The molecular and enzymatic basis of bitter/non-bitter flavor of citrus fruit: evolution of branch-forming rhamnosyltransferases under domestication.

Domestication and breeding of citrus species/varieties for flavor and other characteristics, based on the ancestral species pummelo, mandarin and citron, has been an ongoing process for thousands of years. Bitterness, a desirable flavor characteristic in the fruit of some citrus species (pummelo and grapefruit) and undesirable in others (oranges and mandarins), has been under positive or negative selection during the breeding process of new species/varieties. Bitterness in citrus fruit is determined by the composition of branched-chain flavanone glycosides, the predominant flavonoids in citrus. The flavor-determining biosynthetic step is catalyzed by two branch-forming rhamnosyltransferases that utilize flavanone-7-O-glucose as substrate. The 1,2-rhamnosytransferase (encoded by Cm1,2RhaT) leads to the bitter flavanone-7-O-neohesperidosides whereas the 1,6-rhamnosytransferase leads to the tastelessflavanone-7-O-rutinosides. Here, we describe the functional characterization of Cs1,6RhaT, a 1,6-rhamnosyltransferase-encoding gene directing biosynthesis of the tasteless flavanone rutinosides common to the non-bitter citrus species. Cs1,6RhaT was found to be a substrate-promiscuous enzyme catalyzing branched-chain rhamnosylation of flavonoids glucosylated at positions 3 or 7. In vivo substrates include flavanones, flavones, flavonols and anthocyanins. Cs1,6RhaT enzyme levels were shown to peak in young fruit and leaves, and gradually subside during development. Phylogenetic analysis of Cm1,2RhaT and Cs1,6RhaT demonstrated that they both belong to the branch-forming glycosyltransferase cluster, but are distantly related and probably originated separately before speciation of the citrus genome. Genomic data from citrus, supported by a study of Cs1,6RhaT protein levels in various citrus species, suggest that inheritance, expression levels and mutations of branch-forming rhamnosyltransferases underlie the development of bitter or non-bitter species/varieties under domestication.

ANTHOCYANIN1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the ANTHOCYANIN FRUIT phenotype of tomato.

Anthocyanins are flavonoid metabolites contributing attractive colors and antioxidant qualities to the human diet. Accordingly, there is a growing interest in developing crops enriched with these compounds. Fruits of the cultivated tomato, Solanum (S.) lycopersicum, do not normally produce high levels of anthocyanins. However, several wild tomato species yield anthocyanin-pigmented fruits, and this trait has been introgressed into the cultivated tomato. Two genes encoding homologous R2R3 MYB transcription factors, termed ANT1 and AN2, were previously genetically implicated in anthocyanin accumulation in tomato fruit peels of the ANTHOCYANIN FRUIT (AFT) genotype originating from S. chilense. Here we compared transgenic tomato plants constitutively over-expressing the S. lycopersicum (35S::ANT1 ( L ) ) or the S. chilense (35S::ANT1 ( C )) allele, and show that each displayed variable levels of purple pigmentation in vegetative as well as reproductive tissues. However, 35S::ANT1 ( C ) was significantly more efficient in producing anthocyanin pigments, attributed to its gene coding-sequence rather than to its transcript levels. These results expand the potential of enhancing anthocyanin levels through engineering coding-sequence polymorphisms in addition to the transcriptional alterations commonly used. In addition, a segregating population obtained from a recombinant genotype revealed that the native ANT1, and not AN2, is fully associated with the AFT phenotype and that ANT1 alone can generate the characteristic phenotype of anthocyanin accumulation in AFT fruits. Our results therefore provide further support to the hypothesis that ANT1 is the gene responsible for anthocyanin accumulation in fruits of the AFT genotype.