The upstream oxylipin profile of Arabidopsis thaliana:: a tool to scan for oxidative stresses
The upstream oxylipin profile of Arabidopsis thaliana:: a tool to scan for oxidative stresses
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DOI：
10.1111/j.1365-313x.2004.02223.x
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发表时间：
2004-11-01
期刊：
影响因子：
7.2
通讯作者：
Triantaphylidès, C
中科院分区：
文献类型：
--
作者：
Montillet, JL;Cacas, JL;Triantaphylidès, C
各种生理失衡导致活性氧（ROS）过量产生和/或脂氧合酶（LOX）活性增加，这两种事件均以多不饱和脂肪酸（PUFA）的脂质过氧化作用结束。除了对这一过程进行量化之外，还需要开发工具，以确定其发展和本地化的主要原因。评估9 LOX、13 LOX和ROS介导的膜结合和游离PUFA过氧化反应的生化方法得到了改进。该含量测定是基于通过直相和手性相高效液相色谱法分析来自PUFA氢过氧化物的羟基脂肪酸。除了18：2和18：3 PUFA的过氧化的上游产物外，还表征了来自16：3的产物，并定量了其稳态水平。此外，ROS介导的过氧化异构体18：3的相对量在叶片中是恒定的，这一观察使我们能够规避常规实验中用于区分和定量9 LOX、13 LOX和ROS介导的过程的手性分析。该方法已成功地应用于破译拟南芥叶片的脂质过氧化反应提交生物和非生物胁迫。我们提供了酶和非酶脂质过氧化过程的相对时间的证据。无论胁迫的性质如何，13 LOX途径都被早期激活，导致叶绿体脂质过氧化。镉胁迫下，9 LOX途径增加到13 LOX途径。ROS介导的过氧化主要受光驱动，并且总是作为一个晚期过程出现。
Various physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.