Analysis of regulation mechanism of chloroplstic ascorbate peroxidase isoenzymes

为了探讨叶绿体抗坏血酸过氧化物酶（chlAPX）同工酶的调控机制，我们研究了以下几个方面：（1）我们以前已经证明菠菜叶绿体的基质（sAPX）和类囊体结合（tAPX）抗坏血酸过氧化物酶同工酶是由一个共同的前体mRNA通过选择性剪接产生的。为了探索编码chlAPX同工酶的成熟、功能性mRNA的产生，进行mRNA分析。鉴定了四种mRNA变体，tAPX的一种形式（tAPX-1）和sAPX的三种形式（sAPX-I、-II和-III）。在正常条件下生长的整个菠菜叶中，sAPX和tAPX同工酶的mRNA变体的表达水平几乎相等。(2)研究了菠菜叶片APX同工酶在强光、干旱、盐胁迫、百草枯和脱落酸胁迫下的响应。细胞质APX（cAPX）的稳态转录水平在强光和百草枯处理后显著增加。cAPX的活性增加平行于其转录丰度在强光强度。除chlAPX同工酶活性逐渐下降外，其余同工酶的转录水平和蛋白质水平及活性均无显著变化。(3)从烟草叶片中克隆了一个编码L-半乳糖酸- γ-内酯（GAL）脱氢酶的cDNA克隆。该cDNA克隆含有56，926 Da的蛋白质，前面有一个推定的线粒体靶向信号。事实上，GAL脱氢酶定位于烟草细胞的线粒体中。GAL脱氢酶mRNA在叶、茎和根中以几乎相等的量表达。将部分纯化的重组酶与烟草和其他来源的天然酶进行了比较研究;重组GAL脱氢酶的酶学性质与其他GAL脱氢酶相似。

To explore the regulation mechanism of chloroplastic ascorbate peroxides (chlAPX) isoenzymes, we studied the following heads,(1) We have previously shown that stromal (sAPX) and thylakoid-bound (tAPX) ascorbate peroxidase isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing. To explore the production of mature, functional mRNA encoding chlAPX isoenzymes, mRNA analysis were performed. Four mRNA variants, one form (tAPX-1) for tAPX and three forms (sAPX-I, -II, and -III) for sAPX, were identified. The expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions.(2) We studied the response of each APX isoenzyme in spinach leaves under stress conditions imposed by high-light intensity, drought, salinity, and applications of paraquat and abscisic acid. The steady-state transcript level of cytosolic APX (cAPX) remarkably increased in response to high-light intensity and paraquat treatment. The cAPX activity increased in parallel with its transcript abundance during high-light intensity. The other isoenzymes showed no significant changes in their transcript an protein levels and activities, except for he gradual decrease in the chlAPX isoenzymes activities.(3) A cDNA clone encoding L-galactono- γ-lactone (GAL) dehydrogenase was isolated from tobacco leaves. The cDNA clone contained the protein of 56,926 Da, preceded by a putative mitochondrial targeting signal. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. GAL dehydrogenase mRNA is expressed in the leaves, stems, and roots in almost equal quantities. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources ; The enzymatic properties of recombinant GAL dehydrogenase were similar to those of other GAL dehydrogenases.