Differential accumulation of pelargonidin glycosides in petals at three different developmental stages of the orange-flowered gentian (Gentiana lutea L. var. aurantiaca)

Abstract

Corolla color in Gentiana lutea L. exhibits a yellow/orange variation. We previously demonstrated that the orange petal color of G. lutea L. var. aurantiaca is predominantly caused by newly synthesized pelargonidin glycosides that confer a reddish hue to the yellow background color, derived from the carotenoids. However, the anthocyanin molecules of these pelargonidin glycosides are not yet fully identified and characterized. Here, we investigated the regulation, content and type of anthocyanins determining the petal coloration of the orange-flowered G. lutea L. var. aurantiaca. Anthocyanins from the petals of G. lutea L. var. aurantiaca were characterized and quantified by HPLC-ESI-MS/MS (High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry) coupled with a diode array detector in flowers at three different stages of development (S1, S3 and S5). Eleven pelargonidin derivatives were identified in the petals of G. lutea L. var. aurantiaca for the first time, but quantitative and qualitative differences were observed at each developmental stage. The highest levels of these pelargonidin derivatives were reached at the fully open flower stage (S5) where all anthocyanins were detected. In contrast, not all the anthocyanins were detected at the budlet stage (S1) and mature bud stage (S3) and those corresponded to more complex pelargonidin derivatives. The major pelargonidin derivatives found at all the stages were pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside and pelargonidin 3-O-rutinoside. Furthermore, the expression of DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase), 3GT (UDP-glucose:flavonoid 3-O-glucosyltransferase), 5GT (UDP-glucose:flavonoid 5-O-glucosyltransferase) and 5AT (anthocyanin 5-aromatic acyltransferase) genes was analyzed in the petals of three developmental stages, showing that the expression level of DFR, ANS and 3GT parallels the accumulation of the pelargonidin glucosides. Overall, this study enhances the knowledge of the biochemical basis of flower coloration in Gentiana species, and lays a foundation for breeding of flower color and genetic variation studies on Gentiana varieties.

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.

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).