, Volume 247, Issue 5, pp 1203–1215 | Cite as

Involvement of S-nitrosothiols modulation by S-nitrosoglutathione reductase in defence responses of lettuce and wild Lactuca spp. to biotrophic mildews

  • Tereza Tichá
  • Michaela Sedlářová
  • Lucie Činčalová
  • Zuzana Drábková Trojanová
  • Barbora Mieslerová
  • Aleš Lebeda
  • Lenka Luhová
  • Marek Petřivalský
Original Article


Main conclusion

Resistant Lactuca spp. genotypes can efficiently modulate levels of S-nitrosothiols as reactive nitrogen species derived from nitric oxide in their defence mechanism against invading biotrophic pathogens including lettuce downy mildew.


S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosaB. lactucae and L. sativa cv. UCDM2–G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a two-phase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.


Bremia lactucae Golovinomyces cichoracearum Lettuce downy mildew Lettuce powdery mildew Nitric oxide Pseudoidium neolycopersici 





S-Nitrosoglutathione reductase


Hours post inoculation


Reactive nitrogen species


Reactive oxygen species



This research was supported by the Czech Grant Agency (501/12/0590), by Palacký University in Olomouc (IGA_PrF_2017_016, IGA_PrF_2017_001, IGA_PrF_2018_001), and by Ministry of Education, Youths and Sports, Czech Republic (MSM 6198959215).

Supplementary material

425_2018_2858_MOESM1_ESM.pdf (252 kb)
Supplementary material 1 (PDF 252 kb)


