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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Redox Regulation of a Novel Plastid-Targeted -Amylase of Arabidopsis^1,[W]

Laboratory of Molecular Plant Physiology, Department of Experimental Evolutionary Biology, University of Bologna, I–40126 Bologna, Italy (F.S., P.P., P.T.); and Dipartimento di Biologia, Università degli Studi di Padova, I–35131 Padova, Italy (A.C., F.L.S.)

Nine genes of Arabidopsis (Arabidopsis thaliana) encode for -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 -amylase (At4g17090) and thioredoxin-regulated -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 E_m,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 -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 -amylase of nonphotosynthetic plastids suggesting a redox regulation of starch metabolism in these organelles. In leaves, where chloroplast-targeted -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.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Paolo Trost (trost{at}alma.unibo.it).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079186.

^* Corresponding author; e-mail trost{at}alma.unibo.it; fax 39–051–242576.

Received February 14, 2006; returned for revision April 28, 2006; accepted May 2, 2006.

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