Citrus carotenoid isomerase gene characterization by complementation of the "Micro-Tom" tangerine mutant

Complementation of the "Micro-Tom" tomato tangerine mutant with a Citrus CRTISO allele restores the wild-type fruit carotenoid profile, indicating that the Citrus allele encodes an authentic functional carotenoid isomerase. Citrus fruits are rich in carotenoids; the genus offers a large diversity in composition, yet to be fully explored to improve fruit nutritional quality. As perennial tree species, Citrus lack the resources for functional genetic studies, requiring the use of model plant systems. Here, we used the "Micro-Tom" (MT) tomato carrying the tangerine mutation (t), deficient for the carotenoid isomerase (CRTISO) gene, to functionally characterize the homologous C. sinensis genes. We identified four putative loci in the C. sinensis genome, named CsCRTISO, CsCRTISO-Like 1, CsCRTISO-Like 2, and CsCRTISO-Like 2B, with the latter as a presumed duplication of CRTISO-Like 2. In general, all the Citrus paralogs showed less expression specialization than the tomato ones, with CsCRTISO being the most expressed gene in all tissues analyzed. MT-t plants were successfully complemented with the CsCRTISO, and fruits showed a carotenoid profile similar to the control, indicating that the Citrus allele indeed encodes an authentic functional carotenoid isomerase and that the signal peptide is functional in tomato. MT was silenced using an inverted repeat of a fragment from the Citrus CRTISO resulting in a stronger phenotype than MT-t. MT-t and MT silenced for CRTISO presented an overall decrease in transcript accumulation of all genes from the biosynthesis pathway. The expression of the Citrus CRTISO gene is able to restore the biosynthesis of carotenoids with the appropriate regulation in MT-t. The decrease in transcript accumulation in MT-t and MT-CRTISO-suppressed lines reinforces previous suggestions that transcriptional regulation of the carotenoid biosynthesis involves regulatory loops by intermediate products.