European Journal of Plant Pathology

, Volume 128, Issue 1, pp 7–19 | Cite as

ABA signaling inhibits oxalate-induced production of reactive oxygen species and protects against Sclerotinia sclerotiorum in Arabidopsis thaliana



Oxalic acid is an essential virulence factor of Sclerotinia sclerotiorum that elicits wilting symptoms in infected host plants. Foliar wilting in response to oxalic acid is known to be dependent on an increase in stomatal conductance. To determine whether stomatal regulation controls susceptibility to S. sclerotiorum, abscisic acid-insensitive and open stomata mutants of Arabidopsis thaliana were analyzed. Whereas abscisic acid-insensitive mutants were hypersusceptible to S. sclerotiorum, open stomata mutants were as susceptible as wild type. It was concluded that stomatal regulation does not control susceptibility to S. sclerotiorum because open stomata mutants are known to only impair guard cells whereas abscisic acid-insensitive mutants also affect other cell types. Guard cell-independent processes also control sensitivity to oxalic acid because oxalic acid was more toxic to abscisic acid-insensitive mutants than to open stomata mutants. To explore a possible mechanism of toxicity, production of reactive oxygen species was measured in plant cells after exposure to oxalic acid. Oxalic acid was found to elicit reactive oxygen species production independently of abscisic acid. Nevertheless, cancellation of reactive oxygen species elicitation after co-stimulation of wild-type guard cells with oxalic acid and abscisic acid provided evidence for antagonistic interaction between both molecules.


Disease Guard cell H2O2 Hypersusceptible Stomatal aperture 



abscisic acid




2′,7′-dichlorofluorescein diacetate


programmed cell death


reactive oxygen species



We thank D. Zhang and Y. Kamiya for using their epifluorescence microscopes; G. Tallman for critical reading of the manuscript.


This work was supported by National Sclerotinia Initiative Special Grants program U.S. Department of Agriculture [Grant number 58-5442-7-226].

Supplementary material

10658_2010_9623_MOESM1_ESM.xls (16 kb)
ESM 1 (XLS 15 kb)
10658_2010_9623_MOESM2_ESM.doc (1.3 mb)
Supplemental Figure 1 Images of pathogen-infected wild-type and mutant plants. Pictures were taken 2 days after inoculation with wild-type S. sclerotiorum. Note increased lesion sizes of the abi1 mutant. Original excel file used for figure 1 was included in this supplementary figure 1 to demonstrate that the lesion diameters of abi1 mutant are significant larger than those of Ler. (DOC 1335 kb)
10658_2010_9623_MOESM3_ESM.doc (33 kb)
Supplemental Figure 2 Stomatal apertures (width/length) after inoculation of wild-type (Ler) or abi1-1 mutant plants with S. sclerotiorum. Epidermis was removed from uninfected leaves and measured. Means and standard errors are shown (n > 160). Letters indicate significant differences (P < 0.01). (DOC 33 kb)
10658_2010_9623_MOESM4_ESM.doc (729 kb)
Supplemental Figure 3 Vanadate-sensitive stomatal opening in response to oxalic acid (OA) indicative of P-type ATPase activation. Epidermal strips of Vicia faba were incubated in the presence or absence of 2 mM vanadate (Van) prior to treatment with 10 mM OA (Schwartz et al., 1991). As a positive control, 1 μM fusicoccin (FC), a fungal toxin and activator of the plasma membrane H+-ATPase, was used. Means and standard errors are shown. Asterisks indicate significant differences (P < 0.001). The experiment was repeated six times following published procedures (Guimaraes and Stotz, 2004). (DOC 729 kb)


