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Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis

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Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis
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  Ascorbic Acid Deficiency Activates Cell Death andDisease Resistance Responses in Arabidopsis 1 Valeria Pavet, Enrique Olmos 2  , Guy Kiddle, Shaheen Mowla, Sanjay Kumar 3  , John Antoniw,Marı´a E. Alvarez, and Christine H. Foyer* Centro de Investigaciones en Quı´mica Biolo´gica de Co´rdoba/Consejo Nacional de Investigaciones Cientı´ficasy Te´cnicas, Departamento de Quı´mica Biolo´gica, Facultad de Ciencias Quı´micas, Universidad Nacional deCo´rdoba, Ciudad Universitaria, Cordoba 5000, Argentina (V.P., M.E.A.); and Crop Performance andImprovement Division (E.O., G.K., S.M., S.K., C.H.F.) and Wheat Pathogenesis Program, Plant-PathogenInteractions Division (J.A.), Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controlsthat are influenced by redox regulation. Here we show that the Arabidopsis (  Arabidopsis thaliana ) low vitamin C mutants,  vtc1 and  vtc2 , which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR)proteins, and have enhanced basal resistance against infections caused by  Pseudomonas syringae . The mutants have a delayedsenescence phenotype with smaller leaf cells than the wild type at maturity. The  vtc  leaves have more glutathione than the wildtype, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused tothe nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants.Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1  vtc1  plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the timewhen constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in  vtc plants does not result from activation of early senescence. Moreover, H 2 O 2 -sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold foractivation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program. The complex relationships between programmedcell death (PCD) and natural senescence observedduring leaf development are far from understood.However, one clear distinction is that senescence inleavesisessentiallyreversible,butPCDisnot(Thomaset al., 2003). The genetically programmed cell suicideevents that comprise PCD are triggered by enhancedlevels of reactive oxygen species (ROS; Chen andDickman, 2004; Laloi et al., 2004; Wagner et al., 2004).However, senescence-enhanced genes are also ex-pressed in response to ROS (Navabpour et al., 2003).While the chemical nature of ROS dictates that theyare potentially harmful to cells, plants use ROS assecond messengers in signal transduction cascadesregulating diverse processes such as mitosis, tropisms,and cell death. It is now well accepted that ROS ac-cumulation is crucial to plant development as well asdefense (Foyer and Noctor, 2005a). ROS signal trans-duction will ensue only if ROS escape destruction bycellular antioxidants that determine the lifetime andspecificity of the signal. Ascorbic acid (AA) and glu-tathione are the major redox buffers of the plant cells,and they themselves are also signal-transducing mol-ecules that can either signal independently or furthertransmit ROS signals (Fig. 1). They are thus intrinsic toredox homeostasis and redox-signaling events (Foyerand Noctor, 2005b).ROS production is often genetically programmed,for example, during the hypersensitive response (HR)and PCD following pathogen recognition (Jabs et al.,1996;Levineetal.,1996).Inmammals,themitochondria-controlledPCDresponseinvolvestheproapoptoticBaxfamily of proteins and antiapoptotic Bcl-2 and Bcl-XLfamilies. Although Bax, Bcl-2, and Bcl-XL homologs 1 This work was supported by the Biotechnology and BiologicalSciencesResearch Council(C.F., G.K.,and J.A.);grantsfromAgenciaNacional de Promocio´n Cientı´fica y Tecnolo´gica (BID 1201/OC–ARPICT 01–10123) and Fundacio´n Antorchas and Secretaria de Cienciay Tecnologia/Universidad Nacional de Co´rdoba (to M.E.A.); theDepartment of Biotechnology, Government of India, for a Biotech-nology Overseas Associateship Award (to S.K.); the Royal Society(U.K.) for a short-term fellowship (to S.M.); CONICET for a fellow-ship (to V.P.); and the Spanish Government for a Mobility Grant of Researcher,MinisteriodeEducacionyCiencia(PR2004–0361;toE.O.). 