Investigation into the molecular basis for the absence of dihydromyricetin-derived flavonoids in raspberry

Red raspberry (Rubus idaeus) is considered a health-promoting food with antioxidant properties. Flavonoid-3',5'-hydroxylase (F3'5'H), of the CYP75 family, is a member of the cytochrome P450 superfamily that catalyzes hydroxylation reactions to yield dihydromyricetin (DHM)-derived flavonoids. Raspberry and other Rosaceae family members such as apple (Malus spp.) and peach (Prunus spp.) preferentially accumulate dihydroquercetin (DHQ)-derived flavonoids, while lacking the DHM types. This peculiarity was investigated by comparative genomics and functional genomics of the F3’5’H gene(s). To investigate the molecular basis for the lack of F3’5’H activity in raspberry that could be attributed to aberrations at the transcriptional level, I first validated DNA template quality and a PCR-based strategy for gene cloning in raspberry and found it to be feasible. Numerous PCR efforts failed to detect any F3’5’H transcripts in fruit cDNAs nor could F3’5’H gene(s) be detected in the raspberry genome. Apple and peach whole genome sequence data mining also did not identify F3’5’H. Taken together, I have shown that PCR, anthocyanin profiling, and comparative genomics data are corroborated and collectively make a strong case for the raspberry genome lacking an F3’5’H ortholog. Genetic complementation of the absence of DHM-derived flavonoids in raspberry and apple callus lines was undertaken through inoculation of Agrobacterium tumefaciens harbouring a recombinant vector with grapevine F3’5’H gene driven by the 35S promoter. I established raspberry callus lines derived from leaves as well as apple callus lines derived from fruit. I then showed that both raspberry and apple callus lines are amenable to Agrobacterium-mediated genetic transformation via GUS histochemical assay. I found that light together with nitrate and 2,4-D deficient culture medium rapidly and efficiently induced anthocyanin accumulation in apple callus. While anthocyanins and flavonols were readily detected in treated apple callus lines, only hydroxycinnamic acid derivatives and early pathway intermediates were detected in treated raspberry callus lines. PCR analyses were conducted on putative raspberry transgenic callus lines. It was found that F3’5’H gene expression was detected in actively proliferating raspberry transgenic callus lines, as well as F3’5’H gene insertion(s) in the raspberry transgenic callus genome in the same selected lines.Medicine, Faculty ofMedical Genetics, Department ofGraduat