A simple assay to identify peroxisomal proteins involved in 12-oxo-phytodienoic acid metabolism.

Peroxisomes are vital eukaryotic organelles that house a variety of metabolic functions. To fully define the proteome of plant peroxisomes, we recently performed a proteomic analysis of peroxisomes from etiolated Arabidopsis seedlings, verified the peroxisomal localization of candidate proteins by in vivo targeting analysis of fluorescent proteins, and subjected the T-DNA mutants of the newly confirmed proteins to a series of phenotypic analysis. Our reverse genetics approach revealed the role of a cysteine protease - Response to Drought 21A-like Cysteine Protease1 (RDL1) - in seed germination, indole-3-butyric acid (IBA) β-oxidation and stress response. Here, we developed a quick assay aimed at identifying peroxisomal proteins involved in the metabolism of 12-oxo-phytodienoic acid (OPDA), which is converted to jasmonic acid in the peroxisome through β-oxidation. We performed a survey of the same mutants analyzed in our previous reverse genetics study with this new assay by measuring the response of mutants to OPDA's inhibitory effect on root elongation. Mutants of RDL1 and SERINE CARBOXYPEPTIDASE-LIKE20 (SCPL20) exhibited statistically significant hypersensitivity to OPDA, indicating the potential involvement of these proteins in OPDA metabolism. This convenient assay may be used in the future to rapidly screen for mutants defective in OPDA metabolism or signaling.