Abstract：WRKY transcription factors constitute a large family in plants and play important roles in many different physiological processes. Many members of WRKY genes have been studied in various processes including abiotic and biotic stress tolerance in Arabidopsis thaliana. However, the functions and molecular mechanisms of many WRKY genes are still unknown. In this report, the WRKY72 gene in Arabidopsis, which is a member of group Ⅱb is studied. In order to explore the characteristics of WRKY72 gene and its role in abiotic stress tolerance, various cellular and molecular biology approaches were employed. Subcellular localization using green fluorescence protein reporter gene (GFP) in leaves of Nicotiana benthamiana showed that the WRKY72 transcription factor was localized in the nucleus of leaf epidermal cells. qRT-PCR was used to examine its response to multiple stress and phytohormone treatments at 3 time-points. The results demonstrated that WRKY72 transcription was inhibited by cold, heat, salt, abscisic acid (ABA), methyl viologen (MV), low nitrogen (LN) and glucose (Glc) treatments, which showed that WRKY72 may play a role in these stress signaling pathways. The transcriptional activity of WRKY72 was assessed through in vivo yeast assay, revealing that WRKY72 was a transcriptional repressor. Through a dual luciferase reporter system, WRKY72 was identified to significantly repress the transcription of firefly luciferase gene under the control of tandem repeats of W-box cis-elements, compared to the control. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 and the results showed that WRKY72 interacted with homologous WRKY9, WRKY36, WRKY47, WRKY61 and itself. These indicated that WRKY72 was able to form both heterodimers and homodimers in vivo. Finally, 2 T-DNA insertion mutants of WRKY72 gene were identified through PCR, and semi qRT-PCR assay indicated loss-of-function mutation. Phenotypic assay was done to compare the response of the knock-out mutant to wild-type Col-0 under different abiotic stress conditions at the post-germination level. Root elongation assay showed that the elongation of wrky72 mutant roots were significantly shorter under MV and LN treatments whereas no significant difference was observed under any other stress treatment, suggesting that WRKY72 might participate in the signaling pathways of oxidative stress tolerance and nitrogen absorption and/or utilization. In summary, the experimental evidences provided here support that WRKY72 was a transcriptional repressor and played a role in abiotic stress signaling by forming a complex with homologous WRKY9, WRKY36, WRKY47 and WRKY61. These data have laid a foundation for in-depth dissection of function and molecular mechanism of WRKY72 transcription factor gene in the context of abiotic stress signaling.
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