Abstract
An early event of the incompatible plant–pathogen interactions is an oxidative burst. On one hand, the ROS generated during oxidative burst is advantageous. ROS can serve as secondary messengers mediating defence gene activation and establishment of additional defences. On the other hand, the concentration of ROS must be carefully regulated to avoid undesired cellular cytotoxicity. The major water soluble, low molecular weight antioxidant, ascorbic acid plays a crucial role in ROS balancing (scavenging). The regulation of ascorbate level, therefore, can be an important point of the fine-tuning of ROS level during the early phase of plant–pathogen interaction. To evaluate how this interaction affects the biosynthesis, the recycling, and the level of ascorbate, we challenged Arabidopsis thaliana cells with two different harpin proteins (HrpZpto and HrpWpto). HrpZpto and HrpWpto treatments caused a well-defined ROS peak. The expression of the alternative oxidase (AOX1a) and vtc5, one of the paralog genes that encode the rate limiting enzyme of ascorbate biosynthesis, followed the elevation of ROS. Similarly, the activity of ascorbate peroxidase and galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH), the enzyme catalysing the ultimate, mitochondria coupled step of ascorbate biosynthesis and the level of ascorbate and glutathione also followed the elevation of ROS due to harpin treatment. The enhanced expression of AOX1a, the elevated activity of GLDH, and the increased level of ascorbate and glutathione all can contribute to the mitigation or absence of programmed cell death. Finally, a new function, the fine-tuning of redox balance during plant–pathogen interaction, can be proposed to vtc5.
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References
Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D, Petit J, Beauvoit B, Fernie AR, Rothan C, Baldet P (2007) Silencing of the mitochondrial ascorbate synthesizing enzyme l-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol 145:1408–1422. doi:10.1104/pp.107.106500
Allan AC, Fluhr R (1997) Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. Plant Cell Online 9:1559–1572. doi:10.1105/tpc.9.9.1559
Amirsadeghi S, Robson CA, Vanlerberghe GC (2007) The role of the mitochondrion in plant responses to biotic stress. Physiol Plant 129:253–266. doi:10.1111/j.1399-3054.2006.00775.x
Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399. doi:10.1146/annurev.arplant.55.031903.141701
Baker CJ, Orlandi EW (1995) Active oxygen in plant pathogenesis. Annu Rev Phytopathol 33:299–321. doi:10.1146/annurev.py.33.090195.001503
Baker CJ, O’Neill NR, Keppler LD, Orlandi EW (1991) Early responses during plant bacteria interaactions in tobacco cell suspension. Phytopathology 81:1504–1507
Balogh T, Szarka A (2016) A comparative study: methods for the determination of ascorbic acid in small and middle sized food analytic laboratories. Acta Aliment 1–9. doi:10.1556/AAlim.2015.0017
Barth C, Moeder W, Klessig DF, Conklin PL (2004) The timing of senescence and response to pathogens is altered in the ascorbate-deficient arabidopsis mutant vitamin c-1. Plant Physiol 134:1784–1792. doi:10.1104/pp.103.032185
Bartoli CG, Pastori GM, Foyer CH (2000) Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol 123:335–344
Bindschedler LV, Minibayeva F, Gardner SL, Gerrish C, Davies DR, Bolwell GP (2001) Early signalling events in the apoplastic oxidative burst in suspension cultured French bean cells involve cAMP and Ca2+. New Phytol 151:185–194. doi:10.1046/j.1469-8137.2001.00170.x
Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47:851–863. doi:10.1111/j.1365-313X.2006.02837.x
Bradley DJ, Kjellbom P, Lamb CJ (1992) Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70(1):21–30
Camejo D, Guzmán-Cedeño Á, Moreno A (2016) Reactive oxygen species, essential molecules, during plant–pathogen interactions. Plant Physiol Biochem 103:10–23. doi:10.1016/j.plaphy.2016.02.035
Castro-concha LA, Escobedo RM, Miranda-ham MDL (2006) Measurement of cell viability in in vitro cultures. Methods Mol Biol 318:71–76
Chang JH, Goel AK, Grant SR, Dangl JL (2004) Wake of the flood: ascribing functions to the wave of type III effector proteins of phytopathogenic bacteria. Curr Opin Microbiol 7:11–18. doi:10.1016/j.mib.2003.12.006
Charkowski AO, Alfano JR, Preston G, Yuan J, He SY, Collmer A (1998) The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate. J Bacteriol 180:5211–5217
Chen S, Schopfer P (1999) Hydroxyl-radical production in physiological reactions. A novel function of peroxidase. Eur J Biochem 260:726–735. doi:10.1046/j.1432-1327.1999.00199.x
Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem 278:46869–46877. doi:10.1074/jbc.M307525200
Choi HW, Kim YJ, Lee SC, Hong JK, Hwang BK (2007) Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens. Plant Physiol 145:890–904. doi:10.1104/pp.107.103325
Colville L, Smirnoff N (2008) Antioxidant status, peroxidase activity, and PR protein transcript levels in ascorbate-deficient Arabidopsis thaliana vtc mutants. J Exp Bot 59:3857–3868. doi:10.1093/jxb/ern229
Conklin PL, Williams EH, Last RL (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci USA. 93:9970–9974
Conklin PL, Saracco SA, Norris SR, Last RL (2000) Identification of ascorbic acid-deficient Arabidopsis thaliana mutants. Genetics 154:847–856
Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. Recent Adv Carbohydr Bioeng 3–12. doi:10.1016/S0144-8617(00)00204-6
Cvetkovska M, Vanlerberghe GC (2012) Coordination of a mitochondrial superoxide burst during the hypersensitive response to bacterial pathogen in Nicotiana tabacum. Plant Cell Environ 35:1121–1136. doi:10.1111/j.1365-3040.2011.02477.x
Cvetkovska M, Vanlerberghe GC (2013) Alternative oxidase impacts the plant response to biotic stress by influencing the mitochondrial generation of reactive oxygen species. Plant Cell Environ 36:721–732. doi:10.1111/pce.12009
Desikan R, Hancock JT, Coffey MJ, Neill SJ (1996) Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme. FEBS Lett 382:213–217. doi:10.1016/0014-5793(96)00177-9
Desikan R, Reynolds A, Hancock JT, Neill SJ (1998) Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures. Biochem J 330(Pt 1):115–120
Desikan R, Hancock JT, Ichimura K, Shinozaki K, Neill SJ (2001) Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6. Plant Physiol 126:1579–1587. doi:10.1104/pp.126.4.1579
Dowdle J, Ishikawa T, Gatzek S, Rolinski S, Smirnoff N (2007) Two genes in Arabidopsis thaliana encoding GDP-l-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J 52:673–689. doi:10.1111/j.1365-313X.2007.03266.x
Ferreira AO, Myers CR, Gordon JS, Martin GB, Vencato M, Collmer A, Wehling MD, Alfano JR, Moreno-Hagelsieb G, Lamboy WF, DeClerck G, Schneider DJ, Cartinhour SW (2006) Whole-genome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000, allows de novo reconstruction of the Hrp cis clement, and identifies novel coregulated genes. Mol Plant Microbe Interact 19:1167–1179. doi:10.1094/MPMI-19-1167
Feys BJ, Parker JE (2000) Interplay of signaling pathways in plant disease resistance. Trends Genet 16:449–455. doi:10.1016/S0168-9525(00)02107-7
