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.
Highlights
Plastoglobules (PG) are chloroplast lipoprotein particles surrounded by a lipid monolayer and associated with the thylakoid membrane via the outer lipid leaflet
A N-terminal chloroplast transit peptide of 34 residues was predicted by ChloroP algorithm1
Subcellular localization of Carotenoid cleavage dioxygenase 4 (CCD4) was analyzed by confocal microscopy of protoplasts prepared from A. thaliana ccd4-2 mutants complemented with CCD4-YFP, which were subsequently transformed with a construct encoding FBN1a-CFP
Summary
Plastoglobules (PG) are chloroplast lipoprotein particles surrounded by a lipid monolayer and associated with the thylakoid membrane via the outer lipid leaflet. It was shown that PG contain enzymes such as tocopherol cyclase (VTE1), NAD(P)H dehydrogenase C1 (NDC1), and phytyl ester synthase (PES), as well as others (Zbierzak et al, 2010; Eugeni Piller et al, 2011, 2014; Lippold et al, 2012). PG accumulate large amounts of carotenoid esters and are implicated in carotenoid biosynthesis. Carotenoid biosynthesis enzymes are recruited to chromoplast PG presumably to channel intermediates and streamline carotenoid production (Ytterberg et al, 2006). A homolog of PES1, pale yellow petal 1 (PYP1) has been implicated in the formation of the abundant carotenoid esters in chromoplasts (Ariizumi et al, 2014). Carotenoid biosynthetic enzymes associate with membranes rather than PG (Ruiz-Sola and Rodríguez-Concepción, 2012). It has been shown that carotenoid cleavage dioxygenase 4 (CCD4) is present in the PG proteome (Vidi et al, 2006; Ytterberg et al, 2006; Lundquist et al, 2012) suggesting a role of PG in carotenoid cleavage
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