| Chlorophyll degradation is the most visual representation of plant senescence and fruit ripening. Fruit stay green mutants are valuable resources to study the mechanism of chlorophyll degradation and senescence, while fruit colour mutants are ideal materials for the study of the metabolic regulation of pigment accumulation in fruit. To date, most of color mutants reported in citrus fruits are carotenoid-enriching characteric mutants, but rarely as staygreen mutants, both of which have been widely used to study the regulationary mechanisms underlying pigment metabolism, but seldomly understanding of postharvest traits. Green Ougan is the unique citrus staygreen mutant with important economic value, which is charactered by delay degreened exocarp during ripening stages and storage, and by a better storage property than that in Ougan. Yellow Ponkan is another color mutant with yellowish exocarp when compared with its wide type Ponkan, which showes stronger resistant during postharvest and lower rotting rate than that in wide type.In this study, the mechanisms underlying formation of staygreen phenotype and enhancing storage property of Green Ougan were comprehensively studied at metabolic, protein and transcriptional levels. And also, the mechanisms of formation of yellowish pigmentation and enhanced postharvest resistance characters in Yellowish Ponkan were investigated; In addition, the distinct metabolic profiles of fruits(Yellowish Ponkan and Ponkan) with different harvest maturity during storage were analyzed. The main results are as follows: 1. Molecular mechanism for staygreen phenotype and postharvest traits of Green OuganOur results indicated that Green Ougan fruit is able to start the ripening process and the accumulation of internal nutrient which was characterized by high acid and low sugar. Green Ougan kept stay-green throughtout ripening and storage stages. Green Ougan showed a better storage performance than Ougan as a result of higher juice yield and fruit firmness during postharvest. The above-mentioned results show that the phenotype of Green Ougan belongs to functional stay green. The results of pigment analysis indicated a delayed degradation of chlorophyll and a high accumulation of intermediate metabolites of chlorophylls such as pheophorbide a but a down-regulated accumulation of xanthophylls such as violaxanthin and β-cryptoxanthin in Green Ougan flavedo. Down-regulated accumulation of xanthophylls might lead to the lower ABA content at down-streen pathway in Green Ougan flavedo. The effect of treatments with ethephon, ABA, ethephon+ABA, Na2WO4(ABA synthesis inhibitor), and 1-MCP(inhibitor of ethylene-receptor) on coloring indicated that Green Ougan was able to response to exogenous ethylene signal and then induced a raised synthesis of endogenous ethylene. The promotion of exogenous ABA treaments in the coloring of Ougan but not Green Ougan indicated that content of endogenous ABA might lead to the different pigmentation. Adding ABA could not enhance the effect of ethephon on coloration indicated that ABA and ethylene might have different regulatory mechinsms. Additionally, JA, IAA and other hormones might also be involved in this regulation. Photosynthesis related protein especially photosystem II(PSII) protein showed high level in flavedo of Green Ougan throughtout ripe stages indicated their vital roles in the staygreen event. Digital gene experssion(DEG) analysis showed that expression of the key genes for chlorophyll degradation such as Pa O and PPH and the key genes in chlorophyll cycle NYC1, were significantly down-regulated at coloring stage, while expression of CAO, as well as ELIP and GLK1(both for regulation of chloroplast development) were significantly upregulated at coloring and storage stages. The above differential expressed genes are hints of a abnormal turning between chlorophyll a and b and re-generation of chloroplasts. In summary, the above results suggest that differences of endogenous hormones, assembly and dismantling of plastids, degradation and maintenance of protein in photosystem, chlorophyll degradation and its regulation were likely ot be the cause of the stay green phenotype in Green Ougan. Moreover, we also showed that down-regulation of genes involing in cell wall metabolism and ABA accumulation might also contribute to the delayed senescence status and better postharvest performance in Green Ougan. 2. Molecular mechanism for the yellowish pigmentation and enhanced postharvest resistance in Yellowish PonkanHPLC analysis show that accumulation of xanthophylls such as cryptoxanthin and violaxanthin are reduced in Yelllowish Ponkan. The increase in expression of carotenoid catabolism genes exceeds that of carotenogensis genes in Yellowish Ponkan versus Ponkan might lead to a disequilibrium between catabolism and biosynthesis and furtherly a reduced accumulation of carotenoid. In addition, degree of carotene esterification, abundance of platoglobuli protein(PGL35) and the plastid density in flavedo of yellow Ponkan was significantly down-regulated than those in flavedo of Ponkan, indicating a lower sink capacity of carotenoids in Yellowish Ponkan. These differences might be the cause of the distinct pigmentation between Yellow Ponkan and Ponkan. On the other hand, compared with Ponkan, Yellowish Ponkan showed much stronger resistance against Penicillium digitatum and lower rotting rate during storage. Comparative analysis of secondary metabolites indicated that flavonoids are the most significant differential metabolites between the two materials. Flavonoids especially polymethoxylated flavones showed a significant higher accumulation in Yellow Ponkan throughout ripening stages, storage period and after inoculation than that in Ponkan, might contribute to stronger postharvest resistance in Yellow Ponkan. Moreover, upregulated expression of NECD5 may be cause of enhanced accumulation of abscisic acid in Yellowish Ponkan, while the the abscisic acid content showed positively correlation with the expression of PAL1 gene. Thus, it is hypothesized that changes of pigment in Yellowish Ponkan might upregulate the biosynthesis of flavonoids through increasing the abscisic acid level, which might be contributed to enhanced resistance. The most intense increase of pigment accumulation and reduction of organic acid were occured in fruits during the first 20 days of room temperature storage. After a 50-days storage at room temperature, less mature fruits could catch up with more mature fruits in coloring progress by enhancing the accumulation of carotenoids, mainly xanthophylls. Principal component analyses based on metabolic profiles indicated that the variety is the most important factor to distinguish one flavedo or pulp sample from another. The metabolites which make biggest contribution to principal component in pulp is zeaxanthin, sucrose, glutamine, malic acid, nobiletin; The metabolites which make the biggest contributions to principal component in flavedo is 9-cis-violaxanthin, GABA, gluctose, nobiletin. In addition, both ethephon-dipping and sucrose-injecting treatments on fruits which have been stored for 20 days promoted the accumulation of Vc and amino acids but inhibited that of fatty acids and sugars in the pulp and promoted the degradation of organic acids in the flavedo. |
PDF Full Text Request