Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana

Abstract

AtWRKY30 belongs to a higher plant transcription factor superfamily, which responds to pathogen attack. In previous studies, the AtWRKY30 gene was found to be highly and rapidly induced in Arabidopsis thaliana leaves after oxidative stress treatment. In this study, electrophoretic mobility shift assays showed that AtWRKY30 binds with high specificity and affinity to the WRKY consensus sequence (W-box), and also to its own promoter. Analysis of the AtWRKY30 expression pattern by qPCR and using transgenic Arabidopsis lines carrying AtWRKY30 promoter-β-glucuronidase fusions showed transcriptional activity in leaves subjected to biotic or abiotic stress. Transgenic Arabidopsis plants constitutively overexpressing AtWRKY30 (35S::W30 lines) were more tolerant than wild-type plants to oxidative and salinity stresses during seed germination. The results presented here show that AtWRKY30 is responsive to several stress conditions either from abiotic or biotic origin, suggesting that AtWRKY30 could have a role in the activation of defence responses at early stages of Arabidopsis growth by binding to W-boxes found in promoters of many stress/developmentally regulated genes.

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References

  1. Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Gorlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510

    PubMed  CAS  Google Scholar 

  2. Chen C, Chen Z (2000) Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco. Plant Mol Biol 42:387–396

    PubMed  Article  CAS  Google Scholar 

  3. Ciolkowski I, Wanke D, Birkenbihl RP, Somssich IE (2008) Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Mol Biol 68:81–92

    PubMed  Article  CAS  Google Scholar 

  4. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    PubMed  Article  CAS  Google Scholar 

  5. Cormack RS, Eulgem T, Rushton PJ, Kochner P, Hahlbrock K, Somssich IE (2002) Leucine zipper-containing WRKY proteins widen the spectrum of immediate early elicitor-induced WRKY transcription factors in parsley. Biochim Biophys Acta 1576:92–100

    PubMed  Article  CAS  Google Scholar 

  6. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17

    PubMed  Article  CAS  Google Scholar 

  7. Dong J, Chen C, Chen Z (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51:21–37

    PubMed  Article  CAS  Google Scholar 

  8. Du L, Chen Z (2000) Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis. Plant J 24:837–847

    PubMed  Article  CAS  Google Scholar 

  9. Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signalling. Curr Opin Plant Biol 10:366–371

    PubMed  Article  CAS  Google Scholar 

  10. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206

    PubMed  Article  CAS  Google Scholar 

  11. Fahnenstich H, Scarpeci TE, Valle EM, Flugge UI, Maurino VG (2008) Generation of hydrogen peroxide in chloroplasts of Arabidopsis overexpressing glycolate oxidase as an inducible system to study oxidative stress. Plant Physiol 148:719–729

    PubMed  Article  CAS  Google Scholar 

  12. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signalling networks. Curr Opin Plant Biol 9:436–442

    PubMed  Article  Google Scholar 

  13. Gapper C, Dolan L (2006) Control of plant development by reactive oxygen species. Plant Physiol 141:341–345

    PubMed  Article  CAS  Google Scholar 

  14. Heslop-Harrison J, Heslop-Harrison Y (1985) Germination of stress-tolerant Eucalyptus pollen. J Cell Sci 73:135–157

    PubMed  CAS  Google Scholar 

  15. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300

    PubMed  Article  CAS  Google Scholar 

  16. Jiang M, Zhang J (2003) Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings. Plant, Cell Environ 26:929–939

    Article  CAS  Google Scholar 

  17. Journot-Catalino N, Somssich IE, Roby D, Kroj T (2006) The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant Cell 18:3289–3302

    PubMed  Article  CAS  Google Scholar 

  18. Kalde M, Barth M, Somssich IE, Lippok B (2003) Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signalling pathways. Mol Plant Microbe Interact 16:295–305

    PubMed  Article  CAS  Google Scholar 

  19. Koschmann J, Machens F, Becker M, Niemeyer J, Schulze J, Bülow L, Stahl DJ, Hehl R (2012) Integration of bioinformatics and synthetic promoters leads to discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis thaliana. Plant Physiol. doi:10.1104/pp.112.198259

