed stocks, nd to RIKEN (Jpn) for cdna clones. M-S.R. wishes to thnk the Doctorl School ED129 Sciences de l Environnement d Ile de Frnce for the kind wrd of Bourse Ministérielle (PhD studentship). S.L. thnks Michel Dron (IPS2, Orsy, Frnce) nd the Université de Pris sud for funding towrds his PhD studies. We thnk Alison Bker (University of Leeds, UK) nd Cécile Rynud (IPS2, Orsy, Frnce) for kindly supplying reporter lines for control GFP experiments. We re grteful to Myroslw Miginic-Mslow for discussions nd comments on the mnuscript in preprtion, nd to Jen-Clude Psquet for vluble prcticl dvice nd help. This work ws prtly funded by the Agence Ntionle de l Recherche grnt «Cynthiol» to G.N. Supplementl Informtion Supplementl Figure S1. Genotyping nd nlysis of trnscript bundnce in dhr mutnt lines. Supplementl Figure S2. Photogrphs of dhr mutnts fter three weeks growth. Supplementl Figure S3. Effects of vrious stresses on root growth in dhr mutnts. Supplementl Figure S4. Subcellulr locliztion of DHAR1 nd DHAR3 in roots. Supplementl Figure S5. Subcellulr locliztion of DHARs in the bsence of the ct2 muttion. Supplementl Figure S6. Comprison of DHAR1-GFP signls in leves with positive control for peroxisoml trgeting. Supplementl Figure S7. Phenotypes of Col- nd dhr mutnts 2 d fter sprying with 3-AT. Supplementl Figure S8. Effect of dhr1 dhr2 dhr3 triple muttion on resistnce to Pto DC3 in the Col- nd ct2 bckgrounds. Supplementl Tble S1. Ascorbte nd glutthione redox sttes in Col- nd dhr mutnts. Supplementl Tble S2. Ascorbte nd glutthione redox sttes in Col-, ct2 nd ct2 dhr mutnts. Supplementl Tble S3. T2 segregtion of DHAR-complemented lines growing in vitro on gr. Supplementl Tble S4. T2 segregtion of lesion phenotypes in complemented lines growing in soil. Supplementl Tble S5. Glutthione redox sttes in Col- nd dhr mutnts fter tretment with 3- minotrizole. Supplementl Tble S6. DNA primer sequences used in this study. 22 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

23 Figure legends Figure 1. Functionl chrcteriztion of single, double nd triple dhr mutnts. (A) Extrctble DHAR ctivity in lef extrcts. Plnts were grown three weeks under 16h light/8h drk t n irrdince of 2 µmol.m -2.s -1 nd 65% of reltive humidity. Percentge DHAR ctivity reltive to Col- (dotted line) is indicted t the top of the frme. Dt re mens ± SE of six different extrcts. (B) Fresh weight of Col- nd mutnts. Plnts were smpled in the sme conditions s (A). Vlues re mens ± SE of fifteen plnts. (C) Lef contents of scorbte nd (D) glutthione in Col- nd dhr mutnts. White brs, reduced forms. Blck brs, oxidized forms. Vlues re mens ± SE of three plnts, smpled in the sme conditions s (A) nd (B). The sme letter indictes no significnt difference in t test t P <.5 (for totl lef pools in prts C nd D; redox sttes re shown in Supplementl Tble S1). Figure 2. Mjor ntioxidtive enzyme ctivities (A) nd phenotypes (B) in the ct2 mutnt crrying T- DNA insertions in DHAR1, DHAR2, nd DHAR3, singly or in combintion. (A) Extrctble lef ctivities of dehydroscorbte reductse (DHAR), glutthione reductse (GR), scorbte peroxidse (APX), nd ctlse (CAT) fter three weeks growth t n irrdince of 2 µmol.m -2 s -1 in 16 h photoperiod. Dt re mens ± SE of nine biologicl replictes for DHAR lef ctivity nd six for other enzymes. Different letters indicte significnt differences t P <.5. ND, not detected. (B) Photogrphs of mutnts nd rosette fresh weight, determined in the sme conditions s in (A). The white br indictes 1 cm. For fresh weight, vlues re mens ± SE of fifteen to twentyfour plnts. Figure 3. Effect of dhr muttions on oxidtive stress-triggered chnges in scorbte nd glutthione. White brs, reduced forms. Blck brs, oxidized forms. Vlues re mens ± SE of 3 plnts, smpled 3 weeks t n irrdince of 2 µmol.m -2 s -1 in 16 h photoperiod. Different letters indicte significnt difference in oxidized forms (top row of letters on blck bckground) or totl contents (lower row of letters) t P <.5 with one-wy ANOVA (ANlysis Of VArince) with post-hoc Tukey HSD (Honestly Significnt Difference), test clcultor for compring multiple tretments. Figure 4. Subcellulr locliztion of DHAR-GFP fusion proteins. Full-length sequence of AtDHAR1, AtDHAR2, nd AtDHAR3 were tgged with GFP in N-terminl (GFP- DHAR) or C-terminl (DHAR-GFP) position, nd introduced into the ct2 dhr1 dhr2 dhr3 23 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

