Plant Molecular Biology

, Volume 64, Issue 1–2, pp 1–15 | Cite as

Overexpression of salicylic acid carboxyl methyltransferase reduces salicylic acid-mediated pathogen resistance in Arabidopsis thaliana

  • Yeon Jong Koo
  • Myeong Ae Kim
  • Eun Hye Kim
  • Jong Tae Song
  • Choonkyun Jung
  • Joon-Kwan Moon
  • Jeong-Han Kim
  • Hak Soo Seo
  • Sang Ik Song
  • Ju-Kon Kim
  • Jong Seob Lee
  • Jong-Joo Cheong
  • Yang Do Choi


We cloned a salicylic acid/benzoic acid carboxyl methyltransferase gene, OsBSMT1, from Oryza sativa. A recombinant OsBSMT1 protein obtained by expressing the gene in Escherichia coli exhibited carboxyl methyltransferase activity in reactions with salicylic acid (SA), benzoic acid (BA), and de-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid (dSM-BTH), producing methyl salicylate (MeSA), methyl benzoate (MeBA), and methyl dSM-BTH (MeBTH), respectively. Compared to wild-type plants, transgenic Arabidopsis overexpressing OsBSMT1 accumulated considerably higher levels of MeSA and MeBA, some of which were vaporized into the environment. Upon infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen Golovinomyces orontii, transgenic plants failed to accumulate SA and its glucoside (SAG), becoming more susceptible to disease than wild-type plants. OsBSMT1-overexpressing Arabidopsis showed little induction of PR-1 when treated with SA or G. orontii. Notably, incubation with the transgenic plant was sufficient to trigger PR-1 induction in neighboring wild-type plants. Together, our results indicate that in the absence of SA, MeSA alone cannot induce a defense response, yet it serves as an airborne signal for plant-to-plant communication. We also found that jasmonic acid (JA) induced AtBSMT1, which may contribute to an antagonistic effect on SA signaling pathways by depleting the SA pool in plants.


Arabidopsis Methyl salicylate (MeSA) Plant disease resistance Rice SA carboxyl methyltransferase Salicylic acid (SA) 



This work was supported by grants from the Crop Functional Genomics Center (CG2112 to JJC and CG2111 to JK) and the Korea Research Foundation (KRF-2004-005-F00013 to YDC). Financial support, including graduate research assistantships to YJK and CJ, from the Brain Korea 21 project of the Ministry of Education is also acknowledged. Special thanks are due to Professors In Gyu Hwang and Soon Ok Kim (Seoul National University) for the identification of Golovinomyces orontii.

Supplementary material

11103_2006_9123_Fig8_ESM.gif (36 kb)

Enhanced disease susceptibility of OsBSMT1-overexpressing plants to the avirulent bacterial pathogen Pseudomonas syringae pv. maculicola strain DG6 and their response. (A) Enhanced disease susceptibility of OsBSMT1-overexpressing plants. Wild-type (left) and OsS6 (right) plants were inoculated with P. Syringae DG6. Infected leaves were photographed after 3 days of inoculation. (B) Growth of P. syringae in plants. Wild-type (circles) and OsS6 (triangles) plants were inoculated with P. syringae DG6. On days 2 and 3, P. Syringae grew faster in OsS6 plants than in wild-type plants (P < 0.009, t-test, n = 8). Bars indicate standard deviation. This experiment was repeated two times with similar results. (C) Expression of PR-1 during infection. The fourth or fifth leaves of wild-type and OsS6 plants were infected with P. syringae at OD600 = 0.01 (approximately 5 × 106 cfu/ml) or mock-infected with 10 mM MgSO4. RNA at the indicated time point was isolated, and Northern blot analysis was conducted (GIF 37 kb)

11103_2006_9123_Fig9_ESM.gif (46 kb)

