Faculty of Biology, University of Freiburg, Freiburg, Germany.
Institute for Biology II, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2020;2083:279-291. doi: 10.1007/978-1-4939-9952-1_21.
Metabolite accumulation in plant tissues represents the transient net result of their constant biosynthesis and degradation. For carotenoids, degradation might occur enzymatically by carotenoid cleavage producing plant hormones and volatiles or by nonenzymatic oxidation, both depending on environmental and developmental conditions. Carotenoid biosynthesis is therefore constantly regulated at various levels to attain sufficient carotenoid accumulation, mainly for photosynthesis and photoprotection. Due to the plenitude of carotenoids and their degradation products, it is not feasible to investigate overall carotenoid biosynthetic activity and its regulation by the quantification of all carotenoids including their derivatives. This is an issue encountered in investigations on many other highly branched pathways. We therefore present protocols to determine carotenoid biosynthesis flux in a given plant tissue by HPLC quantification of phytoene, the first pathway-specific intermediate and precursor of all carotenoids synthesized by phytoene synthase (PSY). For this purpose, enzymatic metabolization of phytoene in the tissue under investigation is prevented by treatment with the bleaching herbicide norflurazon, blocking the carotenogenic pathway downstream of PSY. As phytoene is more resistant to oxidation than desaturated carotenoids, the rate of phytoene biosynthesis serves as a good measure for total carotenogenic flux in a given tissue. The method is described for Arabidopsis for two photosynthetically active sample types, namely, seedlings and leaves, as well as for seed-derived callus as nongreen tissue. It should be realizable using only a relatively simple experimental setup and is applicable to other plant tissues as well as to different plant species. Additionally, similar experimental setups could be a useful tool to investigate total flux and turnover rates in other biosynthetic pathways.
植物组织中代谢物的积累代表了它们不断进行生物合成和降解的瞬时净结果。对于类胡萝卜素，降解可能通过类胡萝卜素裂解酶催化产生植物激素和挥发性物质，或者通过非酶促氧化发生，这两者都取决于环境和发育条件。因此，类胡萝卜素生物合成在不同水平上不断受到调节，以达到足够的类胡萝卜素积累，主要是为了光合作用和光保护。由于类胡萝卜素及其降解产物的丰富性，通过定量分析所有包括其衍生物在内的类胡萝卜素来研究整体类胡萝卜素生物合成活性及其调节是不可行的。这是在许多其他高度分支途径的研究中遇到的问题。因此，我们提出了通过 HPLC 定量分析类胡萝卜素生物合成途径中的第一个特异性中间产物和所有类胡萝卜素的前体——phytoene，来确定特定植物组织中类胡萝卜素生物合成通量的方案。为此，通过用漂白除草剂 norflurazon 处理受研究的组织，防止 phyotene 的酶促代谢，从而阻断 PSY 下游的类胡萝卜素生成途径。由于 phyotene 比去饱和类胡萝卜素更能抵抗氧化，因此 phyotene 的生物合成速率可以很好地衡量特定组织中的总类胡萝卜素生成通量。该方法针对拟南芥的两种光合作用活性的样本类型，即幼苗和叶片，以及非绿色组织的种子衍生愈伤组织进行了描述。它应该只需要相对简单的实验设置即可实现，并且适用于其他植物组织以及不同的植物物种。此外，类似的实验设置也可以成为研究其他生物合成途径中总通量和周转率的有用工具。