24 qudruple mutnt. No trnsformnts for GFP-DHAR1 were recovered. The imges correspond to epiderml cells of 1-dy old seedlings. From the left to right : GFP fluorescence (green), chlorophyll utofluorescence (red), bright field nd merged imges. The scle is the sme for ll pnels, with the br top left indicting 1 µm. Figure 5. Dehydroscorbte reductse (DHAR) ctivities in the ct2 dhr1 dhr2 dhr3 line complemented with DHAR1, DHAR2, or DHAR3 (two independent lines ech). Enzyme ctivities were determined in lef extrcts from 15-dy old T2 plnts. Plus or minus indictes presence or bsence of the trnsgene in the plnts in which ctivity ws mesured. The number of plnts ssyed is given bove the columns for ech genotype. Indictes significnt difference t P <.5 from the ctivity in the uncomplemented ct2 dhr1 dhr2 dhr3 mutnt. ND, not detected. Figure 6. Ascorbte nd glutthione in the ct2 dhr1 dhr2 dhr3 line complemented with DHAR1, DHAR2, or DHAR3. White brs, reduced forms. Blck brs, oxidized forms. (A,B) DHAR1 nd DHAR3 complemented lines. (C,D) DHAR2 complemented lines. Plnts were grown on soil from germintion in 16-h/8-h dy/night regime for 3 weeks. Plus or minus indictes presence of bsence of the trnsgene in the plnts in which ctivity ws mesured. Dt re mens ± SE of between three nd nine biologicl repets. Indictes significnt differences in totl scorbte or glutthione contents in mutnts nd complemented lines compred to Col- nd indictes significnt differences compred to ct2 t P <.5. Numbers bove the columns indicte % reduced (1reduced form/totl content). Above these numbers indictes significnt differences compred to Col- nd indictes significnt differences compred to ct2 t P <.5. Figure 7. Influence of dhr muttions on lesions triggered by the ct2 muttion (left) or by the ctlse inhibitor, 3-mino-1,2,4-trizole (3-AT; right). (A) Effect of different dhr mutnt combintions on ct2-triggered lesions. Numbers indicte genotypes s follows: 1, Col-; 2, ct2; 3, ct2 dhr1; 4, ct2 dhr2; 5, ct2 dhr3; 6, ct2 dhr1 dhr2; 7, ct2 dhr1 dhr3; 8, ct2 dhr2 dhr3; 9, ct2 dhr1 dhr2 dhr3. Indictes significnt differences in % of lesions on ct2 bckground mutnt leves compred to Col- nd indictes significnt differences when they were compred to ct2 t P <.5. Vlues re mens ± SE of fifteen plnts. (B) Quntifiction of lesions in the ct2 dhr1 dhr2 dhr3 qudruple mutnt retrnsformed (+) or not (-) with DHAR1, DHAR2, or DHAR3. The number of plnts nlyzed in complemented lines is 24 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

