Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer
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DOI：
10.1111/j.1365-313x.2007.03280.x
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发表时间：
2007-12-01
期刊：
影响因子：
7.2
通讯作者：
Hell, Ruediger
中科院分区：
文献类型：
--
作者：
Meyer, Andreas J.;Brach, Thorsten;Hell, Ruediger
细胞内谷胱甘肽氧化还原缓冲液被认为是生物和非生物胁迫期间传递环境信号的信号转导途径的一部分，因此对于调节代谢和发育是必不可少的。在拟南芥中表达的比率氧化还原敏感的GFP（roGFP）可逆地响应于与H2O2或DTT孵育诱导的氧化还原变化。这些氧化还原变化的动力学分析，结合roGFP 2在体外的详细表征，表明roGFP 2在胞质溶胶中表达的感觉细胞谷胱甘肽缓冲液的氧化还原电位通过谷氧还蛋白（GRX）作为谷胱甘肽和roGFP 2之间的可逆电子流的介体。roGFP 2对谷胱甘肽氧化还原电位的敏感性在体内进行了测试，通过操纵野生型植物的谷胱甘肽（GSH）含量，通过在低GSH突变体的胞质溶胶和野生型植物的内质网（ER）中表达roGFP 2，以及通过创伤作为应激诱导的氧化还原变化的一个例子。如果GSH浓度已知，roGFP 2有助于确定GSH溶液的氧化程度。假设有足够的谷胱甘肽还原酶活性和非限制性NADPH供应，所观察到的roGFP 2在体内几乎完全还原表明2.5 mM细胞溶质谷胱甘肽缓冲液将仅含有25 nM氧化型谷胱甘肽二硫化物（GSSG）。roGFP 2通过GRX对GSSG的高灵敏度使得roGFP 2能够用于真实的实时监测体内应激诱导的氧化还原变化。roGFP 2作为人工GRX靶标的结果进一步表明，氧化还原触发的生物过程的变化可能通过GRX作为介体直接与谷胱甘肽氧化还原电位相关。
The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H2O2 or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mM cytosolic glutathione buffer would contain only 25 nM oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.