Glutathione oxidation in response to intracellular H₂O₂: Key but overlapping roles for dehydroascorbate reductases

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H₂O₂ detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function <i>gstU8, gstU24, gstF8, prxIIE</i> and <i>prxIIF</i> mutants as well as double <i>gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF</i> mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H₂O₂ metabolism.谷胱甘肽是氧化应激过程中的关键分子，在此过程中它可通过多条与过氧化氢（H₂O₂）解毒相关的通路发生潜在氧化。本研究以拟南芥为实验材料，探究了谷胱甘肽S-转移酶（glutathione S-transferases, GST）、谷氧还蛋白依赖型过氧化物氧还蛋白（glutaredoxin-dependent peroxiredoxins, PRXII）以及脱氢抗坏血酸还原酶（dehydroascorbate reductases, DHAR）介导的三条潜在谷胱甘肽氧化通路在氧化应激中的响应特征与功能重要性。我们成功获得了功能缺失型gstU8、gstU24、gstF8、prxIIE、prxIIF单突变体，以及gstU8 gstU24、gstU8 gstF8、gstU24 gstF8、prxIIE prxIIF双突变体。在最优生长条件以及过氧化氢酶抑制诱导的氧化应激环境下，所有突变体株系的表型与谷胱甘肽谱均未出现相较于野生型植株的显著差异。与之相反，同时缺失多种DHAR功能的植株，其由过氧化氢酶缺乏所触发的谷胱甘肽氧化水平显著降低。为评估该效应是直接由DHAR酶活性缺失所介导，还是通过上调谷胱甘肽与抗坏血酸循环相关的其他酶而间接产生，我们检测了谷胱甘肽还原酶（glutathione reductase, GR）的表达水平，以及单脱氢抗坏血酸还原酶（monodehydroascorbate reductases, MDHAR）的表达量与酶活性。未在DHAR功能缺失植株中发现GR或MDHAR表达上调的证据。因此，不同DHAR之间的协同作用似乎是连接抗坏血酸与谷胱甘肽库、并在过氧化氢代谢增强时实现谷胱甘肽相关信号传导所必需的。