Foyer CH, Noctor G (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Physiol 155:2–18. doi:10.1104/pp.110.167569
Fukunaga K, Fujikawa Y, Esaka M (2010) Light regulation of ascorbic acid biosynthesis in rice via light responsive cis-elements in genes encoding ascorbic acid biosynthetic enzymes. Biosci Biotechnol Biochem 74:888–891. doi:10.1271/bbb.90929
Gao Y, Badejo AA, Shibata H, Sawa Y, Maruta T, Shigeoka S, Page M, Smirnoff N, Ishikawa T (2011) Expression analysis of the VTC2 and VTC5 genes encoding GDP-l-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana. Biosci Biotechnol Biochem 75:1783–1788. doi:10.1271/bbb.110320
Garmier M, Priault P, Vidal G, Driscoll S, Djebbar R, Boccara M, Mathieu C, Foyer CH, De Paepe R (2007) Light and oxygen are not required for harpin-induced cell death. J Biol Chem 282:37556–37566. doi:10.1074/jbc.M707226200
Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124:21–29
Haapalainen M, Engelhardt S, Küfner I, Li C-M, Nürnberger T, Lee J, Romantschuk M, Taira S (2011) Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction. Mol Plant Pathol 12:151–166. doi:10.1111/j.1364-3703.2010.00655.x
Haapalainen M, Dauphin A, Li C-M, Bailly G, Tran D, Briand J, Bouteau F, Taira S (2012) HrpZ harpins from different Pseudomonas syringae pathovars differ in molecular interactions and in induction of anion channel responses in Arabidopsis thaliana suspension cells. Plant Physiol Biochem PPB 51:168–174. doi:10.1016/j.plaphy.2011.10.022
Hückelhoven R (2007) Cell wall-associated mechanisms of disease resistance and susceptibility. Annu Rev Phytopathol 45:101–127. doi:10.1146/annurev.phyto.45.062806.094325
Imai T, Niwa M, Ban Y, Hirai M, Ôba K, Moriguchi T (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. doi:10.1007/s11240-008-9466-x
Karpinski S, Gabrys H, Mateo A, Karpinska B, Mullineaux PM (2003) Light perception in plant disease defence signalling. Curr Opin Plant Biol 6:390–396. doi:10.1016/S1369-5266(03)00061-X
Keppler LD (1989) Active oxygen production during a bacteria-induced hypersensitive reaction in tobacco suspension cells. Phytopathology 79:974–978. doi:10.1094/Phyto-79-974
Klessig DF, Durner J, Noad R, Navarre DA, Wendehenne D, Kumar D, Zhou JM, Shah J, Zhang S, Kachroo P, Trifa Y, Pontier D, Lam E, Silva H (2000) Nitric oxide and salicylic acid signaling in plant defense. Proc Natl Acad Sci 97:8849–8855. doi:10.1073/pnas.97.16.8849
Kliebenstein DJ (2004) Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses. Plant Cell Environ. doi:10.1111/j.1365-3040.2004.01180.x
Kovtun Y, Chiu W-L, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci 97:2940–2945. doi:10.1073/pnas.97.6.2940
Krause M, Durner J (2004) Harpin inactivates mitochondria in Arabidopsis suspension cells. Mol Plantmicrobe Interact MPMI 17:131–139
Kuźniak E (2010) The ascorbate–gluathione cycle and related redox signals in plant–pathogen interactions. In: Anjum NA, Chan MT, Umar S (eds) Ascorbate–glutathione pathway and stress tolerance in plants. Springer, Dordrecht, pp 115–136. doi:10.1007/978-90-481-9404-9_4
Kvitko BH, Ramos AR, Morello JE, Oh H-S, Collmer A (2007) Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors. J Bacteriol 189:8059–8072. doi:10.1128/JB.01146-07
Lacomme C, Roby D (1999) Identification of new early markers of the hypersensitive response in Arabidopsis thaliana. FEBS Lett 459:149–153. doi:10.1016/S0014-5793(99)01233-8
