are dissolved in lipid droplets [1–3]. However, once synthesized,
carotenoid amounts are not static. In contrast, they are subject to
constant degradation and their continuous biosynthesis is required
to maintain carotenoid levels present in various tissues, as is characteristic of homeostatic systems [4, 5].
Degradation occurs to a minor proportion through
carotenoid-cleaving enzymes (carotenoid cleavage dioxygenases,
CCDs and nine-cis epoxycarotenoid dioxygenases, NCEDs) generating plant hormones like strigolactones and abscisic acid, volatiles like β-cyclocitral and β-ionone, and apocarotenoids like in
saffron or apocarotenoids which are glycosidically bound [5–
8]. However, the majority of carotenoids are degraded nonenzymatically through oxidation, producing apocarotenoids of various
chain lengths as primary oxidation products which are truncated
further through continued oxidation [9–12]. The terminal fate of
the products of continued apocarotenoid degradation is not known
but subject of current investigations of numerous research groups
in the field of carotenoid research.
Continuous carotenoid biosynthesis is required to compensate
for carotenoid degradation which varies between developmental
stages, tissue types and environmental conditions, each characterized by different rates of phytohormone biosynthesis and carotenoid oxidation. Therefore, carotenoid pathway flux takes place
permanently and is dynamically regulated. However, determination
of pathway flux through integrative quantification of all carotenoidderived products is impossible due to their sheer diversity. One
possible approach for the quantification of carotenoid biosynthetic
flux is to determine the turnover of carotenoids by
14
C pulse chase
assay [13, 14]. The high turnover rate observed for, for example,
β-carotene in these experiments strongly supports a very high
carotenogenic pathway flux in leaves.
Alternatively, inhibition of early pathway enzymes can be used
to determine the amounts of carotenoid biosynthesis intermediates, these accumulating upon pathway inhibition and mirroring
the overall carotenoid biosynthesis rate through the rate-limiting
enzyme phytoene synthase (PSY). A common approach to achieve
this is to inhibit phytoene desaturase (PDS) positioned downstream
of PSY by the application of bleaching herbicides, mainly fluridone
and norflurazon [15–17]. This results in the accumulation of phytoene, an intermediate which is usually instantly further metabolized in the pathway and thus absent in most tissues. As shown
previously, phytoene is practically not metabolized by carotenoid
cleavage enzymes and is also resistant toward oxidation as oxidative
cleavage targets higher desaturated carotenoids such as xanthophylls and β-carotene [10]. Accordingly, the phytoene amount
under such inhibitory conditions is a good measure for the total
carotenoid amount that would have been synthesized during the
time the sample is exposed to the bleaching herbicides [4, 18–20].
280
Julian Koschmieder and Ralf Welsch
carotenoid amounts are not static. In contrast, they are subject to
constant degradation and their continuous biosynthesis is required
to maintain carotenoid levels present in various tissues, as is characteristic of homeostatic systems [4, 5].
Degradation occurs to a minor proportion through
carotenoid-cleaving enzymes (carotenoid cleavage dioxygenases,
CCDs and nine-cis epoxycarotenoid dioxygenases, NCEDs) generating plant hormones like strigolactones and abscisic acid, volatiles like β-cyclocitral and β-ionone, and apocarotenoids like in
saffron or apocarotenoids which are glycosidically bound [5–
8]. However, the majority of carotenoids are degraded nonenzymatically through oxidation, producing apocarotenoids of various
chain lengths as primary oxidation products which are truncated
further through continued oxidation [9–12]. The terminal fate of
the products of continued apocarotenoid degradation is not known
but subject of current investigations of numerous research groups
in the field of carotenoid research.
Continuous carotenoid biosynthesis is required to compensate
for carotenoid degradation which varies between developmental
stages, tissue types and environmental conditions, each characterized by different rates of phytohormone biosynthesis and carotenoid oxidation. Therefore, carotenoid pathway flux takes place
permanently and is dynamically regulated. However, determination
of pathway flux through integrative quantification of all carotenoidderived products is impossible due to their sheer diversity. One
possible approach for the quantification of carotenoid biosynthetic
flux is to determine the turnover of carotenoids by
14
C pulse chase
assay [13, 14]. The high turnover rate observed for, for example,
β-carotene in these experiments strongly supports a very high
carotenogenic pathway flux in leaves.
Alternatively, inhibition of early pathway enzymes can be used
to determine the amounts of carotenoid biosynthesis intermediates, these accumulating upon pathway inhibition and mirroring
the overall carotenoid biosynthesis rate through the rate-limiting
enzyme phytoene synthase (PSY). A common approach to achieve
this is to inhibit phytoene desaturase (PDS) positioned downstream
of PSY by the application of bleaching herbicides, mainly fluridone
and norflurazon [15–17]. This results in the accumulation of phytoene, an intermediate which is usually instantly further metabolized in the pathway and thus absent in most tissues. As shown
previously, phytoene is practically not metabolized by carotenoid
cleavage enzymes and is also resistant toward oxidation as oxidative
cleavage targets higher desaturated carotenoids such as xanthophylls and β-carotene [10]. Accordingly, the phytoene amount
under such inhibitory conditions is a good measure for the total
carotenoid amount that would have been synthesized during the
time the sample is exposed to the bleaching herbicides [4, 18–20].
280
Julian Koschmieder and Ralf Welsch