Mata-Pérez Capilla, Padilla María N, Sánchez-Calvo Beatriz, Begara-Morales Juan C, Valderrama Raquel, Corpas Francisco J, Barroso Juan B
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Experimental Biology, Center for Advanced Studies in Olive Grove and Olive Oils, Faculty of Experimental Sciences, Campus Universitario "Las Lagunillas" s/n, University of Jaén, Jaén, Spain.
Group of Antioxidants, Free Radicals, and Nitric Oxide in Biotechnology, Food, and Agriculture, Department of Biochemistry, Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Methods Mol Biol. 2018;1747:231-239. doi: 10.1007/978-1-4939-7695-9_18.
In the last few years, the role of nitric oxide (NO) and NO-related molecules has attracted attention in the field of plant systems. In this sense, the ability of NO to mediate several posttranslational modifications (NO-PTM) in different biomolecules, such as protein tyrosine nitration or S-nitrosylation, has shown the involvement of these reactive nitrogen species in a wide range of functions in plant physiology such as the antioxidant response or the involvement in processes such as germination, growth, development, or senescence. However, growing interest has focused on the interaction of these NO-derived molecules with unsaturated fatty acids, yielding nitro-fatty acids (NO-FAs). It has recently been shown that these molecules are involved in key signaling pathways in animal systems through the implementation of antioxidant and anti-inflammatory responses. Nevertheless, this interaction has been poorly studied in plant systems. Very recently, the endogenous presence of NO-FAs in the model plant Arabidopsis thaliana has been demonstrated as well as the significant involvement of nitro-linolenic acid (NO-Ln) in the defence response against several abiotic and oxidative stress conditions. In this respect, the detection of NO-FAs in plant systems can be a useful tool to determine the importance of these molecules in the regulation of different biochemical pathways. Using high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), the methods described in this chapter enable the determination of the NO-FA content in a pM range as well as the characterization of these nitrated derivatives of unsaturated fatty acids in plant tissues.
在过去几年中，一氧化氮（NO）及与NO相关的分子在植物系统领域引起了关注。从这个意义上说，NO介导不同生物分子中多种翻译后修饰（NO-PTM）的能力，如蛋白质酪氨酸硝化或S-亚硝基化，已表明这些活性氮物种参与了植物生理学中的广泛功能，如抗氧化反应以及参与种子萌发、生长、发育或衰老等过程。然而，越来越多的研究兴趣集中在这些源自NO的分子与不饱和脂肪酸的相互作用上，从而产生硝基脂肪酸（NO-FAs）。最近的研究表明，这些分子通过实施抗氧化和抗炎反应参与动物系统中的关键信号通路。然而，这种相互作用在植物系统中研究较少。最近，已证明模式植物拟南芥中存在内源性NO-FAs，以及硝基亚麻酸（NO-Ln）在抵御几种非生物和氧化胁迫条件的防御反应中发挥重要作用。在这方面，检测植物系统中的NO-FAs可以作为一种有用的工具，来确定这些分子在调节不同生化途径中的重要性。利用高压液相色谱与三重四极杆质谱联用（LC-MS/MS），本章所述方法能够测定植物组织中低至皮摩尔范围内的NO-FA含量，并对这些不饱和脂肪酸的硝化衍生物进行表征。
