Purification, cloning, and heterologous expression of a catalytically efficient flavonol 3-O-galactosyltransferase expressed in the male gametophyte of Petunia hybrida.
Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.
Miller
K D
KD
Program in Plant Physiology, Washington State University, Pullman, Washington 99164-6340, USA.
Guyon
V
V
Evans
J N
JN
Shuttleworth
W A
WA
Taylor
L P
LP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D063245:Plant Development; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D011058:Pollen / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C117137:UDP-galactose flavonoid 3-O-galactosyltransferase; D005700:Galactosyltransferases