Sparla Francesca, Costa Alex, Lo Schiavo Fiorella, Pupillo Paolo, Trost Paolo
Laboratory of Molecular Plant Physiology, Department of Experimental Evolutionary Biology, University of Bologna, I-40126 Bologna, Italy.
Plant Physiol. 2006 Jul;141(3):840-50. doi: 10.1104/pp.106.079186. Epub 2006 May 12.
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.
拟南芥（Arabidopsis thaliana）的九个基因编码β-淀粉酶同工酶。该家族的六个成员预计为叶绿体外同工酶，三个含有预测的质体转运肽。在后者中，叶绿体靶向的β-淀粉酶（At4g17090）和硫氧还蛋白调节的β-淀粉酶（TR-BAMY；At3g23920；本研究）经实验证明可靶向质体。重组TR-BAMY只有在作为成熟蛋白表达时才具有催化活性，即没有转运肽。成熟的TR-BAMY是一个60 kD的单体，水解可溶性淀粉的最佳活性在pH 6.0至8.0之间。重组TR-BAMY的活性严格依赖于氧化还原电位，Em,7.0为-302±14 mV。拟南芥的硫氧还蛋白f1、m1和y1都能够介导氧化型TR-BAMY的还原激活。位点特异性突变体表明，TR-BAMY的氧化抑制取决于Cys-32和Cys-470之间二硫键的形成。与TR-BAMY的氧化还原依赖性一致，拟南芥叶绿体中的总β-淀粉酶活性部分受氧化还原调节，需要还原条件才能完全激活。在拟南芥中，在叶、根、花、花粉和种子中都检测到了TR-BAMY转录本。TR-BAMY可能是非光合质体中唯一的β-淀粉酶，这表明这些细胞器中淀粉代谢存在氧化还原调节。在叶片中，叶绿体靶向的β-淀粉酶参与黑暗中淀粉的生理降解，而TR-BAMY被认为在特定胁迫条件下参与淀粉降解的氧化还原调节途径。
