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.

Keywords: Arabidopsis thaliana; carotenoid; ccd4; chloroplast; plastoglobule; senescence.

FIGURE 1

Plastoglobule localization of CCD4. (A) Pre-[³⁵S] CCD4-6xHIS (pCCD4) was incubated with isolated Pea chloroplasts from 0 to 10 min in vitro. Upon import, mature [³⁵S] CCD4-6xHIS (CCD4) showed a lower molecular mass due to the removal of the transit peptide and unlike pCCD4 was resistant to thermolysin protease. (B) Colocalization of CCD4-YFP and FBN1a-CFP in isolated protoplasts of Arabidopsis thaliana. (C) Isolation of PG by chloroplast membrane fractionation on a discontinuous sucrose gradient (PG, plastoglobules; Env, envelopes; Thyl, thylakoids). Pure PG fractions (nos. 1 and 2) contain recombinant CCD4-YFP. Tic40, FBN1a and LHCBII are envelope, PG and thylakoid markers, respectively. +, positive control which corresponds to a membrane fraction containing all four markers.

FIGURE 2

Carotenoids accumulated in senescent plastoglobules of ccd4-2. (A) PG were isolated from naturally senescent ccd4-2 and control plants (3 months old) by a sucrose gradient loaded with a chlorophyll-equivalent of 30 mg of membranes. Lipids from the PG fractions were analyzed by untargeted ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-quadrupole time of flight mass spectrometry and chromatograms of detected lipids are shown. (B) Spectrophotometric analysis of PG fractions. Three maxima around 450 nm, typical of carotenoid absorption spectra, were detected in ccd4-2 PG. (C) Quantification of total carotenoid contents in PG fractions. Absorbance at 448 nm was recorded and compared with a calibration curve of β-carotene standard. Carotenoid content is expressed in μg of β-carotene per 30 mg of chlorophyll of total membranes used in the experiment. MGDG, monogalactosyldiacylglycerol; α-T, α-tocopherol; VitK, vitamin K; PC-OH, hydroxy-plastochromanol; PQH2, plastoquinol; PC-8, plastochromanol; PQ-9, plastoquinone; Ctrl, control; Car, β-carotene; Chl, chlorophyll.

FIGURE 3

Complementation of ccd4 mutants with 35S:CCD4-YFP-HA. (A) Relative amounts of CCD4-YFP-HA in complemented lines were assessed by immunoblot analysis with anti-HA antibodies. A representative blot of 16 independent lines is shown, none of which showed a visible phenotype. (B) Complementation analysis of CCD4 complemented lines by dark-induced senescence. Detached leaves were subjected to dark-induced senescence for 10 days and complementation was determined based on the phenotype. Two 35S:CCD4 plants (h and j) were not complemented as they showed a yellowish color similar to ccd4-4. (C) Total chlorophyll and carotenoids were extracted from the leaves shown in (B) (NB: the white asterisks mark the leaves that were excluded for the calculations). Concentrations of pigments were established with a spectrophotometer. Means ± SD were calculated for CCD4 complemented plants (n = 7), WT (n = 3) and ccd4 (n = 2). Statistical significance was determined by a Student’s t-test (unpaired homoscedastic, two-tailed, ^∗P < 0.05, ^∗∗∗P < 0.001). (D) CCD4 gene expression analysis. Transcripts of CCD4 were quantified by qPCR in the WT and in the two complemented lines, i.e., 35S:CCD4.2 and 35S:CCD4.4. The relative expression of CCD4 was normalized to the reference gene ACTIN. Means ± SD (n = 6). Statistical significance between transgenic lines and the WT was determined by a Student’s t-test (unpaired heteroscedastic, two-tailed, ^∗∗P < 0.01, ^∗∗∗P < 0.001).

FIGURE 4

Constitutive expression of CCD4 resulted in a distinct lipid profile during dark-induced senescence in leaves when compared with WT. (A) Detached leaves were placed in darkness for 8 days prior to lipid extraction (n = 5). Lipids were analyzed by ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-quadrupole time of flight mass spectrometry. Discrimination was evaluated with a partial least squares-discriminant analysis in which outlying samples were removed prior to data processing. (B) Compounds responsible for the difference between CCD4 complemented lines and WT are indicated on the loading plot. ^∗Statistically significant according to Student’s t-test (unpaired heteroscedastic, two-tailed). PC, principal component.

FIGURE 5

Reduced level of photosynthesis-related carotenoids during senescence in CCD4 complemented plants. Detached leaves were placed in darkness for 8 days prior to lipid extraction (n = 5). Lipids were separated by UHPLC-DAD following the carotenoid-profiling protocol. Absolute quantification of carotenoids was carried out on the basis of calibration curves obtained with pure standards. Box plots created with SigmaPlot are shown. Statistical differences across genotypes were assessed with a one-way ANOVA. Pairwise multiple comparisons (Holm–Sidak method) revealed significant differences (letters are distributed accordingly, P < 0.05).