Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures ofDaucus carota L.

Abstract

The major anthocyanins accumulated by an Afghan cultivar of Daucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.

Biosynthesis of UDP-xylose: characterization of membrane-bound AtUxs2

“…UDP-xylose is also required for the synthesis of glycoproteins (Zeng et al 1997;Strasser et al 2000) and minor plant metabolites (Rose et al 1996;Martin et al 1997). Immunocytochemical and biochemical studies have provided evidence that the synthesis of glycoprotein (Pagny et al 2003) and polysaccharides (Moore et al 1991;Baydoun and Brett 1997;Bolwell 2000) occurs in the lumen of the Golgi apparatus and is catalyzed by different xylosyltransferases that transfer xylose from UDP-Xyl onto acceptor molecules.…”

Biosynthesis of UDP-Xylose. Cloning and Characterization of a Novel Arabidopsis Gene Family, UXS, Encoding Soluble and Putative Membrane-Bound UDP-Glucuronic Acid Decarboxylase Isoforms

“…UDP-Xyl is a nucleotide sugar required for the synthesis of diverse plant cell wall polysaccharides including xyloglucan (Ray, 1980;Hayashi and Matsuda, 1981;White et al, 1993;Baydoun and Brett, 1997), xylan (Rodgers and Bolwell, 1992), and minor plant metabolites (Rose et al, 1996;Martin et al, 1997). UDP-Xyl is also required for the synthesis of glycoproteins (Zeng et al, 1997;Strasser et al, 2000), animal proteoglycans (Gotting et al, 2000;Kuhn et al, 2001), and fungal polysaccharides (Ankel et al, 1967).…”

Identification and characterization of DcUCGalT1, a galactosyltransferase responsible for anthocyanin galactosylation in purple carrot (Daucus carota L.) taproots

“…The molecular mass of glycosyltransferases generally ranges from 45 to 60 kDa23. That of DcUCGalT1 was calculated to be 49.37 kDa, a little lower than a native UCGalT (52 kDa) from D. carota which was determined using a Sephadex G-75 column20.…”

“…)2223 and kiwifruit ( Actinidia chinensis )24 have been confirmed to be UDP-galactose:cyanidin galactosyltransferase (UCGalT). The UCGalT purified from carrot showed activity for cyanidin, but the gene which encodes this protein has not yet been confirmed20.…”

“…Glycosyltransferases have selective substrate specificities towards aglycones and sugar donors. Although many glycosyltransferases from various species have shown glycosylation activity for anthocyanins in previous investigations, only those from carrots20, peach ( Prunus persica )21, udo ( Aralia cordata Thunb. )2223 and kiwifruit ( Actinidia chinensis )24 have been confirmed to be UDP-galactose:cyanidin galactosyltransferase (UCGalT).…”