PDF(Pubmed)
Abstract：
Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in the detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. In this study, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small-molecule probe contains glutathione, a photoreactive group and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts of different plant species. Purification and mass spectrometry (MS) analysis of labeled proteins from Arabidopsis identified 10 enriched GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST induction in wheat seedlings upon treatment with safeners and in Arabidopsis leaves upon infection with avirulent bacteria. Treatment of Arabidopsis with salicylic acid (SA) analog benzothiadiazole (BTH) induces GST labeling independent of NPR1, the master regulator of SA. Six Phi- and Tau-class GSTs that are induced upon BTH treatment were identified, and their labeling was confirmed upon transient overexpression. These data demonstrate that GST photoaffinity labeling is a useful approach to studying GST induction in crude extracts of different plant species upon different types of stress.
摘要：
谷胱甘肽转移酶（GSTs）代表了一个庞大而多样的酶家族，参与作物中谷胱甘肽缀合对小分子的解毒。杂草和模型植物。这里,我们介绍了一种简单快速的GSTs光亲和标记测定法，以研究各种植物物种中的全球GSTs。小分子探针含有谷胱甘肽，光反应性基团，和一个微型标签，用于通过点击化学耦合到报告标签。在紫外线照射下，该探针快速而稳健地标记不同植物物种的粗蛋白提取物中的GSTs。来自拟南芥的标记蛋白质的纯化和MS分析鉴定了来自Phi（F）和Tau（U）类的十个富集的GST。GSTs的光亲和力标记证明了用安全剂处理后小麦幼苗中的GST诱导，在被无毒细菌感染的拟南芥中。用水杨酸（SA）类似物苯并噻二唑（BTH）处理拟南芥会诱导GST标记，而与水杨酸（SA）的主要调节因子NPR1无关。鉴定了在BTH处理后诱导的六个Phi和Tau类GST，并在瞬时过表达后确认了它们的标记。这些数据表明，GST光亲和标记是研究不同类型胁迫下不同植物物种粗提物中GST诱导的有用方法。
