Comprehensive survey of redox sensitive starch metabolising enzymes in Arabidopsis thaliana

In chloroplasts, the ferredoxin/thioredoxin pathway regulates enzyme activity in response to light by reduction of regulatory disulfides in target enzymes, ensuring coordination between photosynthesis and diurnal metabolism. Although earlier studies have suggested that many starch metabolic enzymes are similarly regulated, redox regulation has only been verified for a few of these in vitro. Using zymograms and enzyme assays, we performed a comprehensive analysis of the redox sensitivity of known starch metabolising enzymes in extracts of Arabidopsis thaliana. Manipulation of redox potentials revealed that several enzymatic activities where activated by reduction at physiologically relevant potentials. Among these where the isoamylase complex AtISA1/AtISA2, the limit dextrinase AtLDA, starch synthases AtSS1 and AtSS3, and the starch branching enzyme AtBE2. The reversibility of the redox reaction was confirmed by enzyme assays for AtLDA, AtSS1 and AtSS3. Analysis of an AtBAM1 knock-out mutant identified an additional redox sensitive beta-amylase activity, which was most likely AtBAM3. A similar requirement for reducing conditions was observed for recombinant chloroplastic alpha-amylase (AtAMY3) activity. This study adds further candidates to the list of reductively activated starch metabolising enzymes and supports the view that redox regulation plays a role in starch metabolism. (C) 2012 Elsevier Masson SAS. All rights reserved.