Department of Life Science, II University of Naples, via Vivaldi 43, 81100 Caserta, Italy.
Mol Biol Rep. 2012 Aug;39(8):7843-53. doi: 10.1007/s11033-012-1627-7. Epub 2012 Apr 28.
Ascorbate is an antioxidant and a cofactor of many dioxygenases in plant and animal cell metabolism. A well-recognized enzyme consuming ascorbate is ascorbate peroxidase (APX), which catalyses the reduction of hydrogen peroxide to water with the simultaneous oxidation of ascorbate with a high specificity. The isolation and characterisation of new Apx cDNAs, could provide new insights about the physiological roles and regulation of these enzymes. In this work chloroplastic (Br-chlApx) and cytosolic (Br-cApx) isoform transcripts were isolated by RT-PCR in Brassica rapa and expression changes were analysed by semi-quantitative RT-PCR performed in different tissues (layer, stalk and florets) at different days (0, 4 and 14 day). The result showed that BrApx isoforms were differentially expressed and the Br-chlApx, in particular in the layer, had the highest expression level and remained unchanged also after 14 day after harvest. In addition, expression changes were compared with total BrAPX activity and the results showed that the activity decreased in all tissues at 14 day after harvest, independently of transcripts. Finally, additional solutes as the substrate of APX ascorbate and its oxidized form, dehydroascorbate, as well as α-tocopherol, the major vitamin E compound that prevents the propagation of lipid peroxidation in thylakoid membranes, were followed. The changes in the BrApx expression, BrAPX activity and metabolites can provide further evidence of the close relationships that exist between antioxidants which compensate for each other and suggest that there are multiple sites of reciprocal control.
抗坏血酸是一种抗氧化剂，也是植物和动物细胞代谢中许多双加氧酶的辅助因子。一种广为人知的消耗抗坏血酸的酶是抗坏血酸过氧化物酶（APX），它能高特异性地催化过氧化氢还原为水，同时将抗坏血酸氧化。分离和鉴定新的 Apx cDNA，可以深入了解这些酶的生理作用和调控机制。在这项工作中，通过 RT-PCR 从芸薹属植物中分离出质体（Br-chlApx）和细胞质（Br-cApx）同工型转录本，并通过半定量 RT-PCR 在不同组织（层、茎和花）的不同时间（0、4 和 14 天）分析表达变化。结果表明，BrApx 同工型表达不同，特别是在层中，Br-chlApx 的表达水平最高，收获后 14 天仍保持不变。此外，还将表达变化与总 BrAPX 活性进行了比较，结果表明，所有组织的活性在收获后 14 天都降低了，与转录本无关。最后，还跟踪了 APX 的其他底物抗坏血酸及其氧化形式脱氢抗坏血酸，以及α-生育酚，作为质体膜中脂质过氧化传播的主要维生素 E 化合物，作为 APX 的辅助因子。BrApx 表达、BrAPX 活性和代谢物的变化进一步证明了抗氧化剂之间存在着密切的关系，它们相互补偿，并暗示存在多个相互控制的位点。
