Chapter 21
Quantification of Carotenoid Pathway Flux in Green
and Nongreen Systems
Julian Koschmieder and Ralf Welsch
Abstract
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.
Key words Phytoene desaturase, Phytoene, Quantitative HPLC, Norflurazon, Seedlings, Callus,
Arabidopsis
1 Introduction
Carotenoids accumulate in all tissues and throughout all developmental stages of a plant’s life. In chloroplasts of photosynthetically
active tissues, carotenoids are constituents of light-harvesting proteins and photosynthetic reaction center while in chromoplasts,
carotenoids accumulate at high levels and are protein-bound,
integrated into proliferated membranes, accumulate as crystals or
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_21,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
279
Quantification of Carotenoid Pathway Flux in Green
and Nongreen Systems
Julian Koschmieder and Ralf Welsch
Abstract
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.
Key words Phytoene desaturase, Phytoene, Quantitative HPLC, Norflurazon, Seedlings, Callus,
Arabidopsis
1 Introduction
Carotenoids accumulate in all tissues and throughout all developmental stages of a plant’s life. In chloroplasts of photosynthetically
active tissues, carotenoids are constituents of light-harvesting proteins and photosynthetic reaction center while in chromoplasts,
carotenoids accumulate at high levels and are protein-bound,
integrated into proliferated membranes, accumulate as crystals or
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_21,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
279