Fruits are the mechanism by which angiosperms disperse seeds and are the result of a tight co-evolution between plants and their seed dispersers [1]. Tomato (Solanum lycopersicum L.) belongs to the Solanaceae (Nightshade) family, which contains many differentiated taxa occurring worldwide. Its fruit type is the berry: red, fleshy and with a pulpy interior rich in seeds [2]. Among the 12 wild relatives of tomato, there is only one (Solanum pimpinellifolium B. Juss.) with a red berry and two with yellow, yellow-green or orange fruits [Solanum cheesmaniae (L. Riley) Fosberg; Solanum galapagense S.C. Darwin and Peralta], whereas all
The pathway of carotenoids starts with the synthesis of phytoene and proceeds along a single path up to lycopene which can be transformed to β-carotene by the action of lycopene β-cyclase or to α-carotene through the sequential action of lycopene ε-cyclase and lycopene β-cyclase. All xanthophylls are produced from these two cyclic precursors following two hydroxylation steps. β,β-Xanthophyll biosynthesis requires hydroxylases belonging to the so-called 'non-heme di-iron' group while the biosynthesis of lutein involves enzymes belonging to the vast group of P450 monooxygenases with different enzymatic specificity due to the distinct rings of α-carotene. Here we report on the isolation and functional characterization of tomato CYP97A29 and CYP97C11 genes encoding the P450 carotenoid β- and ε-hydroxylases. Through a reverse transcription-quantitative real-time PCR analysis of the two P450 and nine other carotenoid biosynthetic genes it was possible to highlight the transcriptional patterns of the 11 genes in root, leaf, petal and fruit at three stages of development and ripening. Finally, the characterization of the two P450 carotenoid (A29 and C11) hydroxylases was complemented by an in planta analysis through the use of transgenic plants. Results of this study have permitted us to model the lutein synthesis in leaf and in fruit of tomato.
CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.