25 indicted on the top of ech br. Indictes significnt differences in % of lesions on complemented line compred to untrnsformed qudruple mutnt. (C) Extent of bleching in Col- nd different dhr mutnt lines cused by exposure to 2 mm 3-AT. 3- AT ws spryed onto the lef surfce of 3 week-old plnts nd lesions were quntified nd smples tken 48 h lter. Indictes significnt effect of 3-AT tretment compred to non-treted Col-; indictes significnt differences between treted dhr mutnts nd Col- t P <.5. Vlues re mens ±SE of thirty plnts. ND, not detected. (D) Lef glutthione content fter 3-AT tretment. White brs, reduced forms. Blck brs, oxidized forms. Dt re mens ±SE of three plnts. For totl glutthione, indictes significnt effect of 3-AT tretment compred to non-treted Col- while indictes significnt difference between dhr mutnts nd Col- t P <.5. Figure 8. Slicylic cid-linked gene expression in Col-, ct2 nd derived lines crrying ll three dhr muttions. PR1, PR2 nd ICS1 trnscripts were quntified by qrt-pcr in three week-old plnts using two reference genes (ACTIN2 nd RCE1). Inset grphs show Col- (left) nd dhr1 dhr2 dhr3 (right). Dt re mens ± SE of three biologicl replictes nd hve been multiplied by 1 for ese of expression. Significnt difference between mutnts nd Col- t P <.5 is indicted by sterisks while dimonds indictes significnt difference between ct2 nd the qudruple mutnt. Figure 9. Detiled nlysis of the effect of dhr muttions on the ct2 phenotype: reltionship to loclized glutthione sttus nd slicylic cid contents. (A) Photogrphs of rosette leves nd trypn blue stining for ded cells in the different mutnts. Brs = 1 cm in lef photogrphs nd 5 mm in trypn blue photogrphs. (B) Lef glutthione contents in leves number 4 nd 5. White brs, GSH. Blck brs, glutthione disulfide (GSSG). Vlues re mens ± SE of three biologicl replictes. (C) Slicylic cid contents in leves 4 nd 5. Vlues re mens ± SE of six biologicl replictes. In (B) nd (C) indictes significnt differences in totl glutthione nd slicylic cid content in mutnts compred to Col- nd + indictes significnt differences between of ct2 dhr mutnts from ct2 t P <.5. For (A), (B) nd (C), plnts were grown for three weeks under 16h light/8h drk t n irrdince of 2 µmol.m -2.s -1 nd 65% of reltive humidity. 25 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

26 Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

27 A Lef DHAR ctivity (nmol.mg -1 protein.min -1 ) % C Lef scorbte (nmol.g -1 FW) B 6 D 9 Fresh weight (mg) 4 2 Lef gutthione (nmol.g -1 FW) 6 3 Col- dhr1 dhr2 dhr3 dhr1 dhr2 dhr1 dhr3 dhr2 dhr3 dhr1 dhr2 dhr3 Col- dhr1 dhr2 dhr3 dhr1 dhr2 dhr1 dhr3 dhr2 dhr3 dhr1 dhr2 dhr3 Figure 1. Functionl chrcteriztion of single, double nd triple dhr mutnts. (A) Extrctble DHAR ctivity in lef extrcts. Plnts were grown three weeks under 16h light/8h drk t n irrdince of 2 µmol.m -2.s -1 nd 65% of reltive humidity. Percentge DHAR ctivity reltive to Col- (dotted line) is indicted t the top of the frme. Dt re mens ± SE of six different extrcts. (B) Fresh weight of Col- nd mutnts. Plnts were smpled in the sme conditions s (A). Vlues re mens ± SE of fifteen plnts. (C) Lef contents of scorbte nd (D) glutthione in Col- nd dhr mutnts. White brs, reduced forms. Blck brs, oxidized forms. Vlues re mens ± SE of three plnts, smpled in the sme conditions s (A) nd (B). The sme letter indictes no significnt difference in t test t P <.5 (for totl lef pools in Downloded from on June 8, Published by prts C nd D; redox sttes re shown in Supplementl Tble S1). Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