2 Present address: Centro de Edafologia y Biologia Aplicada delSegura/Consejo Superior de Investigaciones Cientı´ficas, Depart-ment of Plant Physiology, P.O. Box 164, 30080 Murcia, Spain. 3 Present address: Biotechnology Division,Institute of HimalayanBioresource Technology, P.O. Box 6, Palampur–176 061 (HP), India.* Corresponding author; e-mail christine.foyer@bbsrc.ac.uk; fax0044–1582–763010.The authors responsible for distribution of materials integral to thefindingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(www.plantphysiol.org)are:ChristineH.Foyer (christine.foyer@bbsrc.ac.uk) and Marı´a E. Alvarez (malena@mail.fcq.unc.edu.ar).Article, publication date, and citation informationcan be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.105.067686. Plant Physiology,  November 2005, Vol. 139, pp. 1291–1303, www.plantphysiol.org    2005 American Society of  Plant Biologists 1291  www.plant.orgon September 2, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  have not yet been found in plants, expression of mam-malian Bax causes death, while that of mammalianBcl-XL or Bl-1 suppresses cell death in plant cellschallenged with elicitors, suggesting that elements of mammalian PCD processes are also found in plants(Matsumura et al., 2003). Moreover, mammalian Bcl-2family proteins localize to the mitochondrial, chloro-plast, and nuclear fractions when expressed in plants,where they prevent herbicide and ROS-induced PCD(Chen and Dickman, 2004). Such observations suggestthat mitochondrial and chloroplast ROS accumulationisintrinsic togeneticallyprogrammedPCDeventsandfunction alongside NADPH oxidases and other super-oxide-generating systems on the plasma membrane.Mitochondria also contribute to ROS-dependent geneexpression during pathogen-induced PCD (Maxwellet al., 2002).In addition to redox buffering and ROS detoxifica-tion (Foyer and Noctor, 2005a, 2005b), AA fulfills manyessential roles in plant biology (Arrigoni and de Tullio,2000). In particular, AA modulates growth throughregulation of the cell cycle (Potters et al., 2002, 2004)and through regulation of elongation growth (Fry,1998; Tokuna et al., 2005). AA and hydroxyl radicalsparticipate in the oxidative scission of structural poly-saccharides, promoting cell wall loosening (Fry, 1998).Inaddition,AAisasubstrateforsecretoryperoxidasesinvolved in cell wall stiffening and its presence limitsthe formation of phenolic radical intermediates in wallperoxidase reactions (de Pinto and De Gara, 2004). Figure 1.  The involvement of key re-dox couples in the expression of PRproteins in plant cells. The major sol-uble reductant couples are arrangedaccordingto their midpoint potentials,with principal protein componentswith which they interact indicated.DHAR, Dehydroascorbate reductase;Fd ox , oxidized ferredoxin; Fd red , re-duced ferredoxin; FTR, ferredoxin-thioredoxinreductase;GR,glutathionereductase.Solidarrowsindicateknowninteractions, while broken arrows in-dicate putative components or redoxcouples implicated in the signal trans-duction cascade. Figure 2.  The delayed developmentphenotype of the  vtc1  (vtc1) and  vtc2 (vtc2) mutants compared to the wildtype (WT). The number of weeks aftersowing is indicated. Pavet et al.1292 Plant Physiol. Vol. 139, 2005  www.plant.orgon September 2, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  AA-dependent dioxygenases participate in the syn-thetic pathways of a number of key plant hormonesthatalsoinfluencegrowth(ArrigonianddeTullio,2000).For example, AA is required for the activity of 9-cis-epoxycarotenoid dioxygenase, an enzyme catalyzingtheformationofxanthoxin,theprecursorofabscisicacid.A number of Arabidopsis (  Arabidopsis thaliana ) mu-tants that have low AA (vitamin C;  vtc ) have beenisolated (Conklin et al., 2000). Since the function of most of the genes modified in these mutants is un-known, we have concentrated our efforts on analyzing vtc1  and  vtc2 , which are better characterized. Mostimportantly, the  vtc1  and  vtc2  phenotypes are caused by low AA alone, as shown by studies involving theexpression of the animal AA biosynthetic enzyme L -gulono-1,4-oxidase, which restores wild-type AAlevels and the wild-type phenotype to these mutants(Radzio et al., 2003).  