  1. AbuQamar, S., Chen, X., Dhawan, R., Bluhm, B., Salmeron, J., Lam, S., et al. (2006). Expression profiling and mutant analysis reveals complex regulatory networks involved in Arabidopsis response to Botrytis infection. The Plant Journal, 48, 28–44.CrossRefPubMedGoogle Scholar
  2. Adie, B. A. T., Perez-Perez, J., Perez-Perez, M. M., Godoy, M., Sanchez-Serrano, J.-J., Schmelz, E. A., et al. (2007). ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis. The Plant Cell, 19, 1665–1681.CrossRefPubMedGoogle Scholar
  3. Aihara, K., Byer, K. J., & Khan, S. R. (2003). Calcium phosphate-induced renal epithelial injury and stone formation: involvement of reactive oxygen species. Kidney International, 64, 1283–1291.CrossRefPubMedGoogle Scholar
  4. Anderson, J. P., Badruzsaufari, E., Schenk, P. M., Manners, J. M., Desmond, O. J., Ehlert, C., et al. (2004). Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. The Plant Cell, 16, 3460–3479.CrossRefPubMedGoogle Scholar
  5. Bateman, D. F., & Beer, S. V. (1965). Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis of Sclerotium rolfsii. Phytopathology, 55, 204–211.PubMedGoogle Scholar
  6. Belin, C., de Franco, P. O., Bourbousse, C., Chaignepain, S., Schmitter, J. M., Vavasseur, A., et al. (2006). Identification of features regulating OST1 kinase activity and OST1 function in guard cells. Plant Physiology, 141, 1316–1327.CrossRefPubMedGoogle Scholar
  7. Bessire, M., Chassot, C., Jacquat, A. C., Humphry, M., Borel, S., Petetot, J. M., et al. (2007). A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea. The EMBO Journal, 26, 2158–2168.CrossRefPubMedGoogle Scholar
  8. Boland, G. J., & Hall, R. (1994). Index of plant hosts of Sclerotinia sclerotiorum. Canadian Journal of Plant Pathology, 16, 93–108.CrossRefGoogle Scholar
  9. Cessna, S. G., Sears, V. E., Dickman, M. B., & Low, P. S. (2000). Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. The Plant Cell, 12, 2191–2199.CrossRefPubMedGoogle Scholar
  10. Cruickshank, R. H. (1983). Distinction between Sclerotinia species by their pectic zymograms. Transaction of the British Mycological Society, 80, 117–119.CrossRefGoogle Scholar
  11. de Bary, A. (1886). Ueber einige Sclerotinien and Sclerotienkrankheiten. Botanische Zeitung, 44, 377–474.Google Scholar
  12. Galletti, R., Denoux, C., Gambetta, S., Dewdney, J., Ausubel, F. M., De Lorenzo, G., et al. (2008). The AtrbohD-mediated oxidative burst elicited by oligogalacturonides in Arabidopsis is dispensable for the activation of defense responses effective against Botrytis cinerea. Plant Physiology, 148, 1695–1706.CrossRefPubMedGoogle Scholar
  13. Godoy, G., Steadman, J. R., Dickman, M. B., & Dam, R. (1990). Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. Physiological and Molecular Plant Pathology, 37, 179–191.CrossRefGoogle Scholar
  14. Guimaraes, R. L., & Stotz, H. U. (2004). Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection. Plant Physiology, 136, 3703–3711.CrossRefPubMedGoogle Scholar
  15. Guimarães, R., Chetelat, R., & Stotz, H. (2004). Resistance to Botrytis cinerea in Solanum lycopersicoides is dominant in hybrids with tomato, and involves induced hyphal death. European Journal of Plant Pathology, 110, 13–23.Google Scholar
  16. Guo, X., & Stotz, H. U. (2007). Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on jasmonic acid, salicylic acid, and ethylene signaling. Molecular Plant-Microbe Interactions, 20, 1384–1395.CrossRefPubMedGoogle Scholar
  17. Han, Y., Joosten, H. J., Niu, W. L., Zhao, Z. M., Mariano, P. S., McCalman, M., et al. (2007). Oxaloacetate hydrolase, the C-C bond lyase of oxalate secreting fungi. The Journal of Biological Chemistry, 282, 9581–9590.CrossRefPubMedGoogle Scholar
  18. Hegedus, D. D., & Rimmer, S. R. (2005). Sclerotinia sclerotiorum: When “to be or not to be” a pathogen? FEMS Microbiology Letters, 251, 177–184.CrossRefPubMedGoogle Scholar
  19. Hu, X., Bidney, D. L., Yalpani, N., Duvick, J. P., Crasta, O., Folkerts, O., et al. (2003). Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant Physiology, 133, 170–181.CrossRefPubMedGoogle Scholar
  20. Israelsson, M., Siegel, R. S., Young, J., Hashimoto, M., Iba, K., & Schroeder, J. I. (2006). Guard cell ABA and CO2 signaling network updates and Ca2+ sensor priming hypothesis. Current Opinion in Plant Biology, 9, 654–663.CrossRefPubMedGoogle Scholar
  21. Jonassen, J. A., Cao, L. C., Honeyman, T., & Scheid, C. R. (2003). Mechanisms mediating oxalate-induced alterations in renal cell functions. Critical Reviews in Eukaryotic Gene Expression, 13, 55–72.CrossRefPubMedGoogle Scholar
  22. Kaliff, M., Staal, J., Myrenas, M., & Dixelius, C. (2007). ABA is required for Leptosphaeria maculans resistance via ABI1- and ABI4-dependent signaling. Molecular Plant-Microbe Interactions, 20, 335–345.CrossRefPubMedGoogle Scholar
