title: Redox regulation of a novel plastid-targeted beta-amylase of Arabidopsis.
abstract: Nine genes of Arabidopsis (Arabidopsis thaliana) encode for beta-amylase isozymes. Six members of the family are predicted to be extrachloroplastic isozymes and three contain predicted plastid transit peptides. Among the latter, chloroplast-targeted beta-amylase (At4g17090) and thioredoxin-regulated beta-amylase (TR-BAMY; At3g23920; this work) are experimentally demonstrated to be targeted to plastids. Recombinant TR-BAMY was catalytically active only when expressed as a mature protein, i.e. with no transit peptide. Mature TR-BAMY was a monomer of 60 kD, hydrolyzing soluble starch with optimal activity between pH 6.0 and 8.0. The activity of recombinant TR-BAMY was strictly dependent on redox potential with an Em,7.0 of -302 +/- 14 mV. Thioredoxins f1, m1, and y1 of Arabidopsis were all able to mediate the reductive activation of oxidized TR-BAMY. Site-specific mutants showed that TR-BAMY oxidative inhibition depended on the formation of a disulfide bridge between Cys-32 and Cys-470. Consistent with TR-BAMY redox dependency, total beta-amylase activity in Arabidopsis chloroplasts was partially redox regulated and required reducing conditions for full activation. In Arabidopsis, TR-BAMY transcripts were detected in leaves, roots, flowers, pollen, and seeds. TR-BAMY may be the only beta-amylase of nonphotosynthetic plastids suggesting a redox regulation of starch metabolism in these organelles. In leaves, where chloroplast-targeted beta-amylase is involved in physiological degradation of starch in the dark, TR-BAMY is proposed to participate to a redox-regulated pathway of starch degradation under specific stress conditions.
authors.affiliation: Laboratory of Molecular Plant Physiology, Department of Experimental Evolutionary Biology, University of Bologna, I-40126 Bologna, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Sparla
authors.forename: Alex
authors.initials: A
authors.lastname: Costa
authors.forename: Fiorella
authors.initials: F
authors.lastname: Lo Schiavo
authors.forename: Paolo
authors.initials: P
authors.lastname: Pupillo
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018087:Plastids / Q000201:enzymology*; D016415:Sequence Alignment; D013816:Thermodynamics; D013879:Thioredoxins / Q000378:metabolism; D001614:beta-Amylase / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D004220:Disulfides; D013879:Thioredoxins; D001614:beta-Amylase; C514690:thioredoxin-regulated beta-amylase, Arabidopsis