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Background  Fruit coloration is one of the main quality parameters of  Citrus  fruit primarily determined by genetic factors. The fruit of ordinary sweet orange ( Citrus sinensis ) displays a pleasant orange tint due to accumulation of carotenoids, representing β,β-xanthophylls more than 80% of the total content. ‘Pinalate’ is a spontaneous bud mutant, or somatic mutation, derived from sweet orange ‘Navelate’, characterized by yellow fruits due to elevated proportions of upstream carotenes and reduced β,β-xanthophylls, which suggests a biosynthetic blockage at early steps of the carotenoid pathway.    Results  To identify the molecular basis of ‘Pinalate’ yellow fruit, a complete characterization of carotenoids profile together with transcriptional changes in carotenoid biosynthetic genes were performed in mutant and parental fruits during development and ripening. ‘Pinalate’ fruit showed a distinctive carotenoid profile at all ripening stages, accumulating phytoene, phytofluene and unusual proportions of 9,15,9′-tri- cis-  and 9,9′-di- cis -ζ-carotene, while content of downstream carotenoids was significantly decreased. Transcript levels for most of the carotenoid biosynthetic genes showed no alterations in ‘Pinalate’; however, the steady-state level mRNA of  ζ-carotene isomerase  ( Z-ISO ), which catalyses the conversion of 9,15,9′-tri- cis-  to 9,9′-di- cis -ζ-carotene ,  was significantly reduced both in ‘Pinalate’ fruit and leaf tissues. Isolation of the ‘Pinalate’  Z-ISO  genomic sequence identified a new allele with a single nucleotide insertion at the second exon, which generates an alternative splicing site that alters  Z-ISO  transcripts encoding non-functional enzyme. Moreover, functional assays of citrus Z-ISO in  E.coli  showed that light is able to enhance a non-enzymatic isomerization of tri- cis  to di- cis -ζ-carotene, which is in agreement with the partial rescue of mutant phenotype when ‘Pinalate’ fruits are highly exposed to light during ripening.    Conclusion  A single nucleotide insertion has been identified in ‘Pinalate’  Z-ISO  gene that results in truncated proteins. This causes a bottleneck in the carotenoid pathway with an unbalanced content of carotenes upstream to β,β-xanthophylls in fruit tissues. In chloroplastic tissues, the effects of Z-ISO alteration are mainly manifested as a reduction in total carotenoid content. Taken together, our results indicate that the spontaneous single nucleotide insertion in  Z-ISO  is the molecular basis of the yellow pigmentation in ‘Pinalate’ sweet orange and points this isomerase as an essential activity for carotenogenesis in citrus fruits.

bmc plant biology

A mutant allele of ζ-carotene isomerase (Z-ISO) is associated with the yellow pigmentation of the “Pinalate” sweet orange mutant and reveals new insights into its role in fruit carotenogenesis