Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus.
Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.
Frydman
Ahuva
A
Institute of Horticulture, The Volcani Center, ARO, PO Box 6, Bet-Dagan 50250, Israel.
Weisshaus
Oori
O
Bar-Peled
Maor
M
Huhman
David V
DV
Sumner
Lloyd W
LW
Marin
Francisco R
FR
Lewinsohn
Efraim
E
Fluhr
Robert
R
Gressel
Jonathan
J
Eyal
Yoram
Y
D000595:Amino Acid Sequence; D002957:Citrus / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D005419:Flavonoids / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013649:Taste
D005419:Flavonoids; D010940:Plant Proteins; D012333:RNA, Messenger; D006602:Hexosyltransferases; C493952:flavonoid 1-2 rhamnosyltransferase