  1. Arasimowicz-Jelonek M, Floryszak-Wieczorek J (2016) Nitric oxide in the offensive strategy of fungal and oomycete plant pathogens. Front Plant Sci 7:252CrossRefPubMedPubMedCentralGoogle Scholar
  2. Barroso JB, Corpas FJ, Carreras A, Rodriguez-Serrano M, Esteban FJ, Fernandez-Ocana A, Chaki M, Romero-Puertas MC, Valderrama R, Sandalio LM, Del Río LA (2006) Localization of S-nitrosoglutathione and expression of S-nitrosoglutathione reductase in pea plants under cadmium stress. J Exp Bot 57:1785–1793CrossRefPubMedGoogle Scholar
  3. Barroso J, Valderrama R, Corpas F (2013) Immunolocalization of S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles. Acta Physiol Plant 35:2635–2640CrossRefGoogle Scholar
  4. Begara-Morales JC, Sánchez-Calvo B, Chaki M, Valderrama R, Mata-Pérez C, Padilla MN, Corpas FJ, Barroso JB (2016) Antioxidant systems are regulated by nitric oxide-mediated post-translational modifications (NO-PTMs). Front Plant Sci 7:152CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Chaki M, Fernández-Ocaňa AM, Valderrama R, Carreras A, Esteban FJ, Luque F, MaV Gómez-Rodríguez, Begara-Morales JC, Corpas FJ, Barroso JB (2009) Involvement of reactive nitrogen and oxygen species (RNS and ROS) in sunflower–mildew interaction. Plant Cell Physiol 50:265–279CrossRefPubMedGoogle Scholar
  7. Chaki M, Valderrama R, Fernández-Ocaňa AM, Carreras A, Gómez-Rodríguez MV, Pedrajas JR, Begara-Morales JC, Sánchez-Calvo B, Luque F, Leterrier M, Corpas FJ, Barroso JB (2011) Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings. J Exp Bot 62:1803–1813CrossRefPubMedGoogle Scholar
  8. Corpas FJ, Alché JD, Barroso JB (2013) Current overview of S-nitrosoglutathione (GSNO) in higher plants. Front Plant Sci 4:126PubMedPubMedCentralGoogle Scholar
  9. Díaz M, Achkor H, Titarenko E, Martínez MC (2003) The gene encoding glutathione-dependent formaldehyde dehydrogenase/GSNO reductase is responsive to wounding, jasmonic acid and salicylic acid. FEBS Lett 543:136–139CrossRefPubMedGoogle Scholar
  10. Domingos P, Prado Ana M, Wong A, Gehring C, Feijo JA (2015) Nitric oxide: a multitasked signaling gas in plants. Mol Plant 8:506–520CrossRefPubMedGoogle Scholar
  11. Durner J, Klessig DF (1999) Nitric oxide as a signal in plants. Curr Opin Plant Biol 2:369–374CrossRefPubMedGoogle Scholar
  12. Espunya MC, De Michele R, Gómez-Cadenas A, Martínez MC (2012) S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana. J Exp Bot 63:3219–3227CrossRefPubMedPubMedCentralGoogle Scholar
  13. Feechan A, Kwon E, Yun BW, Wang Y, Pallas JA, Loake GJ (2005) A central role for S-nitrosothiols in plant disease resistance. Proc Natl Acad Sci USA 102:8054–8059CrossRefPubMedPubMedCentralGoogle Scholar
  14. Frungillo L, Skelly MJ, Loake GJ, Spoel SH, Salgado I (2014) S-Nitrosothiols regulate nitric oxide production and storage in plants through the nitrogen assimilation pathway. Nat Commun 5:5401CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gong B, Wen D, Wang X, Wei M, Yang F, Li Y, Shi Q (2015) S-Nitrosoglutathione reductase-modulated redox signaling controls sodic alkaline stress responses in Solanum lycopersicum L. Plant Cell Physiol 56:790–802CrossRefPubMedGoogle Scholar
  16. Gow A, Doctor A, Mannick J, Gaston B (2007) S-nitrosothiol measurements in biological systems. J Chromatogr B 851:140–151CrossRefGoogle Scholar
  17. Hong JK, Yun BW, Kang JG, Raja MU, Kwon E, Sorhagen K, Chu C, Wang Y, Loake GJ (2008) Nitric oxide function and signalling in plant disease resistance. J Exp Bot 59:147–154CrossRefPubMedGoogle Scholar
  18. Janus Ł, Milczarek G, Arasimowicz-Jelonek M, Abramowski D, Billert H, Floryszak-Wieczorek J (2013) Normoergic NO-dependent changes, triggered by a SAR inducer in potato, create more potent defense responses to Phytophthora infestans. Plant Sci 211:23–34CrossRefPubMedGoogle Scholar
  19. Kubienová L, Kopečný D, Tylichová M, Briozzo P, Skopalová J, Šebela M, Navrátil M, Tache R, Luhová L, Barroso JB, Petřivalský M (2013) Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum. Biochimie 95:889–902CrossRefPubMedGoogle Scholar
  20. Kubienová L, Tichá T, Jahnová J, Luhová L, Mieslerová B, Petrřivalský M (2014) Effect of abiotic stress stimuli on S-nitrosoglutathione reductase in plants. Planta 239:139–146CrossRefPubMedGoogle Scholar