28 A 6 B Lef DHAR ctivity (nmol.mg -1 protein.min -1 ) 4 2 b c b c d ND b d ND Col- ct2 ct2 dhr1 2 Lef GR ctivity (nmol.mg -1 protein.min -1 ) 1 b b b ct2 dhr2 ct2 dhr3 ct2 dhr1 dhr2 Lef APX ctivity (nmol.mg -1 protein.min -1 ) ct2 dhr1 dhr3 6 ct2 dhr2 dhr3 ct2 dhr1 dhr2 dhr3 Fresh weight (mg) 4 2 b b b b b b b b Col- ct2 ct2 dhr1 ct2 dhr2 ct2 dhr3 ct2 dhr1 dhr2 ct2 dhr1 dhr3 ct2 dhr2 dhr3 ct2 dhr1 dhr2 dhr3 b 3 Lef CAT ctivity (µmol.mg -1 protein.min -1 ) 2 1 b b b b b b b b Col- ct2 ct2 dhr1 ct2 dhr2 ct2 dhr3 ct2 dhr1 dhr2 ct2 dhr1 dhr3 ct2 dhr2 dhr3 ct2 dhr1 dhr2 dhr3 Figure 2. Mjor ntioxidtive enzyme ctivities (A) nd phenotypes (B) in the ct2 mutnt crrying T-DNA insertions in DHAR1, DHAR2, nd DHAR3, singly or in combintion. (A) Extrctble lef ctivities of dehydroscorbte reductse (DHAR), glutthione reductse (GR), scorbte peroxidse (APX), nd ctlse (CAT) fter three weeks growth t n irrdince of 2 µmol.m -2 s -1 in 16 h photoperiod. Dt re mens ± SE of nine biologicl replictes for DHAR lef ctivity nd six for other enzymes. Different letters indicte significnt differences t P <.5. ND, not detected. (B) Photogrphs of mutnts nd rosette fresh weight, determined in the sme conditions s in (A). Downloded from on June 8, Published by The white br indictes 1 cm. Copyright For fresh 217 weight, Americn vlues Society of re Plnt mens Biologists. ± All SE rights of fifteen reserved. to twenty-four plnts.

29 6 Lef scorbte (nmol.g -1 FW) 4 2 Lef gutthione (nmol.g -1 FW) c b b b b c b c c b b b b bc b bc Col- ct2 ct2 dhr1 ct2 dhr2 ct2 dhr3 ct2 dhr1 dhr2 ct2 dhr1 dhr3 ct2 dhr2 dhr3 ct2 dhr1 dhr2 dhr3 Figure 3. Effect of dhr muttions on oxidtive stress-triggered chnges in scorbte nd glutthione. White brs, reduced forms. Blck brs, oxidized forms. Vlues re mens ± SE of 3 plnts, smpled 3 weeks t n irrdince of 2 µmol.m -2 s -1 in 16 h photoperiod. Different letters indicte significnt difference in oxidized forms (top row of letters on blck bckground) or totl contents (lower row of letters) t P <.5 with one-wy ANOVA (ANlysis Of VArince) with post-hoc Downloded from on June 8, Published by Tukey HSD (Honestly Significnt Difference), test clcultor for compring multiple tretments. Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

30 GFP fluorescence Chlorophyll utofluorescence Bright field Merge DHAR1-GFP DHAR2-GFP GFP-DHAR2 DHAR3-GFP GFP-DHAR3 Figure 4. Subcellulr locliztion of DHAR-GFP fusion proteins. Full-length sequence of AtDHAR1, AtDHAR2, nd AtDHAR3 were tgged with GFP in N-terminl (GFP-DHAR) or C-terminl (DHAR-GFP) position, nd introduced into the ct2 dhr1 dhr2 dhr3 qudruple mutnt. No trnsformnts for GFP-DHAR1 were recovered. The imges correspond to epiderml cells of 1-dy old seedlings. From the left to right : GFP fluorescence (green), chlorophyll utofluorescence (red), bright field nd merged imges. The scle br indictes 1 µm. Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

