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

Abstract

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 M 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, Mg 2؉ , 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.

PROHIBITIN3 Forms Complexes with ISOCHORISMATE SYNTHASE1 to Regulate Stress-Induced Salicylic Acid Biosynthesis in Arabidopsis

“…In addition to identifying the localization of PHB3 in mitochondria (Van Aken et al, 343 2007;Piechota et al, 2010), this work established that there is a pool of PHB3 that resides 344 in chloroplasts, the organelle where SA is made and where ICS1 is targeted (Fragnire et al, 345 2011;Strawn et al, 2007). PHB3 has a hydrophobic region at the N-terminus that likely 346 serves to anchor it to membranes.…”

“…We took advantage of previously constructed sid2-2 plants that contain a 149 complementing ICS1-V5 transgene (Strawn et al, 2007) to identify proteins with which 150 ICS1 forms complexes using affinity purification and proteomics. V5 antibody-conjugated 151 beads were used to immunoprecipitate (IP) proteins from extracts of untreated and UV-C-152 treated sid2-2/ICS1-V5 or wild-type (WT) plants.…”

“…520 ICS1 was detected with the rabbit polyclonal ICS1 antibody UCB68 at a 1:10,000 521 dilution. This antibody was made by immunizing rabbits with purified recombinant mature 522 ICS1 protein (described in Strawn et al, 2007). The titer and specificity of the antibody 523 were tested by immunoblot analysis.…”

Light Regulation and Daytime Dependency of Inducible Plant Defenses in Arabidopsis: Phytochrome Signaling Controls Systemic Acquired Resistance Rather Than Local Defense

“…Metabolizable sugars have been shown to positively influence secondary metabolism and defense gene expression in Arabidopsis, because they promote lignification in dark grown roots and induce PR transcript levels in seedlings (Thibaud et al, 2004;Rogers et al, 2005). As SA biosynthesis via isochorismate synthase occurs in plastids (Strawn et al, 2007), photosynthetic activity might be required to supply reducing equivalents and energy for SA accumulation. At least for HR execution, intact chloroplasts and associated ROS production seem to play an important role (Genoud et al, 2002;Liu et al, 2007).…”

Arabidopsis GH3.12 (PBS3) Conjugates Amino Acids to 4-Substituted Benzoates and Is Inhibited by Salicylate

“…Potential Inhibition of ICS1-catalyzed SA Biosynthesis-To evaluate compounds as inhibitors of the AtICS1-catalyzed conversion of chorismate to isochorismate, a coupled spectrophotometric assay was used (22). The conversion of chorismate to isochorismate was coupled to the oxidation of NADH through the coupling enzymes isochorismate pyruvate lyase (recombinant PchB), which converts isochorismate to pyruvate and SA, and lactic dehydrogenase (Sigma L1254), which converts pyruvate to lactate in an NADH-dependent fashion.…”