High Throughput Analyses of Ascorbate-turnover Enzyme Activities in Rice (Oryza sativa L.) Seedlings

作者：Lin‐Bo Wu, Yanru Feng, Frederike Zeibig, Muhammad Shahedul Alam, Michael Frei · 发表于：BIO-PROTOCOL · 年份：2021 · DOI：10.21769/bioprotoc.4190 · 被引用次数：15 · 研究领域：Plant Stress Responses and Tolerance、Plant Micronutrient Interactions and Effects、Vitamin C and Antioxidants Research

Ascorbate (Vitamin C) fulfills various functions in plant photosynthesis and abiotic stress tolerance. The four key enzymes involved in the ascorbate-turnover pathway are ascorbate peroxidase, ascorbate oxidase, monodehydroascorbate reductase, and dehydroascorbate reductase. Several reports have shown the pivotal roles of these enzymes in plant development and stress tolerance. Therefore, reliable and rapid assay protocols are required for researchers to investigate their enzymatic activities during plant development and stress responses. Previously published methods for analyzing these enzymatic activities rely on cuvette spectrophotometers, which can only handle one sample per test, leading to a prolonged investigation. In this protocol, we employed a 96-well microplate reader to analyze at least eight samples with two technical replicates simultaneously. We analyzed two rice (Oryza sativa L.) genotypes with distinct ascorbate oxidase and dehydroascorbate reductase activities to demonstrate the assay process, including plant growth, sample preparation, reaction setup, and data analysis. Our protocol provides a high throughput method for investigating ascorbate turnover-related enzymatic activities in plants., [抽象的] 抗坏血酸（维生素 C）在植物光合作用和非生物胁迫耐受性方面具有多种功能。参与抗坏血酸转换途径的四种关键酶是抗坏血酸过氧化物酶、抗坏血酸氧化酶、单脱氢抗坏血酸还原酶和脱氢抗坏血酸还原酶。一些报告显示了这些酶在植物发育和胁迫耐受性中的关键作用。因此，研究人员需要可靠和快速的检测方案来研究它们在植物发育和应激反应过程中的酶活性。以前发表的分析这些酶活性的方法依赖于比色皿分光光度计，每次测试只能处理一个样品，导致调查时间延长。在该协议中，我们采用了 96 孔酶标仪同时分析了至少 8 个样本，同时进行了两次技术复制。我们分析了两种...