Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development

Abstract

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953-2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants.

Fig. 1.

Isolation of chromoplasts from six crop species. (A) Images of fruits and vegetables used for chromoplast isolation. (B) Chromoplasts were isolated by sucrose gradient centrifugation as described in Materials and Methods. Intact chromoplasts were collected from layers between 17/30% and 30/50% and washed extensively to remove contaminated proteins (this figure is available in colour at JXB online).

Fig. 2.

One-dimensional SDS-PAGE gels of chromoplast proteins from various fruits and vegetables. M, membrane fraction; S, stromal fraction; T, total chromoplast (this figure is available in colour at JXB online).

Fig. 3.

Functional category of plastidial proteins identified from six crop species according to MapMan.

Fig. 4.

Simplified scheme of carotenoid biosynthetic pathway. The enzymes identified in the chromoplast proteomes are boxed. The crop species representing by numbers that contain the particular carotenoid pathway proteins are aligned next to the enzymes in the pathway: 1, watermelon; 2, tomato; 3, carrot; 4, cauliflower; 5, papaya; 6, pepper. ABA, abscisic acid; CCD, carotenoid cleavage dioxygenase; CCS, capsanthin/capsorubin synthase; CMK, 4-(cytidine 5’-diphospho)-2-C-methyl-d-erythritol kinase; CrtISO, ζ-carotene isomerase/carotenoid isomerase; CYP97A, β-carotene hydroxylase (P450); CYP97C, ε-carotene hydroxylase (P450); DXR, 1-deoxy-d-xylulose-5-phosphate reductase; DXS, 1-deoxy-d-xylulose-5-phosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, hydroxymethylbutenyl diphosphate synthase; HYD, β-carotene hydroxylase gene; IDI, isopentenyl diphosphate isomerase; LCY-b, lycopene β-cyclase; LCY-e, lycopene ε-cyclase; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase; NCED, 9-cis-epoxycarotenoid dioxygenase; PDS, phytoene desaturase; PSY, phytoene synthase; VDE, violaxanthin de-epoxidase; ZDS, ζ-carotene desaturase; ZEP, zeaxanthin epoxidase; Z-ISO, ζ-carotene isomerase.