SA and BTH insensitivity of OsBSMT1 plants. (A) Northern blot analysis of PR-1 gene expression. Treatment with SA, BTH, or dSM-BTH was performed by spraying wild-type, OsBSMT1, and nahG plants. RNAs were isolated after 1 day of treatment. (B) Metabolism of BTH in plants. In plants, BTH is hydrolyzed to produce dSM-BTH (hydrolyzed BTH) and subsequently conjugated with sugars (Tomlin, 2003). The hydrolyzed BTH is converted to MeBTH by the OsBSMT1 enzyme, as shown in Figure 2. Both BTH and hydrolyzed BTH are known activators of plant disease resistance (Kunz et al., 1997). All experiments were repeated three times with similar results (GIF 47 kb)


  1. Adam L, Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana. Plant J 9:341–356PubMedCrossRefGoogle Scholar
  2. Baldwin IT, Halitschke R, Paschold A, von Dahl CC, Preston CA (2006) Volatile signaling in plant–plant interactions: “talking trees” in the genomics era. Science 311:812–815PubMedCrossRefGoogle Scholar
  3. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88:57–63PubMedCrossRefGoogle Scholar
  4. Chen F, D’Auria JC, Tholl D, Ross JR, Gershenzon J, Noel JP, Pichersky E (2003) An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense. Plant J 36:577–588PubMedCrossRefGoogle Scholar
  5. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  6. Coquoz J-L, Buchala A, Métraux J-P (1998) The biosynthesis of salicylic acid in potato plants. Plant Physiol 117:1095–1101PubMedCrossRefGoogle Scholar
  7. D’Auria JC, Chen F, Pichersky E (2002) Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. Plant Physiol 130:466–476PubMedCrossRefGoogle Scholar
  8. Dean JV, Mohammed LA, Fitzpatrick T (2005) The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures. Planta 221:287–296PubMedCrossRefGoogle Scholar
  9. Delaney TP, Uknes S, Vernooij B, Friedrich L, Weymann K, Negrotto D, Gaffney T, Gut-Rella M, Kessmann H, Ward E, Ryals J (1994) A central role of salicylic acid in plant disease resistance. Science 266:1247–1250CrossRefPubMedGoogle Scholar
  10. Dempsey DA, Shah J, Klessig DF (1999) Salicylic acid and disease resistance in plants. Crit Rev Plant Sci 18:547–575CrossRefGoogle Scholar
  11. Dewdney J, Reuber TL, Wildermuth MC, Devoto A, Cui J, Stutius LM, Drummond EP, Ausubel FM (2000) Three unique mutants of Arabidopsis identify eds loci required for limiting growth of biotrophic fungal pathogen. Plant J 24:205–218PubMedCrossRefGoogle Scholar
  12. Doares SH, Harváez-Vásquez J, Conconi A, Ryan CA (1995) Salicylic acid inhibits synthesis of proteinase inhibitors in tomato leaves induced by systemin and jasmonic acid. Plant Physiol 108:1741–1746PubMedGoogle Scholar
  13. Doherty HM, Selvendran RR, Bowles DJ (1988) The wound response of tomato plants can be ingibited by aspirin and related hydroxyl-benzoic acid. Physiol Mol Plant Pathol 33:377–384CrossRefGoogle Scholar
  14. Dong X (2004) NPR1, all things considered. Curr Opin Plant Biol 7:547–552PubMedCrossRefGoogle Scholar
  15. Effmert U, Saschenbrecker S, Ross J, Negre F, Fraser CM, Noel JP, Dudareva N, Piechulla B (2005) Floral benzenoid carboxyl methyltransferases: from in vitro to in planta function. Phytochemistry 66:1211–1230PubMedCrossRefGoogle Scholar
  16. Engelberth J, Schmelz EA, Alborn HT, Cardoza YJ, Huang J, Tumlinson JH (2003) Simultaneous quantification of jasmonic acid and salicylic acid in plants by vapor-phase extraction and gas chromatography-chemical ionization-mass spectrometry. Anal Biochem 312:242–250PubMedCrossRefGoogle Scholar
  17. Forouhar F, Yang Y, Kumar D, Chen Y, Fridman E, Park SW, Chiang Y, Acton TB, Montelione GT, Pichersky E, Klessig DF, Tong L (2005) Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity. Proc Natl Acad Sci USA 102:1773–1778PubMedCrossRefGoogle Scholar
  18. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J (1993) Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261:754–756CrossRefPubMedGoogle Scholar
