State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai 200433, China.
J Integr Plant Biol. 2010 Apr;52(4):400-9. doi: 10.1111/j.1744-7909.2010.00921.x.
Vitamin C (L-ascorbic acid, AsA) has important antioxidant and metabolic functions in both plants and animals. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR, EC 1.8.5.1). To analyze the physiological role of DHAR catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, we examined whether increasing the level of AsA through enhanced AsA recycling would limit the deleterious effects of oxidative stress. A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-dhar gene was introduced into Arabidopsis thaliana. Transgenic plants were biochemically characterized and tested for responses to oxidative stress. Western blot indicated that the dhar-transgene was successfully expressed. In homozygous T(4) transgenic seedlings, DHAR overexpression was increased up to 1.5 to 5.4 fold, which enhanced foliar ascorbic acid levels 2- to 4.25-fold and ratio of AsA/DHA about 3- to 16-fold relative to wild type. In addition, the level of glutathione, the reductant used by DHAR, also increased as did its redox state. When whole plants were treated with high light and high temperature stress or in vitro leaf discs were subjected to 10 muM paraquat, transgenic plants showed a larger AsA pool size, lower membrane damage, and a higher level of chlorophyll compared with controls. These data suggested that increasing the plant vitamin C content through enhanced ascorbate recycling could limit the deleterious effects of environmental oxidative stress.
维生素 C（L-抗坏血酸，AsA）在植物和动物中都具有重要的抗氧化和代谢功能。一旦被使用，抗坏血酸可以在脱氢抗坏血酸还原酶（DHAR，EC 1.8.5.1）催化的反应中从其氧化形式再生。为了分析 DHAR 催化 DHA 还原为抗坏血酸在环境胁迫适应中的生理作用，我们研究了通过增强抗坏血酸循环是否会限制氧化应激的有害影响来增加 AsA 水平。将由双 CaMV35S 启动子融合到 Myc-dhar 基因的嵌合构建体导入拟南芥。对转基因植物进行了生化特征分析，并进行了氧化应激反应测试。Western blot 表明 dhar-转基因成功表达。在纯合 T(4)转基因幼苗中，DHAR 过表达增加了 1.5 至 5.4 倍，叶片抗坏血酸水平增加了 2 至 4.25 倍，AsA/DHA 比值增加了 3 至 16 倍。此外，DHAR 使用的还原剂谷胱甘肽的水平也增加了，其氧化还原状态也增加了。当整个植物受到高光和高温胁迫处理或体外叶片圆盘受到 10 μM 百草枯处理时，与对照相比，转基因植物表现出更大的 AsA 池大小、更低的膜损伤和更高的叶绿素水平。这些数据表明，通过增强抗坏血酸循环增加植物维生素 C 含量可以限制环境氧化应激的有害影响。
