The many effects of salicylic acid (SA) application on plants have been documented for nearly half a century. They include such diverse processes as flowering, stomatal closure, seed germination, adventitious root initiation and thermogenesis. The role(s) of endogenous SA in disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in disease resistance, several SA-binding proteins (SABPs) were identified and characterized. These include catalase, ascorbate peroxidase, carbonic anhydrase (SABP3), and methyl salicylate esterase (SABP2), all of which appear to be involved in resistance. To identify additional SABPs, including the SA receptor, affinity chromatography was performed using a novel SA matrix. Preliminary experiments identified many soluble and membrane-bound/enriched candidate SABPs (cSABPs).

To confirm and extend the findings from SA affinity chromatography, we proposed to use two powerful, high-throughput, but less proven, approaches. They are the Arabidopsis protein micro-array (developed by Popescu, Snyder, and Dinesh-Kumar) and a yeast three hybrid system, which uses a small hybrid ligand. During the course of the grant a third screen was developed.

It utilizes a SA analogs 4-azido SA (4AzSA) or 3-aminoethyl SA (3AESA), in conjunction with either a photo-affinity labeling technique or surface plasmon resonance (SPR)-based technology, to identify and evaluate cSABPs from Arabidopsis. The photo-affinity labeling and SPR-based approaches are more sensitive than the traditional approach for identifying plant SA-binding activity using size exclusion chromatography with radiolabeled SA, as many of these proteins exhibited little to no SA-binding activity in such an assay. These novel approaches therefore complement conventional techniques and have help dissect the complex SA signaling network in plants by identification of more than two dozen new SABPs (see table under Resource) as well as multiple candidates that have yet to be characterized. Some of these proteins bind SA with high affinity, while the affinity others exhibit is low. Given that SA levels vary greatly even within a particular plant species depending on subcellular location, tissue type, developmental stage, and with respect to both time and location after an environmental stimulus such as infection, the presence of SABPs exhibiting a wide range of affinities for SA may provide great flexibility and multiple mechanisms through which SA can act.

The current view is that hormones in plants, as well as in animals, exert their effect(s) by binding to one or a small number of receptors. The identification of more than 30 SABPs using traditional purification approaches and genome-wide, high-throughput screens argues that SA exerts its effects via multiple targets rather than one or a few receptors. Might this be a paradigm shift beyond SA for other plant hormones or perhaps even animal hormones?