    PubMed  Google Scholar 

  20. Lippok B, Birkenbihl RP, Rivory G, Brümmer J, Schmelzer E, Logemann E, Somssich IE (2007) Expression of AtWRKY33 encoding a pathogen- or PAMP-responsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements. Mol Plant Microbe Interact 20:420–429

    PubMed  Article  CAS  Google Scholar 

  21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    PubMed  Article  CAS  Google Scholar 

  22. Lu D, Lin W, Gao X, Wu S, Cheng C, Avila J, Heese A, Devarenne TP, He P, Shan L (2011) Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity. Science 332:1439–1442

    PubMed  Article  CAS  Google Scholar 

  23. Mao G, Meng X, Liu Y, Zheng Z, Chen Z, Zhang S (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23(4):1639–1653

    PubMed  Article  CAS  Google Scholar 

  24. McInnis SM, Desikan R, Hancock JT, Hiscock SJ (2006) Production of reactive oxygen species and reactive nitrogen species by angiosperm stigmas and pollen: potential signalling crosstalk? New Phytol 172:221–228

    PubMed  Article  CAS  Google Scholar 

  25. Murata Y, Pei ZM, Mori IC, Schroeder J (2001) Abscisic acid activation of plasma membrane Ca(2 +) 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. Plant Cell 13:2513–2523

    PubMed  CAS  Google Scholar 

  26. Nylander M, Svensson J, Palva ET, Welin BV (2001) Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant Mol Biol 45:263–279

    PubMed  Article  CAS  Google Scholar 

  27. Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, Park CY, Jeong JC, Moon BC, Lee JH, Yoon HW, Lee SH, Chung WS, Lim CO, Lee SY, Hong JC, Cho MJ (2004) Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiol 135:2150–2161

    PubMed  Article  CAS  Google Scholar 

  28. Pei ZM, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406:731–734

    PubMed  Article  CAS  Google Scholar 

  29. Rober M, Geider K, Muller-Rober B, Willmitzer L (1996) Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates. Planta 199:528–536

    PubMed  Article  CAS  Google Scholar 

  30. Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15:5690–5700

    PubMed  CAS  Google Scholar 

  31. Salinas-Mondragon RE, Garciduenas-Pina C, Guzman P (1999) Early elicitor induction in members of a novel multigene family coding for highly related RING-H2 proteins in Arabidopsis thaliana. Plant Mol Biol 40:579–590

    PubMed  Article  CAS  Google Scholar 

  32. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. CSHL Press, Cold Spring Harbor, New York

    Google Scholar 

  33. Scarpeci TE, Marro ML, Bortolotti S, Boggio SB, Valle EM (2007) Plant nutritional status modulates glutamine synthetase levels in ripe tomatoes (Solanum lycopersicum cv. Micro-Tom). J Plant Physiol 164:137–145

    PubMed  Article  CAS  Google Scholar 

  34. Scarpeci TE, Zanor MI, Carrillo N, Mueller-Roeber B, Valle EM (2008) Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes. Plant Mol Biol 66:361–378

    PubMed  Article  CAS  Google Scholar 

  35. Sgro G, Ficarra F, Dunger G, Scarpeci T, Valle EM, Orellano E, Gottig N, Ottado J (2012) Contribution of a harpin protein from Xanthomonas axonopodis pv. citri to pathogen virulence. Mol Plant Pathol 13:1047–1059

    PubMed  Article  CAS  Google Scholar 

  36. Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479

    PubMed  Article  CAS  Google Scholar 

  37. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414:562–565

    PubMed  Article  CAS  Google Scholar 

  38. Yamamoto YY, Ichida H, Matsui M, Obokata J, Sakurai T, Satou M, Seki M, Shinozaki K, Abe T (2007) Identification of plant promoter constituents by analysis of local distribution of short sequences. BMC Genomics 8:67

    PubMed  Article  Google Scholar 

  39. Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Tomo Y, Hayami N, Terada T, Shirouzu M, Tanaka A, Seki M, Shinozaki K, Yokoyama S (2005) Solution structure of an Arabidopsis WRKY DNA binding domain. Plant Cell 17:944–956

    PubMed  Article  CAS  Google Scholar 

  40. Yu D, Chen C, Chen Z (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13:1527–1540

    PubMed  CAS  Google Scholar 

  41. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song CP (2001) Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol 126:1438–1448

    PubMed  Article  CAS  Google Scholar 

  42. Zou C, Sun K, Mackaluso JD, Seddon AE, Jin R, Thomashow MF, Shiu SH (2011) Cis-regulatory code of stress-responsive transcription in Arabidopsis thaliana. Proc Natl Acad Sci USA 108:14992–14997

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

The work described in this article was performed with the financial support of the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina. Bernd Mueller-Roeber thanks the Fond der Chemischen Industrie for funding (No. 0164389).

