Mechanisms For Differential Accumulation Of Carotenoids In Flesh Of Red-fleshed And Ordinary Oranges

The Cara Cara, a red-fleshed mutant originated from Washington navel orange (C. sinensis Osbeck), as the unique navel orange accumulating high amounts of linear carotenoids (phytoene, phytofluene, lycopene) and β-carotene in flesh compared with the ordinary Newhall oranges. While ordinary orange cannot accumulate red lycopene. So the mechanism of difference of carotenoid accumulation in red-fleshed mutant (Cara Cara) and ordinary (Newhall) was our key point. The main results are as follows:We measured the content and component of carotenoid in flesh of Cara Cara and Newhall orange during development and maturation. Significant difference was present in content and component of carotenoid in the two cultivars. The content of total carotenoid of Cara Cara was markedly higher than Newhall, respectively 45.03 μg/g FW and 2.09 μg/g FW, but the content of β,β-xanthophylls were 1.99 μg/g FW and 1.99 μg/g FW, the large difference was high accumulation of phytoene, phytofluene and lycopene in Cara Cara orange. During development, the total carotenoid of Cara Cara was 19-56 times higher than that of Newhall. Meanwhile, big difference was present in the change of component and content of carotenoid. The linear carotenoid appeared to increase quickly in Cara Cara. Though no lycopene was generated in Newhall, where the pattern was similar to Cara Cara, the content was apparently lower.The transcriptional levels of carotenogenic genes were analysed, the mRNA levels of carotenogenic genes was corresponding with the change of content of carotenoid. Expression levels ofDXR、PDS、ZDS、LCYE were slightly higher in flesh of Cara Cara than Newhall, which cannot completely explain the mechanism of differential carotenoid accumulation. Maybe other reason can cause the large accumulation of carotenoid in flesh of Cara Cara orange.Carotenoid biosynthesis was verified to occur in chromoplast, whose number, size and color and development have been elaborated to regulate on the accumulation of carotenoid. Plastid development and interconversion were observed and analysed. Two types of chromoplasts were generated in flesh of Cara Cara, one was red flattened sheets (10-25 μn), and another was spherical (1-2 μm) compared to Newhall, which contain only spherical chromoplast. Large difference was present in the size, color and structural change, which was well corresponding with the high levels of lycopene. Plastid development in two cultivars was different by observing the inner structure. Spherical chromoplasts accumulate yellow carotenoids, whose increase was the result of increase of plastoglobulis. But similar pattern in the two cultivars was present in development of plastid, which came from the conversion of chloroplast. The pattern of development of flattened sheets was proplastid-amyloplast-chromoplast. Flattened sheets chromoplasts generated increasing undulating membranes and large lipid bodies, whose size ranged from 5-10μm to 10-25 μm. This was closely related to sharp increase of high levels of linear carotenoids in Cara Cara orange.Significant difference was present in plastid development of the two cultivars. So the two types of chromoplasts in Cara Cara were separated and analysed comparably. Further, we analysed the differential proteome. Content of carotenoid in flattened sheets chromoplasts was largely higher than spherical chromoplasts. Phytoene, phytofluene, lycopene and β-carotene in flattened sheets chromoplasts was respectively 21.9,21.1,12.5 and 19.0 times higher than spherical chromoplasts. But content of xanthophylls had no significant difference between the two types. It noted that flattened sheets chromoplasts can accumulate large linear carotenoid and β-carotene, and spherical chromoplasts can accumulate yellow xanthophylls. By analysis of the proteome, big significant differential proteins were present in the two types of chromoplasts in Cara Cara, which was focused on fatty acid, amino acid, protein, transportation and stress. Between the two cultivars the differential proteins were concentrated on stress, Carbohydrate energy metabolism and regulation and protein.In conclusion, significant difference was present in plastid structure and development. The generation of red flattened sheets chromoplasts caused large accumulation of carotenoid. Lycopene was synthesized and stored in undulating membranes, and phytoene and phytofluene may be synthesized in big lipid bodies and plastoglobulis.