Fig 1.
Isolation of tomato fruit PG from chloroplast and chromoplast.
(A) Isolated chloroplasts were fractionated on a sucrose step gradient (5, 15, 20, 38, 45% sucrose). PG (plastoglobules); EN (envelope); TM (thylakoid membranes). (B) Uneven fractions from 1 to 29 were subjected to SDS-PAGE followed by immunoblotting using antibodies against FBN1A (PG marker), TOC75 (envelope marker), and LHCB2 (thylakoid marker). (C) Isolated chromoplasts from red tomato fruit were fractionated on a sucrose step gradient (5, 15, 20, 38, 45% sucrose). PG (plastoglobules); CR (carotenoid crystals); EN (envelope). (D) Uneven fractions 1 to 29 were subjected to SDS-PAGE followed by immunoblotting using antibodies FBN1A (PG marker), TOC75 (envelope marker), and LHCB2 (thylakoid marker).
Fig 2.
Experimental design used for tomato fruit PG proteome analysis.
PGs from mature green and red fruit were isolated separately by sucrose gradient flotation. The two independent replicate PG fractions from mature green and red fruit were analyzed by nano-liquid chromatography (nanoLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS / MS) for peptide identification. All identified peptides (Total Peptides) were further processed by Scaffold, MASCOT, and MaxQuant software to obtain corresponding individual proteins (Individual proteins). The number of individual proteins collected after merging two independent replicates (Total proteins). The Total proteins were filtered using the Uniport, TargetP, SUBA4, and TAIR databases to identify plastid protein (Plastid protein). Plastid proteins were filtered using known chloroplast and chromoplast proteome based on the existing literatures resulting in PG core proteins (PG core proteins). The new PG protein candidates (New candidates) were identified by exclusion of curated stromal, thylakoid and envelope proteins from plastid proteins (Plastid proteins) using PPDB and STRING databases as well as subcellular localization studies in the literature.
Table 1.
Proteome of tomato fruit chloroplast and chromoplast plastoglobules.
Fig 3.
Carotenoid biosynthetic enzymes enriched in tomato PG chromoplasts.
The carotenoid biosynthetic enzymes heatmap was generated from peptide counts obtained from PG isolated from chloroplasts and chromoplasts, respectively.
Fig 4.
Interaction network of chromoplast PG proteins.
The tomato chromoplast PG proteome analysis using the STRING software identified three clusters. Pear, (cluster 1) enriched in carotenoid biosynthetic enzymes; Red, (cluster 2) enriched in prenyl quinone metabolism and regulation; green, (cluster 3) enriched in chloroplast senescence and thylakoid membrane dismantling; blue, proteins not associated with the network.
Fig 5.
Lycopene and β-Carotene accumulate high levels in chromoplast PG.
(A) Total carotenoids were extracted from equal volumes of gradient fractions containing chloroplast (Chl) or chromoplast (Chr) PG. Lycopene was quantified. (B) Quantification of lutein, β-carotene, and violaxanthin/neoxanthin. All values in the Fig are the mean of 3 biological replicates (n = 3). Statistical differences were assessed with students’ t test and p values are indicated.
Fig 6.
Reduced levels of prenyl quinones in chromoplast PG.
(A) The total prenyl quinones were extracted from equal volumes of gradient fractions containing chloroplast (Chl) and chromoplast (Chr) PG, PC-8, plastochromanol; PC-OH, hydroxy-plastochromanol; PQ-9, plastoquinone; PQH2-9, plastoquinol and PQ-OH, hydroxy-plastoquinone were quantified (B) Quantification of phylloquinone. (C) Quantification of tocopherols. All values in the Fig are the mean of 3 biological replicates (n = 3). Statistical differences were assessed with student’s t test and p values are indicated.
Fig 7.
Carotenoid biosynthetic pathway in tomato chromoplast PG.
Chromoplast plastoglobules recruit the carotenoid biosynthetic pathway enzymes: phytoene synthase 1 (PSY1), phytoene desaturase (PDS), z-carotene desaturase (ZDS), and carotenoid isomerase (CRTISO) and lycopene β-cyclase (LYC-β) to promote carotenoid biosynthesis. Lipoxygenase (LOX) may associate with PG, FBNs have structural functions and may contribute to carotenoid sequestration in PG. Phytyl ester synthase (PES) synthesizes phytyl esters and triacyl glycerol during thylakoid dismantling and Activity of BC1 complex kinase 1 (ABC1K1) is implicated in regulation of prenyl lipid metabolism.