| Carotenoids are important secondary metabolites for enhancing of maket value and neturient quality. Carotenoid biosynthesis is believed to occur in chromoplasts which are non-photoynthetic plastids often present in flowers and fruits. The study of mechasim undelying chromoplast differentiation during citrus ripening progress is valuable for both academic sense and applied value. In this thesis, we used several tools, including cytobiology, molecular biology, biochemistry, and proteome analysis, to understand the mechasims of chromoplast characterization and plastid differentiation. The main results were described as follows:1:Chromoplast ultrastructure and understanding of regulation in diverse red-and yellow-fleshed citrus fruits during ripeningThe chromoplast ultrastructure of six red-and yellow-fleshed citrus varieties during ripening was observed by light and transmission electron microscopy, revealing the region-specific formation of two different chromoplast types:globular and crystalloid chromoplasts. The plastid differentiation pattern found in both red-and yellow-colored edible pulps was amyloplast-to-chromoplast transition. We report, for the first time, that red-fleshed mutants of sweet orange were assigned in the membranes of the segment and sac rather than the juice, showing two different chromoplast development patterns. Results of high-performance liquid chromatography (HPLC) analysis of carotenoids indicated that the accumulation of excessive lycopene or P-carotene probably promotes the formation of crystalloid chromoplasts, while the accumulation of lutein and β-violaxanthin was associated with the formation of globular chromoplasts. Real-time PCR analysis of carotenoid metabolic gene expression revealed an increased expression of carotenogenic and storage genes, and a declined expression of CCD4a, along with highly expressed HYD following globular chromoplast differentiation, indicating that the accumulation of violaxanthin and/or lutein is under complex co-regulation. Additionally, the greatly different expression pattern of CCD4b between the two chromoplast types implies that CCD4b might play an important role in the formation of crystalloid chromoplasts. Unlike plastid differentiation in citrus flavedo, the chromoplast differentiation provides a new patternfor further research.2:A proteome-based analysis of the chromoplasts isolated from sweet orange fruits (Citrus sinensis [L.] osbeck) reveals the possible mechanisms underlying chromoplast differentiation(1) Firstly, we report a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation. GeLC-MS/MS shotgun was used to identify the proteins of pooled chromoplast sample. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (-60%) of the chromoplast proteome of sweet orange are constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (Plastid-lipid-associated protein) and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplasts proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.(2) iTRAQ-based comparative proteomics analysis of amyloplast-to-chromoplast transition and chromoplast senescence in Citrus pulp uncovers a complex array of change in impacting metabolic reorganization coupled with plastoglobule formation during fruit ripeningFruit maturation process is accompanied by the transition from an amyloplast metabolic state in an unripening fruit pulp to a typical chromoplast metabolic state in citrus pulp. This process was characterized by following features:the starch granules were significantly degradated and meanwhile the plastoglobules were formed, as well as the internal membrane system was reorganized within plastids. Here, we examined proteome dynamics of this plastid differentiation during fruit ripening. A comparative proteome analysis (iTRAQ) was performed to identify differentially expressed proteins in plastids at three stages of citrus pulp ripening (unripening, breaker, and ripen), and its senescence process (ripen and over-ripen). Stringent curation and bioinformatics processing of the data from three independent replicates indentified 1,905 proteins, among which 1,386 proteins were predicted to be plastid-localized proteins and 1,016 were quantified by spectral counting. The obtained iTRAQ quantification procedures have been subsequently validated by western blotting analysis of five proteins representative of distinct metabolic or regulatory pathways. Among the main changes of the amyloplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts:(a) enzymes involved in plastoglobules formation and protein degradation were strongly up-regulated, (b) several enzymes in ribosome metabolism (involving in protein synthesis in plastid) were significantly decreased, and (c) enzymes, especially in signaling, cell and DNA& RNA remained unchanged in relative abundance. Of particular note, stability in protein import, loss of ribosome assembly, and build-up of chromoplast proteases might be associated with the remodelling of protein systems and might have a crucial role in governing chromoplast biogenesis, With regarding to chromoplast senescence, most proteins assigned to redox & stress, cell, transport, and TCA/org were significantly increased in protein abundance. By contrast, protein involved in protein synthesis, major CHO metabolism, and vitamin metabolism were reduced during senescence but plastoglobule-localized protein remain unchanged. Taken together, the data provide new insights on the chromoplast differentiation from the amyloplast and chromoplast senescence progress, which complement our knowledge of the plant plastid proteome.3. The function of ABC1Ks in the plastoglobules development(1) Structural diffenretiation of the chromoplast is characterized by a sharp and continuous increase of plastoglobules. This is coincident with a significant increase of several plastoglobule-localized proteins, such as ABCK1,3,7, and 9 involved in plastoglobule development. The functional examination were performed after introducing CitABCK1,3, and 7 into tomato, citrus callus and Arabidopsis. This work is under the way.(2) Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening revealing the multi-phosphorylated modification of plastoglobule-localized FBN1Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post-translational modifications, such as phosphorylation, of their resident proteins. We report a proteome-wide mapping of in vivo phosphorylation sites in chromoplast-enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide-based affinity chromatography for phosphoprotein enrichment with LC-MS/MS. A total of 109 plastid-localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. Based on Motif-X analysis, two distinct types of phosphorylation sites, one as proline-directed phosphorylation motif and the other as casein kinase Ⅱ motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids,～22% of them are novel based on BLAST search using the public databases PhosPhAt and P3DB. A close comparative analysis showed that ～50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high-level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteinsimplicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening. Of particular note, CitFBN1 is multi-phosphorylated with high confidence, which would be an interesting point for the further research. |
