Study and Manipulation of the Flavanoid Biosynthetic Pathway in Citrus for Flavor Engineering and Seedless Fruit

The proposal was aimed to identify and functionally characterize key genes/enzymes in the citrus flavanone neohesperidoside biosynthetic pathway and to use them as tools for metabolic engineering to decrease bitterness levels in grapefruit. The proposed section on fruit seediness was dropped as suggested by the reviewers of the proposal. Citrus flavor and aroma is composed of complex combinations of soluble and volatile compounds. The former includes mainly sugars, acids and flavanones, a subgroup of flavonoids that includes bitter compounds responsible for the bitter flavor of grapefruit and pummelo. Bitter species contain mostly bitter flavanone neohesperidosides, while non-bitter species contain mostly tasteless flavanone rutinosides. Both flavanone versions are diglycosides consisting of a rhamnose-glucose oligosaccharide a-linked at position 7 to the flavanone skeleton. However, in the bitter neohesperidosides the rhamnose is attached at position 2 of the glucose moiety, while in the tasteless rutinosides the rhamnose is attached at position 6 of the glucose moiety. Thus, the position of the rhamnose moiety, determined by the specificity of the last enzymes in the pathway- rhamnosyltransferase (1,2 or 1,6 specificity), is the determinant of the bitter flavor. Flavanones, like all flavonoids are synthesized via one of the branches of the phenylpropanoid pathway; the first committed step is catalyzed by the enzyme Chalcone synthase (CHS) followed by Chalcone isomerase (CHI). During the course of the work a key gene/enzyme in the biosynthesis of the bitter flavanones, a 1,2 rhamnosyltransferase (1,2RT), was functionally characterized using a transgenic cell-culture biotransformation system, confirming that this gene is a prime candidate for metabolic engineering of the pathway. This is the first direct functional evidence for the activity of a plant recombinant rhamnosyltransferase, the first confirmed rhamnosyltransferase gene with 1,2 specificity and the second confirmed rhamnosyltransferase gene altogether in plants. Additional genes of the flavanone pathway that were isolated during this work and are potential tools for metabolic engineering include (I) A putative 1,6 rhamnosyltransferase (1,6RT) from oranges, that is presumed to catalyze the biosynthesis of the tasteless flavanones. This gene is a prime candidate for use in future metabolic engineering for decreased bitterness and is currently being functionally characterized using the biotransformation system developed for characterizing rhamnosyltransferases. (2) A putative 7-0-glucosyltransferase presumed to catalyze the first glycosylation step of the flavanone aglycones. Silencing of gene expression in grapefruit was attempted using three genes: (1) The "upstream" flavonoid biosynthesis genes CHS and CHI, by antisense and co-suppression; and (2) The "downstream" 1,2R T, by an RNAi approach. CHS and CHI silencing resulted in some plants with a dramatically decreased level of the bitter flavanone neohesperidoside naringin in leaves. We have yet to study the long-term effect of silencing these genes on tree physiology, and on the actual bitterness of fruit. The effect of 1,2RT silencing on naringin content in grapefruit has yet to be examined, but a slow growth phenotype for these plants was noted. We speculate that silencing of the final glycosylation step of the flavanones delays their evacuation to the vacuole, resulting in accumulation of flavanones in the cytoplasm, causing inhibitory effects on plant growth. This speculation is yet to be established at the product level. Future metabolic engineering experiments are planned with 1,6RT following functional characterization.