vtc1  is relatively well studied,harboring a point mutation in the AA biosyntheticenzyme GDP-Man pyrophosphorylase (Conklin et al.,2000). This mutant was instrumental in the character-ization of the pathway of AA synthesis in plants(Conklin et al., 1999). In contrast,  vtc2  (At4g26850) har- borsaless-characterizedmutationinanunknownpro-tein (for gene database information, see, for example,http://mips.gsf.de/projects/plants).The  vtc1  mutation confers sensitivity to ozone andother abiotic stresses, such as freezing and UV-B irra-diation (Conklin et al., 1996), but it enhances pathogenresistance (Barth et al., 2004). We have previously Table I.  Comparative developmental changes in vtc1-1, vtc2-1, and wild-type shoot biomass, leaf antioxidants, chlorophyll, and soluble protein  FW, Fresh weight. Plant Age Biomass GSH Glutathione-Disulfide GSH-to-GSSG Ratio AA Chlorophyll Protein g  2 1 FW nmol g  2 1 FW nmol g  2 1 FW nmol g  2 1 FW   m g g  2 1 FW mg  2 1 FW  Week 2 vtc2-1  0.004 6 0.0002 235 6 12 27 6 1 9:1 624 6  21 1,287 6 29 11 ( 6 0.4) vtc1 - 1  0.003 6 0.0001 368 6 25 11 6 1 33:1 1,154 6  54 921 6 13 13 ( 6 0.3)Col-0 0.006 6 0.0003 272 6 45 17 6 3 16:1 3,395 6  57 1,064 6 12 13 ( 6 0.5)Week 4 vtc2 - 1  0.09 6 0.01 598 6 20 37 6 0.7 16:1 1,037 6  67 1,186 6 11 10 ( 6 0.3) vtc1 - 1  0.31 6 0.01 546 6 16 8 6 0.5 68:1 1,278 6  67 1,045 6 7 9 ( 6 0.2)Col-0 0.52 6 0.02 424 6 31 13 6 1 33:1 3,885 6  100 894 6 6 9 ( 6 0.3)Week 6 vtc2 - 1  0.95 6 0.15 338 6 9 30 6 2 11:1 639 6  41 786 6 15 9 ( 6 0.2) vtc1 - 1  1.17 6 0.01 441 6 16 8 6 1 55:1 1,382 6  43 970 6 5 10 ( 6 0.2)Col-0 2.39 6 0.08 326 6 26 12 6 3 27:1 4,648 6  112 894 6 6 10 ( 6 0.4)Week 8 vtc2 - 1  4.62 6 0.31 293 6 10 9 6 1 31:1 375 6  26 996 6 29 7 ( 6 0.2) vtc1 - 1  5.38 6 0.12 312 6 19 15 6 1 20:1 1,464 6  156 1,048 6 10 9 ( 6 0.3)Col-0 8.03 6 0.15 211 6 16 17 6 3 12:1 3,322 6  123 895 6 18 9 ( 6 0.2)Week 10 vtc2 - 1  6.63 6 0.14 279 6 6 7 6 1 60:1 – 1,073 6 39 7 ( 6 0.2) vtc1 - 1  17.2 6 0.12 298 6 8 24 6 4 12:1 – 1,146 6 15 5 ( 6 0.4)Col-0 16.2 6 0.11 183 6 11 12 6 2 15:1 – 680 6 17 3 ( 6 0.1) Figure 3.  The appearance of the apical meristem isdelayed in the  vtc1  mutant compared to the wildtype. The days to flowering were compared in  vtc1 ( n ) and wild-type plants ( : ). The experiment wasperformed using 100 plants and repeated three timeswith similar results. Ascorbate Influences Innate Immune ResponsesPlant Physiol. Vol. 139, 2005 1293  www.plant.orgon September 2, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  shown that AA deficiency in the  vtc1  mutant led tothe differential expression of 171 genes, a substantialnumber of which encode pathogenesis-related (PR)proteinssuchasPR1,PR2,andPR5(Kiddleetal.,2003;Pastori et al., 2003). Moreover, PR proteins accumu-lated more rapidly in  vtc1  than the wild type whenchallengedwith Pseudomonassyringae (Barthetal.,2004).However, when grown under optimal growth condi-tions,  vtc1  shows no evident indications of increasedoxidative stress in terms of tissue H 2 O 2  content or theredoxstateofkeyindicatorpoolssuchasAA(Veljovic- Jovanovic et al., 2001).LikeAA (1–5 m molmg 2 1 freshweight or m mol mg 2 1 chlorophyll), glutathione is an abundant plant metab-olite (100–300 nmol mg 2 1 fresh weight or nmol mg 2 1 chlorophyll) that has many diverse and importantfunctions (Noctor and Foyer, 1998), including signaltransduction (Noctor et al., 2002; Gomez et al., 2004).As an indicator of the general cellular thiol-disulfideredox balance, the reduced glutathione (GSH)-oxidizedglutathione (GSSG) couple is well suited to the role of redox sensor. Cytosolic thiol-disulfide