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J. Biol. Chem., Vol. 282, Issue 8, 5919-5933, February 23, 2007

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Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses^*

Marcus A. Strawn, Sharon K. Marr, Kentaro Inoue, Noriko Inada¹, Chloe Zubieta^¶, and Mary C. Wildermuth²

From the Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, California 94720-3102, the Department of Plant Sciences, University of California at Davis, Davis, California 95616-8780, and the ^¶Joint Center for Structural Genomics, Stanford University, Menlo Park, California 94025

Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (K_eq = 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent K_m of 41.5 µM and k_cat = 38.7 min^-1 for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg²⁺, or redox and that it is remarkably active at 4 °C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.

Received for publication, May 31, 2006 , and in revised form, December 19, 2006.

^* This work was supported using startup funds provided by the University of California at Berkeley. 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 Fig. 1S and Table 1S.

¹ Present address: Dept. of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma-shi, Nara 630-0101, Japan.

² To whom correspondence should be addressed: Dept. of Plant and Microbial Biology, 111 Koshland Hall, University of California at Berkeley, Berkeley, CA 94720-3102. Tel.: 510-643-4861; Fax: 510-642-4995; E-mail: wildermuth{at}nature.berkeley.edu.

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