Lee J, Klusener B, Tsiamis G, Stevens C, Neyt C, Tampakaki AP, Panopoulos NJ, Nöller J, Weiler EW, Cornelis GR, Mansfield JW, Nürnberger T (2001) HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro. Proc Natl Acad Sci USA 98:289–294. doi:10.1073/pnas.011265298
Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593
Lindeberg M, Cartinhour S, Myers CR, Schechter LM, Schneider DJ, Collmer A (2006) Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains. Mol Plant Microbe Interact 19:1151–1158. doi:10.1094/MPMI-19-1151
Mannervik B (2001) Measurement of glutathione reductase activity. Curr Protoc Toxicol Chapter 7(Unit7):2. doi:10.1002/0471140856.tx0702s00
Millar AH, Mittova V, Kiddle G, Heazlewood JL, Bartoli CG, Theodoulou FL, Foyer CH (2003) Control of ascorbate synthesis by respiration and its implications for stress responses. Plant Physiol 1(133):443–447. doi:10.1104/pp.103.028399
Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498. doi:10.1016/j.tplants.2004.08.009
Montillet J-L, Chamnongpol S, Rustérucci C, Dat J, van de Cotte B, Agnel J-P, Battesti C, Inzé D, Van Breusegem F, Triantaphylidès C (2005) Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves. Plant Physiol 138:1516–1526. doi:10.1104/pp.105.059907
Mou Z, Fan W, Dong X (2003) Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell 113(7):935–944
Mur LAJ, Kenton P, Lloyd AJ, Ougham H, Prats E (2008) The hypersensitive response; the centenary is upon us but how much do we know? J Exp Bot 59:501–520. doi:10.1093/jxb/erm239
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
Oba K, Fukui M, Imai Y, Iriyama S, Nogami K (1994) l-Galactono-gamma-lactone dehydrogenase: partial characterization, induction of activity and role in the synthesis of ascorbic acid in wounded white potato tuber tissue. Plant Cell Physiol 35:473–478
Pavet V, Olmos E, Kiddle G, Mowla S, Kumar S, Antoniw J, Alvarez ME, Foyer CH (2005) Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis. Plant Physiol 139:1291–1303. doi:10.1104/pp.105.067686
Polidoros AN, Mylona PV, Arnholdt-Schmitt B (2009) Aox gene structure, transcript variation and expression in plants. Physiol Plant 137:342–353. doi:10.1111/j.1399-3054.2009.01284.x
Preston G, Huang HC, He SY, Collmer A (1995) The HrpZ proteins of Pseudomonas syringae pvs. syringae, glycinea, and tomato are encoded by an operon containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not soybean. Mol Plant Microbe Interact 8:717–732
Rasmusson AG, Soole KL, Elthon TE (2004) Alternative NAD(P)H dehydrogenases of plant mitochondria. Annu Rev Plant Biol 55:23–39. doi:10.1146/annurev.arplant.55.031903.141720
Reboutier D, Frankart C, Briand J, Biligui B, Rona J-P, Haapalainen M, Barny M-A, Bouteau F (2007) Antagonistic action of harpin proteins: HrpWea from Erwinia amylovora suppresses HrpNea-induced cell death in Arabidopsis thaliana. J Cell Sci 120:3271–3278. doi:10.1242/jcs.011098
Remans T, Smeets K, Opdenakker K, Mathijsen D, Vangronsveld J, Cuypers A (2008) Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227:1343–1349. doi:10.1007/s00425-008-0706-4
Simons BH (1999) Enhanced expression and activation of the alternative oxidase during infection of arabidopsis with Pseudomonas syringae pv tomato. Plant Physiol 120:529–538. doi:10.1104/pp.120.2.529
Stahl RL, Liebes LF, Farber CM, Silber R (1983) A spectrophotometric assay for dehydroascorbate reductase. Anal Biochem 131:341–344. doi:10.1016/0003-2697(83)90180-X
Sun A, Nie S, Xing D (2012) Nitric oxide-mediated maintenance of redox homeostasis contributes to NPR1-dependent plant innate immunity triggered by lipopolysaccharides. Plant Physiol 160:1081–1096. doi:10.1104/pp.112.201798