  19. Greenberg JT, Silverman FP, Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5. Genetics 156:341–350PubMedGoogle Scholar
  20. Görlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U, Kogel KH, Oostendorp M, Staub T, Ward E, Kessmann H, Ryals J (1996) Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8:629–643PubMedCrossRefGoogle Scholar
  21. Gupta V, Willits MG, Glazebrook J (2000) Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses; evidence for inhibition of jasmonic acid signaling by SA. Mol Plant–Microbe Interact 13:503–511PubMedGoogle Scholar
  22. Harms K, Ramirez I, Peña-Cortés H (1998) Inhibition of wound-induced accumulation fo allene oxide synthase transcripts in flax leaves by aspirin and salicylic acid. Plant Physiol 118:1057–1065PubMedCrossRefGoogle Scholar
  23. Huang J, Cardoza YJ, Schmelz EA, Raina R, Engelberth J, Tumlinson JH (2003) Differential volatile emissions and salicylic acid levels from tobacco plants in response to different strains of Pseudomonas syringae. Planta 217:767–775PubMedCrossRefGoogle Scholar
  24. Kachroo P, Shanklin J, Shah J, Whittle EJ, Klessig DF (2001) A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc Natl Acad Sci USA 98:9448–9453PubMedCrossRefGoogle Scholar
  25. Kloek AP, Verbsky ML, Sharma SB, Schoelz JE, Vogel J, Klessig DF, Kunkel BN (2001) Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. Plant J 26:509–522PubMedCrossRefGoogle Scholar
  26. Knudsen JT, Tollsten L, Bergstrom LG (1993) Floral scents—a checklist of volatile compounds isolated by headspace techniques. Phytochemistry 33:253–280CrossRefGoogle Scholar
  27. Kohler A, Schwindling S, Conrath U (2002) Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding and infiltration of water into leaves requires the NPR1/NIM1 gene in arabidosis. Plant Physiol 128:1046–1056PubMedCrossRefGoogle Scholar
  28. Kumar D, Klessig DF (2003) High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity. Proc Natl Acad Sci USA 100:16101–16106 PubMedCrossRefGoogle Scholar
  29. Kunz W, Schurter R, Maetzke T (1997) The chemistry of benzothiadiazole plant activators. Pestic Sci 50:275–282CrossRefGoogle Scholar
  30. Lee H-I, Leon J, Raskin I (1995) Biosynthesis and metabolism of salicylic acid. Proc Natl Acad Sci USA 92:4076–4079PubMedCrossRefGoogle Scholar
  31. Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16:319–331PubMedCrossRefGoogle Scholar
  32. Lou Y-G, Du M-H, Turlings TCJ, Cheng J-A, Shan W-F (2005) Exogenous application of jasmonic acid induces volatile emissions in rice and enhances parasitism of Nilaparvata lugens eggs by the parasitoid Anagrus nilaparvatae. J Chem Ecol 31:1985–2002PubMedCrossRefGoogle Scholar
  33. MoriY, SatoY, Takamatsu S (2000) Evolutionary analysis of the powdery mildew fungi nucleotide sequences of the nuclear ribosomal DNA. Mycologia 92:74–93CrossRefGoogle Scholar
  34. Murry JM, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325CrossRefGoogle Scholar
  35. Negre F, Kolosova N, Knoll J, Kish CM, Dudareva N (2002) Novel S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers. Arch Biochem Biophys 406:261–270PubMedCrossRefGoogle Scholar
  36. Obara N, Hasegawa M, Kodama O (2002) Induced volatiles in elicitor-treated and rice blast fungus-inoculated rice leaves. Biosci Biotechnol Biochem 66:2549–2559PubMedCrossRefGoogle Scholar
  37. Petersen M, Brodersen P, Naested H, Andreasson E, Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin MJ, Parker JE, Sharma SB, Klessig DF, Martienssen R, Mattsson O, Jensen AB, Mundy J (2000) Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103:1111–1120PubMedCrossRefGoogle Scholar
  38. Pott MB, Pichersky E, Piechulla B (2002) Evening specific oscillations of scent emission, SAMT enzyme activity, and SAMT mRNA in flowers of Stephanotis floribunda. J Plant physiol 159:925–934CrossRefGoogle Scholar