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Correspondence to Estela M. Valle.

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Supplementary Fig. 1

DNA binding assay of recombinant AtWRKY30 protein to 32P-labelled SP promoter fragment (0.28 kb) encompassing four W-boxes (Fig. 1). Total recombinant AtWRKY30 protein extract (40 μg) from E. coli was used for DNA binding assays with 0.28 kb 32P-labelled promoter fragment (+). As a control, total protein extract (40 μg) from E. coli that did not produce recombinant AtWRKY30 protein was used (-). FP: free probe. (TIFF 2760 kb)

Supplementary Fig. 2

GUS activity in LP #1 transgenic plants. (a) Scheme of plasmid pBI101 with 1.96 kb (LP) DNA fragment from the AtWRKY30 promoter cloned upstream of the β-glucuronidase gene. (b) Leaves were subjected to biotic, abiotic and hormone treatments. LP #1 plants were treated with a solution containing: 20 mM H2O2, 100 mM NaCl, 200 mM mannitol or 100 μM sodium arsenate all in 0.005 % (v/v) Silwet L-77, and incubated for 4 h, or kept without watering for 15 days and then re-watered (indicated as drought). Biotic treatments were assayed by syringe infiltration with X. axonopodis or P. syringae pv. tomato DC3000 (108 cfu ml−1) or by vacuum infiltrating leaves with bacterial (A. tumefaciens) elicitor. The left half of a leaf infiltrated with a solution without A. tumefaciens is also shown. The arrows indicate the site of infiltration using a syringe with the X. axonopodis or P. syringae bacterial suspension or with MgCl2 (control) and the wounded tissue in the A. tumefaciens treated leaf. For hormone treatments, leaves were incubated with 5 mM ethephon, 100 μM ACC or 100 μM MeJA. Representative images are shown. (TIFF 15192 kb)

Supplementary Fig. 3

GUS staining detected in SP #1 line. (a) Scheme of plasmid pBI101 with the 0.28 kb (SP) DNA fragment from the AtWRKY30 promoter cloned upstream of β-glucuronidase gene. (b) AtWRKY30 promoter activity was followed by histochemical localization of GUS activity in leaves or cauline leaves after 24 h of 200 mM mannitol or 100 mM NaCl treatment. Representative images are shown. (TIFF 6932 kb)

Supplementary Fig. 4

Fresh weight (in mg) of seedlings after growth on (a) 1 μM MV or (b) 150 mM NaCl; shown are data for 35S::W30-1, 35S::W30-8, 35S::W30-21, empty vector control (EV) and wild type (Col-0) plants. Age-matched seeds of all lines were germinated in parallel on 0.5x MS agar plates containing either MV or NaCl and scoring was carried out after 10 days of treatment. Error bars represent the SE over three replicate experiments, each including at least 30 plants of each line. Experimental data were subjected to One-way ANOVA test. Significant difference between Col-0 and 35S::W30 plants (P < 0.05) is indicated by an asterisk. (TIFF 7202 kb)

Supplementary Fig. 5

GUS staining detected in LP #1 line during germination. Seeds from LP #1 line were germinated and grown in MS 0.5x agar plates (a), MS 0.5x agar plates supplemented with 0.5 μM MV (b) or 100 mM NaCl (c). GUS staining was carried out when seedlings were four, six and eight-day-old. Representative images are shown. (TIFF 8387 kb)

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Scarpeci, T.E., Zanor, M.I., Mueller-Roeber, B. et al. Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana . Plant Mol Biol 83, 265–277 (2013). https://doi.org/10.1007/s11103-013-0090-8

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Keywords

  • Antioxidant response
  • Chloroplast
  • Germination
  • Oxidative stress
  • Stress signaling