  23. Kim, K. S., Min, J. Y., & Dickman, M. B. (2008). Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development. Molecular Plant-Microbe Interactions, 21, 605–612.CrossRefPubMedGoogle Scholar
  24. Koornneef, M., Leon-Kloosterziel, K. M., Schwartz, S. H., & Zeevaart, J. A. D. (1998). The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopsis. Plant Physiology & Biochemistry, 36, 83–89.CrossRefGoogle Scholar
  25. Kwak, J. M., Mori, I. C., Pei, Z.-M., Leonhardt, N., Torres, M. A., Dangl, J. L., et al. (2003). NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. The EMBO Journal, 22, 2623–2633.CrossRefPubMedGoogle Scholar
  26. Lee, S., Choi, H., Suh, S., Doo, I. S., Oh, K. Y., Choi, E. J., et al. (1999). Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiology, 121, 147–152.CrossRefPubMedGoogle Scholar
  27. Leung, J., Bouvier-Durand, M., Morris, P.-C., Guerrier, D., Chefdor, F., and Giraudat, J. (1994). Arabidopsis ABA response gene ABI1: Features of a calcium-modulated protein phosphatase. Science 264, 1448–1452.Google Scholar
  28. Leung, J., Merlot, S., & Giraudat, J. (1997). The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. The Plant Cell, 9, 759–771.CrossRefPubMedGoogle Scholar
  29. Lopez, M. A., Bannenberg, G., & Castresana, C. (2008). Controlling hormone signaling is a plant and pathogen challenge for growth and survival. Current Opinion in Plant Biology, 11, 420–427.CrossRefPubMedGoogle Scholar
  30. Melotto, M., Underwood, W., Koczan, J., Nomura, K., & He, S. Y. (2006). Plant stomata function in innate immunity against bacterial invasion. Cell, 126, 969–980.CrossRefPubMedGoogle Scholar
  31. Merlot, S., Mustilli, A. C., Genty, B., North, H., Lefebvre, V., Sotta, B., et al. (2002). Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation. The Plant Journal, 30, 601–609.CrossRefPubMedGoogle Scholar
  32. Merlot, S., Leonhardt, N., Fenzi, F., Valon, C., Costa, M., Piette, L., et al. (2007). Constitutive activation of a plasma membrane H+-ATPase prevents abscisic acid-mediated stomatal closure. The EMBO Journal, 26, 3216–3226.CrossRefPubMedGoogle Scholar
  33. Mohr, P. G., & Cahill, D. M. (2003). Abscisic acid influences the susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv. tomato and Peronospora parasitica. Functional Plant Biology, 30, 461–469.CrossRefGoogle Scholar
  34. Murata, Y., Pei, Z.-M., Mori, I. C., & Schroeder, J. (2001). Abscisic acid activation of plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. The Plant Cell, 13, 2513–2523.CrossRefPubMedGoogle Scholar
  35. Mustilli, A.-C., Merlot, S., Vavasseur, A., Fenzi, F., and Giraudat, J. (2002). Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14, 3089–3099.Google Scholar
  36. Nilson, S. E., & Assmann, S. M. (2007). The control of transpiration. Insights from Arabidopsis. Plant Physiology, 143, 19–27.CrossRefPubMedGoogle Scholar
  37. Noyes, R. D., & Hancock, J. G. (1981). Role of oxalic acid in the Sclerotinia sclerotiorum wilt of sunflower Helianthus annuus. Physiological Plant Pathology, 18, 123–132.Google Scholar
  38. Pei, Z.-M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G. J., et al. (2000). Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature, 406, 731–734.CrossRefPubMedGoogle Scholar
  39. Purdy, L. H. (1979). Sclerotinia sclerotiorum: history, diseases and symptomatology, host range, geographic distribution, and impact. Phytopathology, 69, 875–880.CrossRefGoogle Scholar
  40. Riganti, C., Gazzano, E., Polimeni, M., Costamagna, C., Bosia, A., & Ghigo, D. (2004). Diphenyleneiodonium inhibits the cell redox metabolism and induces oxidative stress. The Journal of Biological Chemistry, 279, 47726–47731.CrossRefPubMedGoogle Scholar
  41. Thordal-Christensen, H., Zhang, Z., Wei, Y., & Collinge, D. B. (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. The Plant Journal, 11, 1187–1194.CrossRefGoogle Scholar
  42. Ton, J., & Mauch-Mani, B. (2004). (beta)-amino-butyric acid-induced resistance against necrotrophic pathogens is based on ABA-dependent priming for callose. The Plant Journal, 38, 119–130.CrossRefPubMedGoogle Scholar
  43. Torres, M. A., Dangl, J. L., & Jones, J. D. (2002). Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci U S A, 99, 517–522.CrossRefPubMedGoogle Scholar
  44. Torres, M. A., Jones, J. D. G., & Dangl, J. L. (2005). Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nature Genetics, 37, 1130–1134.CrossRefPubMedGoogle Scholar
  45. Wang, Z., Mao, H., Dong, C., Ji, R., Cai, L., Fu, H., et al. (2009). Overexpression of Brassica napus MPK4 enhances resistance to Sclerotinia sclerotiorum in oilseed rape. Molecular Plant Microbe Interactions, 22, 235–244.CrossRefPubMedGoogle Scholar

Copyright information

© KNPV 2010

Authors and Affiliations

  1. 1.E306 Beadle CenterUniversity of Nebraska-LincolnLincolnUSA
  2. 2.Julius-von-Sachs-Institute für Biowissenschaften, Pharmazeutische Biologie, BiozentrumUniversitaet WuerzburgWuerzburgGermany

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