31 4 Lef DHAR ctivity (nmol.g -1 FW.min -1 ) Col- 6 ct ND ND ND ND ND ND ND ND ND ct2 dhr1 dhr2 dhr DHAR DHAR1-2 DHAR2-1 DHAR DHAR DHAR3-2 Figure 5. Dehydroscorbte reductse (DHAR) ctivities in the ct2 dhr1 dhr2 dhr3 line complemented with DHAR1, DHAR2, or DHAR3 (two independent lines ech). Enzyme ctivities were determined in lef extrcts from 15-dy old T2 plnts. Plus or minus indictes presence or bsence of the trnsgene in the plnts in which ctivity ws mesured. The number of plnts ssyed is given bove the columns for ech genotype. Indictes significnt difference t P<.5 from the ctivity in the uncomplemented ct2 dhr1 dhr2 dhr3 mutnt. ND, not detected. Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

32 A % C % Lef scorbte (nmol.g -1 FW) 4 2 Lef scorbte (nmol.g -1 FW) 4 2 B Lef glutthione (nmol.g -1 FW) % D Lef glutthione (nmol.g -1 FW) % ct2 Col- ct2 dhr1 dhr2 dhr DHAR1-1 DHAR1-2 DHAR3-1 DHAR3-2 Col- ct2 ct2 dhr1 dhr2 dhr DHAR2-1 DHAR2-2 Figure 6. Ascorbte nd glutthione in the ct2 dhr1 dhr2 dhr3 line complemented with DHAR1, DHAR2, or DHAR3. White brs, reduced forms. Blck brs, oxidized forms. (A,B) DHAR1 nd DHAR3 complemented lines. (C,D) DHAR2 complemented lines. Plnts were grown on soil from germintion in 16-h/8-h dy/night regime for 3 weeks. Plus or minus indictes presence of bsence of the trnsgene in the plnts in which ctivity ws mesured. Dt re mens ± SE of between three nd nine biologicl repets. Indictes significnt differences in totl scorbte or glutthione contents in mutnts nd complemented lines compred to Col- nd indictes significnt differences compred to ct2 t P <.5. Numbers bove the columns indicte % reduced (1reduced form/totl content). Above these numbers indictes significnt differences compred to Col- nd indictes significnt differences compred to ct2 t Downloded P <.5. from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

33 A 2 C 1 Lesions (% rosette re) B Lesions (% rosette re) ND 15 ND Col- ct2 ct2 dhr1 dhr2 dhr3 DHAR DHAR DHAR DHAR DHAR DHAR3-2 Bleching (% rosette re) D Lef gutthione (nmol.g -1 FW) ND + 3AT (48 H) Col- -3-AT Col- dhr1 dhr2 dhr3 dhr1 dhr2 dhr1 dhr3 dhr2 dhr3 dhr1 dhr2 dhr3 Figure 7. Influence of dhr muttions on lesions triggered by the ct2 muttion (left) or by the ctlse inhibitor, 3-minotrizole (right) (A) Effect of different dhr mutnt combintions on ct2-triggered lesions. Numbers indicte genotypes s follows: 1, Col-; 2, ct2; 3, ct2 dhr1; 4, ct2 dhr2; 5, ct2 dhr3; 6, ct2 dhr1 dhr2; 7, ct2 dhr1 dhr3; 8, ct2 dhr2 dhr3; 9, ct2 dhr1 dhr2 dhr3. Indictes significnt differences in % of lesions on ct2 bckground mutnt leves compred to Col- nd indictes significnt differences when they were compred to ct2 t P <.5. Vlues re mens ± SE of fifteen plnts. (B) Quntifiction of lesions in the ct2 dhr1 dhr2 dhr3 qudruple mutnt retrnsformed (+) or not (-) with DHAR1, DHAR2, or DHAR3. The number of plnts nlyzed in complemented lines is indicted on the top of ech br. indictes significnt differences in % of lesions on complemented line compred to untrnsformed qudruple mutnt. (C) Extent of bleching in Col- nd different dhr mutnt lines cused by exposure to 2 mm 3- minotrizole (3-AT). 3-AT ws spryed onto the lef surfce of 3 week-old plnts nd lesions were quntified nd smples tken 48 h lter. indictes significnt effect of 3-AT tretment compred to non-treted Col-; indictes significnt differences between treted dhr mutnts nd Col- t P <.5. Vlues re mens ±SE of thirty plnts. ND, not detected. (D) Lef glutthione content fter 3-AT tretment. White brs, reduced forms. Blck brs, oxidized forms. Dt re mens ±SE of three plnts. For totl glutthione, indictes significnt effect of 3-AT tretment compred to non-treted Col- while indictes significnt difference between dhr mutnts nd Col- t P < AT (48 H) Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

