Pummelo (Citrus maxima) is one of important fruit trees, which belongs to Citrus species. The fruits of different pummelo cultivars have different colors and differ in the contents of carotenoid. Our results clearly showed that ‘Huangjinmiyou’ (HJMY) has the highest content of β-carotene, followed by ‘Hongroumiyou’ (HRMY) and ‘Guanximiyou’ (GXMY). Lycopene is dominantly accumulated in HRMY. However, the molecular mechanism underlying the carotenoid accumulation in pummelo flesh is not fully understood. In this study, we used the RNA-Seq technique to investigate the candidate genes of carotenoid metabolism in the flesh of pummelo cv. GXMY and its mutants HRMY and HJMY in three development periods of fruit. After data assembly and bioinformatic analysis, a total of 357 genes involved in biosynthesis of secondary metabolites were isolated, of which 12 differentially expressed genes (DEGs) are involved in carotenoid biosynthesis. Among these 12 DEGs, phytoene synthase (PSY2), lycopene β-cyclase (LYCB2), lycopene Ɛ-cyclase (LYCE), carotenoid cleavage dioxygenases (CCD4), 9-cis-epoxycarotenoid dioxygenase (NCED2), aldehyde oxidase 3 (AAO3), and ABA 8′-hydroxylases (CYP707A1) are the most distinct DEGs in three pummelo cultivars. The co-expression analysis revealed that the expression patterns of several transcription factors such as bHLH, MYB, ERF, NAC and WRKY are highly correlated with DEGs, which are involved in carotenoid biosynthesis. In addition, the expression patterns of 22 DEGs were validated by real-time quantitative PCR (RT-qPCR) and the results are highly concordant with the RNA-Seq results. Our results provide a global vision of transcriptomic profile among three pummelo cultivars with different pulp colors. These results would be beneficial to further study the molecular mechanism of carotenoid accumulation in pummelo flesh and help the breeding of citrus with high carotenoid content.

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Granulation is a physiological disorder of juice sacs in citrus fruits, which develops through secondary cell wall formation. However, the synergistic changes in the cytoplasm of juice sac cells remain largely unknown. This study investigated the dynamic ultrastructure of juice sacs of “Guanxi” pummelo fruits by transmission electron microscopy and determined their cell wall material, soluble sugar, and organic acid contents. The results showed that lignin and hemicellulose are accumulated in juice sacs isolated from dorsal vascular bundles, while lignin and cellulose contribute to the granulation of juice sacs isolated from septal vascular bundles. The significant differences in lignin, cellulose, and hemicellulose contents between the two types of juice sacs began to be observed at 30 days of storage. Fructose levels were elevated in juice sacs isolated from the dorsal vascular bundles from 10 to 60 days. Sucrose contents significantly decreased in juice sacs isolated from the septal vascular bundles from 30 to 60 days. Meanwhile glucose, citric acid, and malic acid contents exhibited no apparent changes in both types of juice sacs. Based on the comprehensive analysis of the ultrastructure of both types of juice sacs, it was clearly found that plasma membrane ruptures induce cell wall material synthesis in intracellular spaces; however, cell wall substance contents did not significantly increase until the number of mitochondria sharply increased. In particular, sucrose contents began to decrease significantly just after the mitochondria amount largely increased in juice sacs isolated from the septal vascular bundles, indicating that mitochondria play a key role in regulating carbon source sugar partitioning for cell wall component synthesis.