Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Abstract

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O-β-D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D<-galactopyranosylcyanidin (1), 3-O-[β-D- xylopyranosyl-(1→2)-β-D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)-β-D-glucopyranosyl-(1→6)-[β-D- xylopyranosyl-(1→2)-]β-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)-β-D-glucopyranosyl-(1→6)-[β- D-xylopyranosyl-(1→2)-]β-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)-β-D-glucopyranosyl-(1→6)- [β-D-xylopyranosyl-(1→2)-]β-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-β- D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]-β-D-glucopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)-]β-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)-β-D-glucopyranosyl-(1→6)-β-D-galactopyranosyl]cyanidin (8), and 3-O-(β-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.