## 3 Discussion  
Citrus fruits are ornamental and edible. Color is one of the main economic traits in citrus production. Red-fleshed citrus is more popular with planters and consumers than wild-type citrus due to its high economic and health value. In this study, we investigated the biochemical and molecular changes underlying the characteristic pigmentation of R-HJH. To the best of our knowledge, this is the first time that this kind of bud mutant has been reported in HJH. Carotenoids are responsible for flesh color and nutritional quality in horticultural crops such as tomato and citrus. In this study, a comparative analysis of carotenoid content and composition in the juice sacs and albedo tissue of R-HJH and wild-type HJH was performed. Wild-type HJH contained a normal level of carotenoids, while the mutant R-HJH contained over three to four times the amount of carotenoids in the albedo tissue and juice sacs. Higher-than-normal accumulation of carotenoids in mutant citrus fruits has also been reported in the orange (C. _sinensis_) varieties Shara, Cara Cara and Hong Anliu, as well as the pummelo (C. _grandis_) varieties Huangjinmiyou and Hongroumiyou (red-fleshed pummelo) [8; 9; 10; 18]. Based on data reported in these literature results, the total carotenoids in the juice sacs of R-HJH were lower than those in Huangjinmiyou and Hongroumiyou but still higher than those in the juice sacs of the red oranges Cara Cara and Hong Anliu. However, the color of the albedo tissue was red in R-HJH, while it was still white in red-fleshed pummelo, similar to normal pummelo. Little attention has been given to the nutritional value of the albedo tissue due to its nonedible nature in freshly consumed cultivars of pumelo. However, the whole fruits of wild-type HJH have been used in traditional Chinese herbal medicines [19]. The total carotenoid content of the albedo tissue in R-HJH was significantly higher than that in wild-type HJH, and there was a slight decrease in this content in the albedo tissue compared with the juice sacs in R-HJH. The same trend was observed for lycopene, \(\beta\)-carotene and \(\gamma\)-carotene. Phytoene, a colorless C40 carotenoid intermediate, was found to be enriched in the juice sacs and albedo of R-HJH. However, it was not present in the juice sacs or albedo of wild-type HJH. Phytoene has been reported to be the precursor of caroten and is converted into lycopene through successive desaturation reactions [20]. Based on these biochemical analyses, we found that the massive accumulation of linear carotenoids, such as lycopene and phytoene, the precursor of caroten, led to an attractive red color in the juice sacs and albedo of R-HJH. Another interesting aspect of this study was the observation of the occurrence of a significant decrease in the flavone, coumarin and volatile contents in R-HJH. Plant secondary metabolites, such as carotenoids, volatile terpenoids, limonoids and ABA, belong to a large class of terpenoids and share the common precursor pyruvate. The content of these metabolites was closely related to the competitive ability of the relative metabolic pathways for their same precursor compounds. Similar results have been reported inother red-fleshed mutants, such as Cara Cara, red-Anliu oranges and red-fleshed Guanxi pumpmelo. Cara Cara and red-Anliu oranges showed increased contents of carotenoids and the limonoid aglycone and decreased contents of abscisic acid (ABA) catabolites, monoterenoid volatiles, and sesquiterpenoid volatiles [21]. [PERSON] et al. reported that the ABA contents in red-Anliu orange and red-fleshed Guanxi pumpmelo were significantly lower than those in their corresponding wild types [22]. With the development of molecular biotechnology, the carotenoid biosynthetic and metabolism pathways in citrus have been clarified, and some key genes have been cloned successfully. This study provides a global view of the gene expression changes in the red-fleshed mutant R-HJH compared to wild-type HJH.