Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth

Abstract

Grape hyacinth (Muscari) is an important ornamental bulbous plant with an extraordinary blue colour. Muscari armeniacum, whose flowers can be naturally white, provides an opportunity to unravel the complex metabolic networks underlying certain biochemical traits, especially colour. A blue flower cDNA library of M. armeniacum and a white flower library of M. armeniacum f. album were used for transcriptome sequencing. A total of 89 926 uni-transcripts were isolated, 143 of which could be identified as putative homologues of colour-related genes in other species. Based on a comprehensive analysis relating colour compounds to gene expression profiles, the mechanism of colour biosynthesis was studied in M. armeniacum. Furthermore, a new hypothesis explaining the lack of colour phenotype of the grape hyacinth flower is proposed. Alteration of the substrate competition between flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) may lead to elimination of blue pigmentation while the multishunt from the limited flux in the cyanidin (Cy) synthesis pathway seems to be the most likely reason for the colour change in the white flowers of M. armeniacum. Moreover, mass sequence data obtained by the deep sequencing of M. armeniacum and its white variant provided a platform for future function and molecular biological research on M. armeniacum.

Keywords: Colour pigmentation; Muscari armeniacum; cyanidin; delphinidin; flower development; transcriptome analysis..

Fig. 1.

A diagram of the putative anthocyanin metabolic process in blue or white M. armeniacum flowers. (A) Mature inflorescence of M. armeniacum. Arrows represent small flower buds just before bloom. (B) Flower bud of M. armeniacum just before bloom used in deep sequencing. (C) Mature inflorescence of M. armeniacum f. album. (D) Flower bud of M. armeniacum f. album just before bloom used in deep sequencing. The scale bar=2mm in A and C, and 1mm in B and D. (E) The putative anthocyanin metabolic process in blue M. armeniacum flowers. (F) The putative anthocyanin metabolic process in white M. armeniacum flowers. (G) Flavonoid composition obtained by HPLC from blue and white flowers of M. armeniacum. ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; Ca, catechin; CHI, chalcone isomerase; CHS, chalcone synthase; Cy, cyanidin; Del, delphinidin; DFR, dihydroflavonol 4-reductase; DHM, dihydromyricetin; Ep, epicatechin; Er, eriodictyol; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; Km, kaempferol; LAR, leucoanthocyanidin reductase; UFGT, anthocyanidin 3-O-glucosyltransferase. (This figure is available in colour at JXB online.)

Fig. 2.

Schematic of physiological and metabolic data related to flower colour development of M. armeniacum. (A) A detailed part of the Del and Cy metabolic subnetwork showing the subset of nodes or metabolites that constitute the process. Enzyme names and expression patterns are indicated at the side of each step. The expression pattern of each uni-transcript is shown on two grids, with the left one representing the RPKM value of blue flowers, and the right one representing the relative log2 (expression ratio) of white flowers. The grids with eight different grey scale levels show the absolute expression magnitude of blue flowers, with the RPKM values 0–10, 10–20, 20–40, 40–80, 80–160, 160–320, 320–640, and 640–1280 represented by grey scale levels 1–8, respectively. (B) Transcript accumulation measurements of colour-related genes involved in the anthocyanin metabolic process. (C) Correlation of gene expression results obtained from q-PCR analysis and RNA-Seq for colour-related genes in blue and white flowers. ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; UFGT, anthocyanidin 3-O-glucosyltransferase. (This figure is available in colour at JXB online.)

Fig. 3.

A model for the process of Del elimination in the white flowers of M. armeniacum. When DFR is suppressed, the substrates used for Del synthesis are then available for synthesis of myricetin and kaempferol. Moreover, an increase of flavonol production occurs through the up-regulation of FLS, furthering the process of blue pigmentation elimination in the white flowers of M. armeniacum. The global output from the minimal anthocyanin subnetwork in flowers of M. armeniacum was considered to be 100% and was used to define the relative level of each product. The black boxes indicate the genes or the compounds which had a higher relative abundance in white flowers of M. armeniacum than that in blue flowers. The grey boxes indicate the genes or the compounds which had a lower abundance in white flowers than that in blue flowers. CHI, chalcone isomerase; CHS, chalcone synthase; Cy, the global output from the minimal cyanidin subnetwork; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase. (This figure is available in colour at JXB online.)

Fig. 4.

A model for Cy elimination in white flowers of M. armeniacum. The fluxes through Cy metabolism were limited. The multishunt process in downstream reactions further promoted Cy turnover and degradation in white flowered grape hyacinth. The global output from the minimal anthocyanin subnetwork in flowers of M. armeniacum was considered as 100% and was used to define the relative level of each product. The black boxes indicate the genes or the compounds which had a higher relative abundance in white flowers of M. armeniacum than that in blue flowers. The grey boxes indicate the genes or the compounds which had a lower abundance in white flowers than that in blue flowers. ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; CHS, chalcone synthase; Del, the global output from the minimal delphinidin subnetwork; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; UFGT, anthocyanidin 3-O-glucosyltransferase. (This figure is available in colour at JXB online.)