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

KEY MESSAGE: 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.

Carotenoid biosynthesis and quality characteristics of new hybrids between tangor (Citrus reticulata x C. sinensis) cv. 'Murcott' and sweet orange (C. sinensis) cv. 'Pêra'.

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. 'Murcott' and sweet orange cv. 'Pêra'. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly ß-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250-426 µg/g dry weight [dw]) and ß-cryptoxanthin (81-125 µg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 µg/g dw) and ß-cryptoxanthin (5-22 µg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 µg/g dw versus 158-250 µg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content.