  21. Lamotte O, Bertoldo JB, Besson-Bard A, Rosnoblet C, Aimé S, Hichami S, Terenzi H, Wendehenne D (2015) Protein S-nitrosylation: specificity and identification strategies in plants. Front Chem 2:114CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lebeda A, Mieslerová B (2011) Taxonomy, distribution and biology of lettuce powdery mildew (Golovinomyces cichoracearum sensu stricto). Plant Pathol 60:400–415CrossRefGoogle Scholar
  23. Lebeda A, Sedlářová M, Petřivalský M, Prokopová J (2008) Diversity of defence mechanisms in plant–oomycete interactions: a case study of Lactuca spp. and Bremia lactucae. Eur J Plant Pathol 122:71–89CrossRefGoogle Scholar
  24. Lebeda A, Mieslerová B, Petrželová I, Korbelová P, Česneková E (2012) Patterns of virulence variation in the interaction between Lactuca spp. and lettuce powdery mildew (Golovinomyces cichoracearum). Fungal Ecol 5:670–682CrossRefGoogle Scholar
  25. Lebeda A, Mieslerová B, Petrželová I, Korbelová P (2013) Host specificity and virulence variation in populations of lettuce powdery mildew pathogen (Golovinomyces cichoracearum s. str.) from prickly lettuce (Lactuca serriola). Mycol Prog 12:533–545CrossRefGoogle Scholar
  26. Lebeda A, Křístková E, Kitner M, Mieslerová B, Jemelková M, Pink DAC (2014a) Wild Lactuca species, their genetic diversity, resistance to diseases and pests, and exploitation in lettuce breeding. Eur J Plant Pathol 138:597–640CrossRefGoogle Scholar
  27. Lebeda A, Mieslerová B, Petřivalský M, Luhová L, Špundová M, Sedlářová M, Nožková-Hlaváčková V, Pink DAC (2014b) Resistance mechanisms of wild tomato germplasm to infection of Oidium neolycopersici. Eur J Plant Pathol 138:69–596Google Scholar
  28. Lee U, Wie C, Fernandez BO, Feelisch M, Vierling E (2008) Modulation of nitrosative stress by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis. Plant Cell 20:786–802CrossRefPubMedPubMedCentralGoogle Scholar
  29. Leterrier M, Chaki M, Airaki M, Valderrama R, Palma JM, Barroso JB, Corpas FJ (2011) Function of S-nitrosoglutathione reductase (GSNOR) in plant development and under biotic/abiotic stress. Plant Signal Behav 6:789–793CrossRefPubMedPubMedCentralGoogle Scholar
  30. Leterrier M, Airaki M, Palma JM, Chaki M, Barroso JB, Corpas FJ (2012) Arsenic triggers the nitric oxide (NO) and S-nitrosoglutathione (GSNO) metabolism in Arabidopsis. Environ Pollut 166:136–143CrossRefPubMedGoogle Scholar
  31. Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS (2001) A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature 410:490–494CrossRefPubMedGoogle Scholar
  32. Malik SI, Hussain A, Yun B-W, Spoel SH, Loake GJ (2011) GSNOR-mediated de-nitrosylation in the plant defence response. Plant Sci 181:540–544CrossRefPubMedGoogle Scholar
  33. MieslerovÁ B, Lebeda A, Kennedy R (2004) Variation in Oidium neolycopersici development on host and non-host plant species and their tissue defence responses. Ann Appl Biol 144:237–248CrossRefGoogle Scholar
  34. Moore KP, Mani AR (2002) Measurement of protein nitration and S-nitrosothiol formation in biology and medicine. Methods Enzymol 359:256–268CrossRefPubMedGoogle Scholar
  35. Mur LAJ, Mandon J, Persijn S, Cristescu SM, Moshkov IE, Novikova GV, Hall MA, Harren FJM, Hebelstrup KH, Gupta KJ (2013) Nitric oxide in plants: an assessment of the current state of knowledge. AoB Plants 5:pls052CrossRefPubMedGoogle Scholar
  36. Parra L, Maisonneuve B, Lebeda A, Schut J, Christopoulou M, Jeuken M, McHale L, Truco M-J, Crute I, Michelmore R (2016) Rationalization of genes for resistance to Bremia lactucae in lettuce. Euphytica 210:309–326CrossRefGoogle Scholar
  37. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedPubMedCentralGoogle Scholar
  38. Pietrowska E, Różalska S, Kaźmierczak A, Nawrocka J, Małolepsza U (2015) Reactive oxygen and nitrogen (ROS and RNS) species generation and cell death in tomato suspension cultures–Botrytis cinerea interaction. Protoplasma 252:307–319CrossRefPubMedGoogle Scholar
  39. Piterková J, Petřivalský M, Luhová L, Mieslerová B, Sedlářová M, Lebeda A (2009) Local and systemic production of nitric oxide in tomato responses to powdery mildew infection. Mol Plant Pathol 10:501–513CrossRefPubMedGoogle Scholar
  40. Piterková J, Hofman J, Mieslerová B, Sedlářová M, Luhová L, Lebeda A, Petřivalský M (2011) Dual role of nitric oxide in Solanum spp.–Oidium neolycopersici interactions. Environ Exp Bot 74:37–44CrossRefGoogle Scholar
