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

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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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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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Authors and Affiliations

  1. Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina

    Telma E. Scarpeci, María I. Zanor & Estela M. Valle

  2. Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 24-25, Haus 20, 14476, Potsdam-Golm, Germany

    Bernd Mueller-Roeber

  3. Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany

    Bernd Mueller-Roeber

Authors
  1. Telma E. Scarpeci
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  2. María I. Zanor
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  3. Bernd Mueller-Roeber
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  4. Estela M. Valle
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Corresponding author

Correspondence to Estela M. Valle.

Electronic supplementary material

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11103_2013_90_MOESM1_ESM.tif

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)

11103_2013_90_MOESM2_ESM.tif

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)

11103_2013_90_MOESM3_ESM.tif

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)

11103_2013_90_MOESM4_ESM.tif

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)

11103_2013_90_MOESM5_ESM.tif

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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