status appearstobeimportantinregulatingtheexpressionofPRgenesthrough the nonexpressor of PR protein 1 (NPR1) path-way(Mou etal.,2003).NPR1is anintrinsiccomponentof the salicylic acid (SA)-triggered systemic acquiredresistance(SAR)responsetobioticattack.Theredoxde-pendence of the NPR1 pathway implies that biotic orabiotic stimuli that perturb the cellular redox state canup-regulatedefense genes via the NPR1 pathway (Mouetal.,2003).Suchredox-linkedeffectsexplain,forexample,PRgeneexpressioninresponsetoUV-Bexposure(Greenand Fluhr, 1995) and in catalase-deficient mutants(Chamnongpoletal.,1996).Figure1illustratestheredoxrelationships between the different components impli-catedintheregulationofNPR1movementfromthecy-tosoltothenucleustoelicitSARandPRgeneexpression.The redox poise of the plant cell at any moment intime is largely dictated by three redox pools, namely,pyridinenucleotides,AA,andglutathione(Fig.1).Thehigh cellular AA content dictates that it is the majorlow-  M r  antioxidant and redox buffer of plant cells(Foyer,2004). It istherefore logical topose thequestionof whether low redox buffering capacity alone, asoccurs during AA deficiency, is sufficient to triggera redox-sensitive pathway leading to enhanced basalresistance. To address this question, we have analyzedSAR and pathogen resistance in two AA-deficientArabidopsis mutants,  vtc1  and  vtc2.  We provide evi-dencethatadecreaseinoverallcellularredox-bufferingcapacity in these mutants, resulting from a diminishedAA pool, together with an enhanced GSH-to-GSSGratio, reduces the threshold for local PCD and causesmovement of NPR1 into the nucleus, triggering sys-temic resistance responses in the absence of enhancedROS levels or external cues. RESULTSAA Deficiency Retards Growth and Senescence andEnhances Leaf GSH-to-GSSG Ratios In this study, the Columbia (Col-0) wild type and the vtc1 and vtc2 mutantsweregrownina10-hphotoperiod Figure 4.  The appearance of individual dead cells (microlesions) in therosette leaves of wild-type (A),  vtc1  (B), and  vtc2  (C) plants duringdevelopment. Cell death was monitored using autofluorescence (top) orlactophenolbluestaining(bottom)ofleaftissues.Toaidclarity,examplesof individualdeadcellsaremarkedwithfinearrowheads,whilelargepatchesof dead cells aremarked by thick arrowheads in the bottom images. Figure5.  Thenumberofdeadcellspresentin vtc1 andwild-typeleavesthrough development. Values were calculated from leaves stained withlactophenol blue as shown in Figure 4, and data represent themeans 6 SE  of five separate experiments involving 25 samples per line. Pavet et al.1294 Plant Physiol. Vol. 139, 2005  www.plant.orgon September 2, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.  in controlled environment chambers with filtered airto remove atmospheric ozone. In these conditions, the vtc1  (Fig. 2) and  vtc2  (data not shown; see Radzioet al.,2003) mutants were smaller at equivalent stages indevelopment than wild-type plants. This phenotypewas clearly evidenced at 2 weeks and was maintainedthroughout the plant growth cycle (Fig. 2). As shownin Table I, the  vtc1  and  vtc2  rosettes accumulate bio-mass at a much slower rate than the wild type. The vtc1  leaves had about 70% less AA than the wild typethroughout development. The  vtc2  mutants studiedhere had even lower leaf AA content for most of thegrowing period, with leaf AA reaching maximumvalues of about 25% of those of the wild type at4 weeks (Table I). In addition, the  vtc1  leaves had 1.3 to1.6 times more glutathione than the wild type. More-over, the GSH-to-GSSG ratios of   vtc1  rosette leaveswerealmostdoublethosemeasuredinthewildtypeatall stages of development. The  vtc2  rosette leaves alsotended to have more leaf glutathione than the wildtype, with much higher GSH-to-GSSG ratios later inrosettedevelopment(TableI).Wild-type, vtc1 ,and vtc2 leaves had similar amounts of chlorophyll and leaf protein at equivalent time points in the growth phaseup to 10 weeks of growth. Thereafter, leaf chlorophylland soluble protein declined in wild-type plants, butin  