04646naa a2200337 a 450000100080000000500110000800800410001902200140006002400350007410000180010924502260012726000090035350008320036252027290119465000170392365000110394065300150395165300200396665300110398665300170399765300290401465300130404365300280405670000130408470000140409770000180411170000130412970000190414270000180416177301290417910606612022-02-24       2019    bl uuuu     u00u1 u    #d  a1471-22297 a10.1186/s12870-019-2078-22DOI1 aRODRIGO, M.J.  aA 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.h[electronic resource]  c2019  aArticle history: Received: 17 June 2019 / Accepted: 16 October 2019 / Published online: 4 November 2019. Funding text: Financial support of the research grants AGL2012?34576 and AGL2015? 70218 (Ministerio Economía y Competitividad, Spain), and RTI2018-095131-BI00 (Ministerio Ciencia, Innovacion y Universidades, Spain). Work in the group of JH was supported by Israel Science Foundation Grant 850/13. MJR, LZ, JH and OD are members of Eurocaroten (COST_Action CA15136). MJR and LZ belong to CaRed (Spanish Carotenoid Network BIO2017?90877-REDT, Minis-terio de Ciencia, Innovación y Universidades, Spain). Authors acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). Supplementary information accompanies this paper.  aAbstract. 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.  © 2019 The Author(s).  aCAROTENOIDES  aCITRUS  aCarotenoid  aGENE EXPRESSION  aMUTANT  aPigmentation  aPLATAFORMA AGROALIMENTOS  aRIPENING  aZeta-carotene isomerase1 aLADO, J.1 aALÓS, E.1 aALQUÉZAR, B.1 aDERY, O.1 aHIRSCHBERG, J.1 aZACARÍAS, L.  tBMC Plant Biology, 4 November 2019, Volume 19, Issue 1, Article number 465. OPEN ACCESS. Doi: DOI: 10.1186/s12870-019-2078-2