  39. Qin G, Gu H, Zhao Y, Ma Z, Shi G, Yang Y, Pichersky E, Chen H, Liu H, Chen Z, Qu L-J (2005) An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development. Plant Cell 17:2693–2704PubMedCrossRefGoogle Scholar
  40. Reuber TL, Plotnikova JM, Dewdney J, Rogers EE, Wood W, Ausubel FM (1998) Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants. Plant J 16:473–485PubMedCrossRefGoogle Scholar
  41. Ross JR (2002) S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase (SAMT), an enzyme involved in floral scent and plant defense in Clarkia breweri. PhD Thesis. University of Michigan, Ann ArborGoogle Scholar
  42. Ross JR, Nam KH, D’Auria JC, Pichersky E (1999) S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. Arch Biochem Biophys 367:9–16PubMedCrossRefGoogle Scholar
  43. Schuurink RC, Haring MA, Clark DG (2006) Regulation of volatile benzenoid biosynthesis in petunia flowers. Trends Plant Sci 11:20–25PubMedCrossRefGoogle Scholar
  44. Seo HS, Song JT, Cheong JJ, Lee YH, Lee YW, Hwang IG, Lee JS, Choi YD (2001) Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses. Proc Natl Acad Sci USA 98:4788–4793PubMedCrossRefGoogle Scholar
  45. Seskar M, Shulaev V, Raskin I (1998) Endogenous methyl salicylate in pathogen-inoculated tobacco plants. Plant Physiol 116:387–392CrossRefGoogle Scholar
  46. Shulaev V, Silverman P, Raskin I (1997) Airborne signaling by methyl salicylate in plant pathogen resistance. Nature 385:718–721CrossRefGoogle Scholar
  47. Spoel SH, Koornneef A, Claessens SM, Korzelius JP, Van Pelt JA, Mueller MJ, Buchala AJ, Metraux JP, Brown R, Kazan K, Van Loon LC, Dong X, Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15:760–770PubMedCrossRefGoogle Scholar
  48. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTALX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  49. Tomlin (2003) Plant activator acibenzolar-S-methyl. In: The Pesticide Manual (eds) British crop protection council. British Crop Protection Council Publications, Hampshire, pp 9–10Google Scholar
  50. Van Poecke RMP, Posthumus MA, Dicke M (2001) Herbivore-induced volatile production by Arabidopsis thaliana leads to attraction of the parasitoid Cotesia rubecula: chemical, behavioral, and gene expression analysis. J Chem Ecol 27:1911–1928PubMedCrossRefGoogle Scholar
  51. Van Wees SC, Glazebrook J (2003) Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. Phaseolicola is due to degradation products of salicylic acid. Plant J 33:733–742PubMedCrossRefGoogle Scholar
  52. White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols (ed) A guide to methods and applications. Academic Press Inc., New York, pp 315–322Google Scholar
  53. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414:562–565PubMedCrossRefGoogle Scholar
  54. Yalpani N, León J, Lawton MA, Raskin I (1993) Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiol 103:315–321PubMedGoogle Scholar
  55. Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, Noel JP (2003) Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell 15:1704–1716PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Yeon Jong Koo
    • 1
  • Myeong Ae Kim
    • 1
  • Eun Hye Kim
    • 2
  • Jong Tae Song
    • 3
  • Choonkyun Jung
    • 1
  • Joon-Kwan Moon
    • 1
  • Jeong-Han Kim
    • 1
  • Hak Soo Seo
    • 4
  • Sang Ik Song
    • 2
  • Ju-Kon Kim
    • 2
  • Jong Seob Lee
    • 5
  • Jong-Joo Cheong
    • 1
  • Yang Do Choi
    • 1
  1. 1.School of Agricultural BiotechnologySeoul National UniversitySeoul Korea
  2. 2.Division of BioscienceMyongji UniversityYongin Korea
  3. 3.Division of Plant BiosciencesKyungpook National UniversityDaegu Korea
  4. 4.Department of Plant ScienceSeoul National UniversitySeoul Korea
  5. 5.School of Biological SciencesSeoul National UniversitySeoul Korea

Personalised recommendations