vtc1  and  vtc2  they remained at similar values tothose observed at 8 weeks (Table I). On the other hand,the  vtc1  plants were delayed in flowering compared tothe wild type (Fig. 3), as did  vtc2  (see Fig. 2). Together,these features show that  vtc1  and  vtc2  rosettes senescelater than the wild-type leaves. Cell Death and Microscopic Lesions Are Evident inNaive  vtc1  Leaves from Early in Rosette Development Macroscopicspontaneouslesionswerenotobservedin naive  vtc1  leaves from plants grown on either shortor long days (Fig. 2), but a few whole chlorotic leavesper plant appeared after 10 weeks, and these, by eye,might easily be mistakenfor a symptom ofsenescence.However, in contrast to Col-0 rosette leaves (Fig. 4A),we consistently detected the presence of individualdead cells early in the development of   vtc1  (Fig. 4B)and  vtc2  (Fig. 4C) rosette leaves at the microscopiclevel. Small foci of collapsed cells were found all overthe leaf surface and preferentially in the mesophylllayer of   vtc1  and  vtc2  leaves (Fig. 4, B and C). By8 weeks, the areas of dead cells had expanded in  vtc2 leaves to form larger foci with clear patches of deadcells detectable by autofluorescence or lactophenol blue staining (Fig. 4C). Even the young (4–6 weeks) vtc1  and  vtc2  rosettes had a small numberof dead cellsas detected by autofluorescence (Fig. 4, B and C) orlactophenol blue staining (Figs. 4 and 5). Individualdead cells were also detected in wild-type leaves, buttheir appearance was delayed compared to  vtc1  and vtc2  (Figs. 4 and 5). Enhanced Resistance to  P. syringae  Reveals That  vtc1 and  vtc 2 Mutations Limit Bacterial Proliferationand Cell Death Expansion To evaluate how  vtc1  controls cell death expansiononce it is initiated by an exogenous stimulus, wechallenged leaf tissues with high doses of the biotro-phic bacterium  P. syringae  pv tomato DC3000 ( Pst ).This virulent pathogen proliferates in the intercellularspaces of leaf tissues of naive wild-type plants causingdiseasewithspreading,chloroticlesions(Whalenetal.,1991). However, proliferation is restricted when plantsorchestrate SAR (Uknes et al., 1992; Cameron et al.,1994). We monitored the development of pathogen-induced lesions in 8-week-old wild-type and  vtc1 and  vtc2  plants infiltrated with a high titer of bacteria(5 3 10 6 cfu/mL). Largepatches of necrotic tissue wereevident 5 d postinfection in inoculated wild-typeleaves, while only a few small groups of dead cells(without spreading necrosis) were found in inoculated vtc1  tissues (Fig. 6). Thus,  vtc1  mutation does not leadto cell death propagation once it is initiated by  Pst inoculation. Like  vtc1 ,  vtc2  leaves exhibited individualcollapsed cells in the young (4–6 weeks) rosettes.Spontaneous cell death is therefore more prolific in vtc1  than  vtc2  leaves, whereas cell death patches were Figure 6.  Pathogen-induced macroscopic and microscopic cell deathsymptoms are greatly decreased in  Pst  -infected  vtc1  leaves comparedto  Pst  -infected wild-type leaves. A and B, Lactophenol blue staining of 7-week-old wild-type and  vtc1  leaves before (A) and after (B) pathogeninoculation (5  3  10 6 cfu/mL). The black arrows in A indicate thepresence of individual dead cells in  vtc1  but not wild type (WT) in theabsence of inoculation. Attached leaves were infiltrated with  Pst   atsingle-point sites (black arrowheads; B [top]), and pathogen-inducedcell death was analyzed at 5 d postinfiltration. Middle section, Thewhole leaves show that patches of dead cells occur around inoculationsitesinwildtypebutnotin vtc1 .Magnificationoftheinfiltrationsites(B[bottom])showsmassivecellularcollapse(indicatedbyfatarrowheads)inthewild-typeplants,whereasonlyafewsmallgroupsofdeadcellsin vtc1  (thin arrows). Bars, 50  m m. Ascorbate Influences Innate Immune ResponsesPlant Physiol. Vol. 139, 2005 1295  www.plant.orgon September 2, 2014 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2005 American Society of Plant Biologists. All rights reserved.
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