Changes in Carotenoid Content and Biosynthetic Gene Expression in Juice Sacs of Four Orange Varieties (Citrus sinensis) Differing in Flesh Fruit Color

The contribution of carotenoid composition to the color range of the fruit juice sacs of four orange varieties (Citrus sinensis) differing in flesh color, namely, Shamouti (normal orange color), Sanguinelli (“blood cultivar” purple color), Cara Cara navel (pink-reddish), and Huang pi Chen (yellowish color), was investigated. To this end, qualitative and quantitative analyses of carotenoid contents were first performed by high-performance liquid chromatography (HPLC) using a C30 column and a photodiode array detector in February, at a late developmental fruit stage. Concomitantly, transcript levels of Dxs, the gene controlling the first step of the MEP pathway, and six genes involved in β,β-xanthophyll biosynthesis (Psy, Pds, Zds, Lcy-b, Hy-b, and Zep) were determined in August, November, and February. Transcript level measurement was carried out by real-time RT-PCR on total RNA from juice sacs. The four orange varieties displayed different carotenoid profiles. Shamouti and Sanguinelli oranges accumulated mainly β,β-xanthophylls as expected in typically colored oranges, whereas Cara Cara navel orange accumulated linear carotenes in addition to cis-violaxanthin. Huang pi Chen fruit flesh orange was characterized by a strong reduction of total carotenoid content. Whereas gene expression was relatively low and similar in August (before color break) in all four varieties, in November (during color break), Dxs, Zds, Hy-b, and Zep expression was higher in Cara Cara and Huang pi Chen oranges. The β,β-xanthophyll accumulation observed in February in Shamouti and Sanguinelli oranges was apparently related to the increase of transcript levels of all measured genes (i.e., Dxs, Psy, Pds, Zds, Hy-b, and Zep) except Lcy-b. At this time, however, transcript levels in Cara Cara were rather similar to those found in Sanguinelli, although both showed different carotenoid compositions. The Huang pi Chen phenotype correlated with lower expression of Dxs and Psy genes. These results revealed a general pattern of transcript change in juice sacs of citrus fruit, characterized by an apparent coordination of Dxs and Psy expression and a general increase in mRNA levels of carotenoid biosynthetic genes. These transcript changes correlated well with the β,β-xanthophyll accumulation, the normal carotenoid set, observed in Shamouti and Sanguinelli oranges and suggest that the preferential accumulation of linear carotenes in Cara Cara navel and the practical absence of carotenoids in Huang pi Chen oranges were not predominantly due to changes in regulation of carotenoid biosynthetic genes at the transcriptional level.