Szarka A (2013) Quantitative data on the contribution of GSH and Complex II dependent ascorbate recycling in plant mitochondria. Acta Physiol Plant 35:3245–3250. doi:10.1007/s11738-013-1359-x
Szarka A, Tomasskovics B, Bánhegyi G (2012) The ascorbate-glutathione-α-tocopherol triad in abiotic stress response. Int J Mol Sci 13:4458–4483. doi:10.3390/ijms13044458
Szarka A, Bánhegyi G, Asard H (2013) The inter-relationship of ascorbate transport, metabolism and mitochondrial, plastidic respiration. Antioxid Redox Signal 19:1036–1044. doi:10.1089/ars.2012.5059
Thoma I, Loeffler C, Sinha AK, Gupta M, Krischke M, Steffan B, Roitsch T, Mueller MJ (2003) Cyclopentenone isoprostanes induced by reactive oxygen species trigger defense gene activation and phytoalexin accumulation in plants. Plant J 34:363–375
Torres MA (2010) ROS in biotic interactions. Physiol Plant 138:414–429. doi:10.1111/j.1399-3054.2009.01326.x
Unger C, Kleta S, Jandl G, Tiedemann AV (2005) Suppression of the defence-related oxidative burst in bean leaf tissue and bean suspension cells by the necrotrophic pathogen Botrytis cinerea. J Phytopathol 153:15–26. doi:10.1111/j.1439-0434.2004.00922.x
Urzica EI, Adler LN, Page MD, Linster CL, Arbing MA, Casero D, Pellegrini M, Merchant SS, Clarke SG (2012) Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-l-galactose phosphorylase. J Biol Chem 287:14234–14245. doi:10.1074/jbc.M112.341982
Vanlerberghe GC (2013) Alternative oxidase: a mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants. J Mol Sci, Int. doi:10.3390/ijms14046805
Vidal G, Ribas-Carbo M, Garmier M, Dubertret G, Rasmusson AG, Mathieu C, Foyer CH, De Paepe R (2007) Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco. Plant Cell 19:640–655. doi:10.1105/tpc.106.044461
Wolff SP (1994) Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol 233:182–189. doi:10.1016/S0076-6879(94)33021-2
Wu Y-X, Von Tiedemann A (2001) Physiological effects of azoxystrobin and epoxiconazole on senescence and the oxidative status of wheat. Pestic Biochem Physiol 71:1–10. doi:10.1006/pest.2001.2561
Zhang W, Lorence A, Gruszewski HA, Chevone BI, Nessler CL (2009) AMR1, an Arabidopsis gene that coordinately and negatively regulates the mannose/l-galactose ascorbic acid biosynthetic pathway. Plant Physiol 150:942–950. doi:10.1104/pp.109.138453
Zsigmond L, Tomasskovics B, Deák V, Rigó G, Szabados L, Bánhegyi G, Szarka A (2011) Enhanced activity of galactono-1,4-lactone dehydrogenase and ascorbate-glutathione cycle in mitochondria from complex III deficient Arabidopsis. Plant Physiol Biochem 49:809–815. doi:10.1016/j.plaphy.2011.04.013
Acknowledgements
We thank Dr. Alen Collmer for his generous gift of the harpin-containing plasmids. Á.C. and P.H. thank Dr. Veronika Deák to her support. This project is supported by the New Hungary Development Plan (Project ID: TÁMOP-4.2.1/B-09/1/KMR-2010-0002). This work was financially supported by the National Scientific Research Fund Grants (OTKA 105416) and MedinProt Protein Excellence foundation.
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Communicated by E. Kuzniak-Gebarowska.
Á. Czobor and P. Hajdinák contributed equally to the manuscript.
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Czobor, Á., Hajdinák, P. & Szarka, A. Rapid ascorbate response to bacterial elicitor treatment in Arabidopsis thaliana cells. Acta Physiol Plant 39, 62 (2017). https://doi.org/10.1007/s11738-017-2365-1
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DOI: https://doi.org/10.1007/s11738-017-2365-1