34 3 Trnscrpt bundnce Reltive to ACT2 nd RCE PR1 8 Trnscrpt bundnce Reltive to ACT2 nd RCE ICS1 Col- dhr1 dhr2 dhr3 ct2 ct2 dhr1 dhr2 dhr3 Trnscrpt bundnce Reltive to ACT2 nd RCE PR2 Figure 8. Slicylic cid-linked gene expression in Col-, ct2 nd derived lines crrying ll three dhr muttions. PR1, PR2 nd ICS1 trnscripts were quntified by qrt-pcr in three week-old plnts using two reference genes (ACTIN2 nd RCE1). Inset grphs show Col- (left) nd dhr1 dhr2 dhr3 (right). Dt re mens ± SE of three biologicl replictes nd hve been multiplied by 1 for ese of expression. Significnt difference between mutnts nd Col- t P<.5 is indicted by sterisks while dimonds Downloded from on June 8, Published by indictes significnt difference between ct2 nd the qudruple mutnt. Copyright 217 Americn Society of Plnt Biologists. All rights reserved.

35 A B C Phenotype nd trypn blue stining in different leves Glutthione (nmol.g -1 FW) GSH ct2 GSSG Slicylic cid (µg.g -1 FW) ct ct2 dhr ct2 dhr ct2 dhr ct2 dhr ct2 dhr ct2 dhr ct2 dhr1 dhr ct2 dhr1 dhr ct2 dhr1 dhr ct2 dhr1 dhr ct2 dhr2 dhr ct2 dhr2 dhr ct2 dhr1 dhr2 dhr3 6 4 ct2 dhr1 dhr2 dhr Col Col- 4 2 Figure 9. Detiled nlysis of the effect of dhr muttions on the ct2 phenotype: reltionship to loclized glutthione sttus nd slicylic cid contents. (A) Photogrphs of rosette leves nd trypn blue stining for ded cells in the different mutnts. Brs = 1 cm in lef photogrphs nd 5 mm in trypn blue photogrphs. (B) Lef glutthione contents in leves number 4 nd 5. White brs, GSH. Blck brs, glutthione disulfide (GSSG). Vlues re mens ± SE of three biologicl replictes. (C) Slicylic cid contents in leves 4 nd 5. Vlues re mens ± SE of six biologicl replictes. In (B) nd (C) indictes significnt differences in totl glutthione nd slicylic cid content in mutnts compred to Col- nd indictes significnt differences between of ct2 dhr mutnts from ct2 t P <.5. For (A), (B) nd (C) Plnts were grown for three weeks under 16h light/8h drk t n irrdince of 2 µmol.m -2.s -1 nd 65% of reltive humidity. Downloded from on June 8, Published by Copyright 217 Americn Society of Plnt Biologists. All rights reserved.