Regulation of enzymes involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with ultraviolet light and fungal elicitors.

The accumulation of anthocyanins in cell cultures of Daucus carota L. and the enzymes involved in their biosynthesis were investigated under growth in the dark, continuous irradiation with UV light, incubation with elicitors from Pythium aphanidermatum, and elicitor treatment of UV-irradiated cells. Upon UV irradiation, anthocyanin accumulation was strongly enhanced, and the enzymes of the phenylpropanoid and flavonoid pathways, including the "late" enzymes cyanidin galactosyltransferase, cyanidin galactoside xylosyltransferase, cyanidin triglycoside sinapoyltransferase and sinapic acid glucosyltransferase, all showed transient increases in their activities. The time courses of the enzyme activities exhibited successive maxima with an ordered sequence corresponding to their position in the biosynthetic pathway, suggesting a coordinated induction of the entire set of enzymes. The key enzymes phenylalanine ammonia-lyase and chalcone synthase are regulated on a transcriptional level. Incubation of dark-grown carrot cells with fungal elicitors led to a rapid and transient induction of phenylalanine ammonia-lyase corresponding to the formation of 4-hydroxybenzoic acid, but the amount of anthocyanin did not increase and there was no enhancement of any of the enzyme activities which are part of the anthocyanin pathway, including the enzymes catalyzing glycosylation and acylation reactions. Treatment with UV light and elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanin biosynthesis was suppressed. These results indicate a coordinated regulation of the enzymes involved in anthocyanin biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the dominating role of the elicitor-signal over the UV stimulus.

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

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.

Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds.

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside ("peonanin"). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.

Anthocyanins from cell suspension cultures of Daucus carota.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2''-O-beta-D-xylopyranosyl-6''-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.