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J. Biol. Chem., Vol. 282, Issue 12, 9260-9268, March 23, 2007

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The EAR-motif of the Cys2/His2-type Zinc Finger Protein Zat7 Plays a Key Role in the Defense Response of Arabidopsis to Salinity Stress^*

Sultan Ciftci-Yilmaz, Mustafa R. Morsy, Luhua Song, Alicia Coutu, Beth A. Krizek, Michael W. Lewis¹, Daniel Warren, John Cushman, Erin L. Connolly, and Ron Mittler²

From the Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada 89557 and the Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.

Received for publication, December 4, 2006 , and in revised form, January 25, 2007.

^* This work was supported by funding from The National Science Foundation (IBN-0420033 and NSF-0431327), The National Institutes of Health IDeA Network of Biomedical Research Excellence (INBRE, RR-03-008), The Nevada Agricultural Experimental Station, and the USC Research and Productive Scholarship Grant. This work was published as publication no. 03055517 of the University of Nevada Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Table S1.

¹ Current address: Dept. of Biology, University of North Carolina, Chapel Hill, NC 27599.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Nevada, Mail Stop 200, Reno, NV 89557. Tel.: 775-784-1384; Fax: 775-784-6911; E-mail: ronm{at}unr.edu.

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