Fig 1.
The gentian phenylpropanoid pathway.
Anthocyanidins (pelargonidin, cyanidin, and delphinidin) are modified by the addition of sugars and other moieties to form anthocyanins in a species-specific manner. Pink-flowered gentians contain gentiocyanin in their petals, and blue-flowered gentians mainly gentiodelphin, whereas orange-flowered gentian accumulates exclusively pelargonidin glycosides. Abbreviations: ANS, anthocyanidin synthase; 5AT, anthocyanin 5-aromatic acyltransferase; CHI, chalcone isomerase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; DHK, Dihydrokaempferol; DHM, Dihydromyricetin; DHQ, Dihydroquercetin; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxlase; F3'5'H, flavonoid 3',5'-hydroxlase; 3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase; 5GT, UDP-glucose:flavonoid 5-O-glucosyltransferase; PAL, phenylalanine ammonia-lyase. Underlined names indicate enzymes/metabolites whose encoding gene expression/accumulation has been investigated in the present study.
Fig 2.
ESI-MS/MS spectra of anthocyanins isolated from petals of Gentiana lutea L. var. aurantiaca.
The number from 1 to 11 corresponded with the anthocyanins characterized and listed in Table 1. The image in L corresponded to the basic structure of the anthocyanin molecule.
Table 1.
Identification of anthocyanins from Gentiana lutea L. var. aurantiaca petals by HPLC-ESI-MS/MS.
Fig 3.
Levels of pelargonidin derivatives identified in leaf and petals of Gentiana lutea L. var. aurantiaca in three different developmental stages by HPLC-ESI-MS/MS analyses.
(A) Data are expressed as average and standard deviation which represent, for each anthocyanin metabolite, the fold over the internal standard (IS), have been obtained by using, at least, 3 independent biological replicates. For more details, see Materials and Methods. (B) Percent relative abundance of each anthocyanin among the three developmental stages (S1, S3, S5) under study. Abbreviations: Pel coum-gluc, Pelargonidin 3-O-(6-p-coumaroyl)glucoside; Pel digluc, Pelargonidin 3,5-O-diglucoside; Pel caff-gluc-mal-gluc, Pelargonidin 3-O-(6-O-caffeoyl-D-glucoside)-5-O-(6-O-malonyl-β-D-glucoside); Pel fer-glucopyr-caff-glucopyr-glucopyr, Pelargonidin 3-O-[2-O-(6-(E)-feruloyl-β-D-glucopyranosyl)-6-O-(E)-caffeoyl-β-D-glucopyranoside]-5-O-(β-D-glucopyranoside); Pel fer-glucopyr-coum-glucopyr-glucopyr, Pelargonidin 3-O-[2-O-(6-(E)-feruloyl-β-D-glucopyranosyl)-6-O-(E)-p-coumaroyl-β-D-glucopyranoside]-5-O-(β-D-glucopyranoside); Pel gluc, Pelargonidin 3-O-glucoside; Pel mal-gluc, Pelargonidin 3-O-(6-O-malonyl-β-D-glucoside); Pel rut, Pelargonidin 3-O-rutinoside; Pel rut-gluc, Pelargonidin 3-O-rutinoside-5-O-β-D-glucoside; Pel mal-gluc-gluc, Pelargonidin 3-O-(6-O-malonyl-β-D-glucoside)-5-β-D-glucoside; Pel coum-gluc-mal-gluc, Pelargonidin 3-O-(6-p-coumaroyl-D-glucoside)-5-(4-O-malonyl-β-D-glucoside).
Fig 4.
Levels of anthocyanin precursors identified in petals of Gentiana lutea L. var. aurantiaca in three different developmental stages by HPLC-ESI-MS/MS analyses.
Data are expressed as average and standard deviation which represent, for each metabolite, the fold over the internal standard (IS), have been obtained by using, at least, three independent biological replicates. For more details, see Materials and Methods.
Fig 5.
Quantitative expression of anthocyanin genes, normalized on the ubiquitin housekeeping gene in leaf and petals of Gentiana lutea L. var. aurantiaca.
qRT-PCT data were calculated from three biological replicates with at least three technical replicates for each biological replicate and with error bars representing the standard deviation. Abbreviations: 5AT, anthocyanin 5-aromatic acyltransferase gene; ANS, anthocyanidin synthase gene; DFR, dihydroflavonol 4-reductase gene; 3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase gene; 5GT, UDP-glucose:flavonoid 5-O-glucosyltransferase gene.
Fig 6.
Integration of transcript-metabolite data involved in G. lutea L. var. aurantiaca anthocyanin metabolism.
(A) Pearson coefficient-based correlation matrix. Legend on the right corresponds to the names of the anthocyanin transcripts and metabolites. Red and blue shaded boxes represent different levels of positive and negative correlations, respectively; white boxes represent no correlation. (B) Anthocyanin transcript/metabolite correlation network using a prefuse force-directed layout (only ρ > 0.65 are shown). Transcripts, anthocyanins and anthocyanin precursors are represented, respectively, as pink rounds, and orange and violet triangles. Blue and red edges refer to negative and positive correlations, respectively. Node size is according to the node strength (ns, representing the average of all the ρs of each node). Lines joining the nodes indicate positive (red) and negative (blue) correlations, of width proportional to each corresponding |ρ|. Abbreviations: ANS, anthocyanidin synthase; 5AT, anthocyanin 5-aromatic acyltransferase; DFR, dihydroflavonol 4-reductase; 3GT, UDP-glucose:flavonoid 3-O-glucosyltransferase gene; 5GT, UDP-glucose:flavonoid 5-O-glucosyltransferase; Pel coum-gluc, Pelargonidin 3-O-(6-p-coumaroyl)glucoside; Pel digluc, Pelargonidin 3,5-O-diglucoside; Pel caff-gluc-mal-gluc, Pelargonidin 3-O-(6-O-caffeoyl-D-glucoside)-5-O-(6-O-malonyl-β-D-glucoside); Pel fer-glucopyr-caff-glucopyr-glucopyr, Pelargonidin 3-O-[2-O-(6-(E)-feruloyl-β-D-glucopyranosyl)-6-O-(E)-caffeoyl-β-D-glucopyranoside]-5-O-(β-D-glucopyranoside); Pel fer-glucopyr-coum-glucopyr-glucopyr, Pelargonidin 3-O-[2-O-(6-(E)-feruloyl-β-D-glucopyranosyl)-6-O-(E)-p-coumaroyl-β-D-glucopyranoside]-5-O-(β-D-glucopyranoside); Pel gluc, Pelargonidin 3-O-glucoside; Pel mal-gluc, Pelargonidin 3-O-(6-O-malonyl-β-D-glucoside); Pel rut, Pelargonidin 3-O-rutinoside; Pel rut-gluc, Pelargonidin 3-O-rutinoside-5-O-β-D-glucoside; Pel mal-gluc-gluc, Pelargonidin 3-O-(6-O-malonyl-β-D-glucoside)-5-β-D-glucoside; Pel coum-gluc-mal-gluc, Pelargonidin 3-O-(6-p-coumaroyl-D-glucoside)-5-(4-O-malonyl-β-D-glucoside).