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

• Published in: The Plant journal : for cell and molecular biology Vol. 40; no. 1; pp. 88 - 100
• Main Authors: Frydman, A, Weisshaus, O, Bar-Peled, M, Huhman, D.V, Sumner, L.W, Marin, F.R, Lewinsohn, E, Fluhr, R, Gressel, J, Eyal, Y
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.10.2004; Blackwell Science; Blackwell Publishing Ltd
• Subjects: Amino Acid Sequence; amino acid sequences; biochemical pathways; Biological and medical sciences; biosynthesis; bitter flavor; bitter-tasting compounds; bitterness; cell suspension culture; Citrus; Citrus - enzymology; Citrus - genetics; Citrus aurantium; Citrus fruits; Citrus maxima; Citrus medica; Citrus paradisi; Citrus reticulata; Citrus sinensis; Cloning, Molecular; complementary DNA; Conserved Sequence; cultured cells; enzyme activity; enzyme substrates; Enzymes; flavanone-7-O-glucoside-1,2-rhamnosyltransferase; flavanones; flavones; flavonoid; flavonoids; Flavonoids - biosynthesis; flavonols; Flavors; Fortunella margarita; fruits (plant anatomy); Fundamental and applied biological sciences. Psychology; Gene expression; genetic transformation; hexosyltransferases; Hexosyltransferases - chemistry; Hexosyltransferases - genetics; Humans; leaves; messenger RNA; Metabolism; Metabolism. Physicochemical requirements; Molecular Sequence Data; naringenin; naringin; neohesperidoside; Nicotiana tabacum; nucleotide sequences; Plant Leaves - enzymology; Plant Leaves - genetics; Plant Leaves - growth & development; Plant physiology and development; plant proteins; Plant Proteins - chemistry; Plant Proteins - genetics; Poncirus trifoliata; pummelos; recombinant fusion proteins; regiospecificity; rhamnosyltransferase; RNA, Messenger - genetics; Sequence Alignment; Sequence Homology, Amino Acid; substrate specificity; Taste; tissue distribution; transgenes; transgenic plants; bitter flavor; Fruit; Enzyme; Citrus fruit; Transferases; Rutaceae; Citrus paradisi; regiospecificity; Biosynthesis; Flavonoid; Flavor; neohesperidoside; Flavanone derivatives; citrus; Gene; Dicotyledones; Angiospermae; Flavone derivatives; Spermatophyta; Glycoside; Transgenic plant; rhamnosyltransferase
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2004.02193.x

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.