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.
Keywords: Arabidopsis; Callus; Norflurazon; Phytoene; Phytoene desaturase; Quantitative HPLC; Seedlings.