  41. Piterková J, Luhová L, Mieslerová B, Lebeda A, Petřivalský M (2013) Nitric oxide and reactive oxygen species regulate the accumulation of heat shock proteins in tomato leaves in response to heat shock and pathogen infection. Plant Sci 207:57–65CrossRefPubMedGoogle Scholar
  42. Rusterucci C, Espunya MC, Díaz M, Chabannes M, Martínez MC (2007) S-Nitrosoglutathione reductase affords protection against pathogens in Arabidopsis, both locally and systemically. Plant Physiol 143:1282–1292CrossRefPubMedPubMedCentralGoogle Scholar
  43. Salgado I, Martínez C, Oliveira H, Frungillo L (2013) Nitric oxide signaling and homeostasis in plants: a focus on nitrate reductase and S-nitrosoglutathione reductase in stress-related responses. Braz J Bot 36:89–98CrossRefGoogle Scholar
  44. Schlicht M, Kombrink E (2013) The role of nitric oxide in the interaction of Arabidopsis thaliana with the biotrophic fungi, Golovinomyces orontii and Erysiphe pisi. Front Plant Sci 4:351CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sedlářová M, Lebeda A, Pink DAC (2001) The early stages of interaction between effective and non-effective race-specific genes in Lactuca sativa, wild Lactuca spp. and Bremia lactucae (race NL16). J Plant Dis Protect 108:477–489Google Scholar
  46. Sedlářová M, Luhová L, Petřivalský M, Lebeda A (2007) Localisation and metabolism of reactive oxygen species during Bremia lactucae pathogenesis in Lactuca sativa and wild Lactuca spp. Plant Physiol Biochem 45:607–616CrossRefPubMedGoogle Scholar
  47. Sedlářová M, Petřivalský M, Piterková J, Luhová L, Kočiřová J, Lebeda A (2011) Influence of nitric oxide and reactive oxygen species on development of lettuce downy mildew in Lactuca spp. Eur J Plant Pathol 129:267–280CrossRefGoogle Scholar
  48. Sedlářová M, Kubienová L, Drábková Trojanová Z, Luhová L, Lebeda A, Petřivalský M (2016) The role of nitric oxide in development and pathogenesis of biotrophic phytopathogens—downy and powdery mildews. Adv Bot Res 77:263–283CrossRefGoogle Scholar
  49. Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, Wang C, Zuo J, Dong X (2008) Plant immunity requires conformational changes of NPR1 via S-nitrosylation and thioredoxins. Science 321:952–956CrossRefPubMedGoogle Scholar
  50. Tichá T, Činčalová L, Kopečný D, Sedlářová M, Kopečná M, Luhová L, Petřivalský M (2017) Characterization of S-nitrosoglutathione reductase from Brassica and Lactuca spp. and its modulation during plant development. Nitric Oxide 68:68–76CrossRefPubMedGoogle Scholar
  51. Tomanková K, Luhová L, Petřivalský M, Peč P, Lebeda A (2006) Biochemical aspects of reactive oxygen species formation in the interaction between Lycopersicon spp. and Oidium neolycopersici. Physiol Mol Plant Pathol 68:22–32CrossRefGoogle Scholar
  52. Wünsche H, Baldwin IT, Wu J (2011) S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata. J Exp Bot 62:4605–4616CrossRefPubMedPubMedCentralGoogle Scholar
  53. Xu S, Guerra D, Lee U, Vierling E (2013) S-Nitrosoglutathione reductases are low-copy number, cysteine-rich proteins in plants that control multiple developmental and defense responses in Arabidopsis. Front Plant Sci 4:430CrossRefPubMedPubMedCentralGoogle Scholar
  54. Yu M, Lamattina L, Spoel SH, Loake GJ (2014) Nitric oxide function in plant biology: a redox cue in deconvolution. New Phytol 202:1142–1156CrossRefPubMedGoogle Scholar
  55. Yun BW, Feechan A, Yin M, Saidi NB, Le Bihan T, Yu M, Moore JW, Kang JG, Kwon E, Spoel SH (2011) S-Nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature 478:264–268CrossRefPubMedGoogle Scholar
  56. Yun BW, Skelly MJ, Yin M, Yu M, Mun B-G, Lee SU, Hussain A, Spoel SH, Loake GJ (2016) Nitric oxide and S-nitrosoglutathione function additively during plant immunity. New Phytol 211:516–526CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Tereza Tichá
    • 1
  • Michaela Sedlářová
    • 2
  • Lucie Činčalová
    • 1
  • Zuzana Drábková Trojanová
    • 2
  • Barbora Mieslerová
    • 2
  • Aleš Lebeda
    • 2
  • Lenka Luhová
    • 1
  • Marek Petřivalský
    • 1
  1. 1.Department of Biochemistry, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  2. 2.Department of Botany, Faculty of SciencePalacký UniversityOlomoucCzech Republic

Personalised recommendations