Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis

Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL. The redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase (DHAR3) is essential for photo-oxidative stress tolerance in Arabidopsis.叶绿体（Chloroplasts）是光照条件下活性氧（reactive oxygen species）产生的关键位点，因此拥有一套以抗坏血酸（ascorbate）为核心组分的完善抗氧化系统。该系统中的抗坏血酸循环存在多种途径，其中包含脱氢抗坏血酸还原酶（dehydroascorbate reductase, DHAR）介导的反应。本研究以拟南芥（Arabidopsis）为材料，探究了DHAR3在其光氧化胁迫耐受过程中的生理意义。经绿色荧光蛋白（GFP）融合的DHAR3蛋白可靶向定位于拟南芥叶片的叶绿体中。DHAR3基因敲除突变体表现出对高光（high light, HL）胁迫的敏感性。高光胁迫下，突变体与野生型植株的抗坏血酸氧化还原状态无显著差异，但突变体的总抗坏血酸含量显著降低，提示DHAR3至少在一定程度上参与了抗坏血酸的循环过程。dhar3突变体中单脱氢抗坏血酸还原酶（monodehydroascorbate reductase）的活性被激活，这或许可弥补DHAR3缺失带来的功能缺陷。有趣的是，dhar3突变体中谷胱甘肽（glutathione）的氧化过程始终受到抑制。上述研究结果表明，DHAR3可通过协同调控抗坏血酸与谷胱甘肽的氧化还原状态，帮助植株适应高光胁迫环境。叶绿体定位的脱氢抗坏血酸还原酶（DHAR3）对抗坏血酸与谷胱甘肽氧化还原状态的精准调控，是拟南芥耐受光氧化胁迫的核心机制。