A genomic approach to study anthocyanin synthesis and flower pigmentation in passionflowers

Abstract

Most of the plant pigments ranging from red to purple colors belong to the anthocyanin group of flavonoids. The flowers of plants belonging to the genus Passiflora (passionflowers) show a wide range of floral adaptations to diverse pollinating agents, including variation in the pigmentation of floral parts ranging from white to red and purple colors. Exploring a database of expressed sequence tags obtained from flower buds of two divergent Passiflora species, we obtained assembled sequences potentially corresponding to 15 different genes of the anthocyanin biosynthesis pathway in these species. The obtained sequences code for putative enzymes are involved in the production of flavonoid precursors, as well as those involved in the formation of particular ("decorated") anthocyanin molecules. We also obtained sequences encoding regulatory factors that control the expression of structural genes and regulate the spatial and temporal accumulation of pigments. The identification of some of the putative Passiflora anthocyanin biosynthesis pathway genes provides novel resources for research on secondary metabolism in passionflowers, especially on the elucidation of the processes involved in floral pigmentation, which will allow future studies on the role of pigmentation in pollinator preferences in a molecular level.

Figure 1

Schematic representation of the anthocyanin biosynthetic pathway (adapted from [16]). Enzymes are indicated in red, and classes of compounds are in green. Anthocyanidin is further modified with glycosyl, acyl, or methyl groups, resulting in the “decorated” anthocyanin. In this case, UF3GT is responsible for the glycosylation of anthocyanidins. The proposed anthocyanin biosynthetic pathway for Passiflora edulis is highlighted by the colored background. CHS: chalcone sintase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3′H: flavanone 3′-hydroxylase; F3′5′H: flavanone 3′5′-hydroxylase; DFR: dihydroflavonol 4-reductase; LDOX/ANS: leucoanthocyanidin dioxygenase/anthocyanidin synthase; GT: glucosyltransferase; GST: glutathione S-transferase.

Figure 2

A Neighbor-joining phylogenetic tree of chalcone synthase (CHS) amino acids sequences. The cluster containing all anther-specific CHS-like enzymes is highlighted. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Only bootstrap values above 75% are indicated at the nodes. Accession numbers for genes from other species are given in Supplementary data.

Figure 3

Multiple sequence alignment of the Passiflora sequence with some plant DFR sequences. The identical and similar residues are highlighted on a black and gray background, respectively. NADP-binding domain is underlined. Boxed amino acids have been considered to control the substrate specificity of DFR enzyme [40], and the amino acid residue (indicated by an arrowhead) is especially important for this specificity [41]. The alignment was performed using CLUSTALX and BOXSHADE program.

Figure 4

A Neighbor-joining phylogenetic tree of dihydroflavonol 4-reductase (DFR) amino acids sequences. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Only bootstrap values above 75% are indicated at the nodes. Asn-type DFRs, Asp-type DFRs, and DFRs of neither Asn nor Asp-type are indicates in blue, red, and black, respectively [42]. Accession numbers for genes from other species are given in Supplementary data which are available online at doi:10.4061/2011/37157.

Figure 5

A Neighbor-joining phylogenetic tree of glucosyltransferase (GT) amino acids sequences. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Only bootstrap values above 75% are indicated at the nodes. Accession numbers for genes from other species are given in Supplementary data.

Figure 6

A Neighbor-Joining phylogenetic tree of glutathione S-transferase (GST) amino acids sequences with three types representing phi, tau, and zeta classes. Phi and tau are plant-specific GSTs. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Only bootstrap values above 75% are indicated at the nodes. Accession numbers for genes from other species are given in Supplementary data.

Figure 7

Multiple sequence alignment of the R2R3 MYB domains involved in anthocyanin production including the deduced amino acid sequence of Passiflora suberosa. R2R3 repeats refer to two imperfect repeats of the MYB domain. The identical and similar residues are highlighted on a black and gray background, respectively. Red box shows the R/B like bHLH interacting motif in the R3 repeat [45], and arrows indicate four specific residues of maize C1 required for interaction with a bHLH cofactor R [46]. Blue box shows a conserved motif in the R2R3 repeats for eudicots MYB related to the anthocyanin pigments [47]. The alignment was performed using CLUSTALX and BOXSHADE program.

Figure 8

A Neighbor-joining phylogenetic tree of plant R2R3 MYB sequences. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Only bootstrap values above 75% are indicated at the nodes. Accession numbers for genes from other species are given in Supplementary data.

Figure 9

Multiple sequence alignment of the WD40 proteins involved in anthocyanin production, including the deduced amino acid sequence of the Passiflora edulis WD40. The identical and similar residues are highlighted on a black and gray background, respectively. Four conserved WD repeat domain are underlined in red. The alignment was performed using CLUSTALX and BOXSHADE program.

Figure 10

A Neighbor-joining phylogenetic tree of plant WD 40 proteins. Bootstrap values from 1,000 replicates were used to assess the robustness of the trees. Only bootstrap values above 75% are indicated at the nodes. Accession numbers for genes from other species are given in Supplementary data.