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 germina tion in maize and petunia. They exist in planta as both the aglycone and gl ycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3Ga lTase) that is expressed exclusively in the male gametophyte and controls t he formation of a pollen-specific class of glycosylated flavonols, Thus an essential step to understanding flavonol-induced germination is the charact erization of F3GalTase, Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length c DNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTa se cDNA in Escherichia coil yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferase s from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis s howed that the k(cat)/K-m values of rF3GalTase, using kaempferol and querce tin as substrates, approaches that of a catalytically perfect enzyme. rF3Ga lTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. T he biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.