Identification of Plastoglobules as a Site of Carotenoid Cleavage

Abstract

Carotenoids play an essential role in light harvesting and protection from excess light. During chloroplast senescence carotenoids are released from their binding proteins and are eventually metabolized. Carotenoid cleavage dioxygenase 4 (CCD4) is involved in carotenoid breakdown in senescing leaf and desiccating seed, and is part of the proteome of plastoglobules (PG), which are thylakoid-associated lipid droplets. Here, we demonstrate that CCD4 is functionally active in PG. Leaves of Arabidopsis thaliana ccd4 mutants constitutively expressing CCD4 fused to yellow fluorescent protein showed strong fluorescence in PG and reduced carotenoid levels upon dark-induced senescence. Lipidome-wide analysis indicated that β-carotene, lutein, and violaxanthin were the principle substrates of CCD4 in vivo and were cleaved in senescing chloroplasts. Moreover, carotenoids were shown to accumulate in PG of ccd4 mutant plants during senescence, indicating translocation of carotenoids to PG prior to degradation.

Carotenoid cleavage in chromoplasts of white and yellow‐fleshed peach varieties

“…An increasing array of data point at this structure not as a mere storage droplet, but as an active site for prenylquinones and carotenoids metabolism . This possibility is also consistent with recent data showing that the plastoglobule is the site of β‐carotene, lutein, and violaxanthin cleavage by CCD4 in Arabidopsis senescent chloroplasts …”

“…Most activity localized in S5 pellets (material pelleted at 5000 g ), where the bulk of chromoplasts are recovered. When this material was resuspended in ipotonic buffer and further centrifuged, no significant solubilization of activity was found, strengthening previous data pointing at the plastoglobule as the site for carotenoid breakdown . Data refer to a single preparation from Yumyeong fruits harvested in S1 (first exponential growth) phase.…”

“…23,24 The same protocol at 510 nm has been claimed to be effective in discriminating -carotene from its coloured degradation intermediates. 25 Moreover, the plastoglobules in chromoplasts have been conclusively identified as a main site of carotenoid breakdown, 26 confirming the data of previous proteomic analyses. 27 Here we report on the measurement of the rates of -carotene and apocarotenal bleaching in chromoplasts isolated from yellow-and white-fleshed peach fruits harvested at different stages of their developmental cycle.…”

“…29 This possibility is also consistent with recent data showing that the plastoglobule is the site of -carotene, lutein, and violaxanthin cleavage by CCD4 in Arabidopsis senescent chloroplasts. 26 To rule out the trivial possibility that substrate bleaching may be only caused by -carotene adsorption to the chromoplast membrane/plastoglobule, activity was in all cases determined by difference between the decrease in absorbance in native samples and that in parallel samples that had been heat-inactivated immediately before the assay (Fig. 3(A)).…”

“…When this material was resuspended in ipotonic buffer and further centrifuged, no significant solubilization of activity was found, strengthening previous data pointing at the plastoglobule as the site for carotenoid breakdown. 26 Data refer to a single preparation from Yumyeong fruits harvested in S1 (first exponential growth) phase. The whole experiment was repeated twice, obtaining very similar patterns.…”