Evolution of the Subgroup 6 R2R3-MYB Genes and Their Contribution to Floral Color in the Perianth-Bearing Piperales

Abstract

Flavonoids, carotenoids, betalains, and chlorophylls are the plant pigments responsible for floral color. Anthocyanins, a class of flavonoids, are largely responsible for the red, purple, pink, and blue colors. R2R3-MYB genes belonging to subgroup 6 (SG6) are the upstream regulatory factors of the anthocyanin biosynthetic pathway. The canonical members of these genes in Arabidopsis include AtMYB75, AtMYB90, AtMYB113, and AtMYB114. The Aristolochiaceae is an angiosperm lineage with diverse floral groundplans and perianth colors. Saruma henryi exhibits a biseriate perianth with green sepals and yellow petals. All other genera have sepals only, with colors ranging from green (in Lactoris) to a plethora of yellow to red and purple mixtures. Here, we isolated and reconstructed the SG6 R2R3-MYB gene lineage evolution in angiosperms with sampling emphasis in Aristolochiaceae. We found numerous species-specific duplications of this gene lineage in core eudicots and local duplications in Aristolochiaceae for Saruma and Asarum. Expression of SG6 R2R3-MYB genes examined in different developmental stages and plant organs of four Aristolochiaceae species, largely overlaps with red and purple pigments, suggesting a role in anthocyanin and flavonoid synthesis and accumulation. A directed RNA-seq analysis corroborated our RT-PCR analyses, by showing that these structural enzymes activate during perianth development in Aristolochia fimbriata and that the regulatory genes are expressed in correlation with color phenotype. Finally, the reconstruction of the flavonoid and anthocyanin metabolic pathways using predicted peptides from transcriptomic data show that all pivotal enzymes are present in the analyzed species. We conclude that the regulatory genes as well as the biosynthetic pathway are largely conserved across angiosperms. In addition, the Aristolochiaceae emerges as a remarkable group to study the genetic regulatory network for floral color, as their members exhibit an outstanding floral diversity with elaborate color patterns and the genetic complement for SG6 R2R3-MYB genes is simpler than in core eudicot model species.

Keywords: Aristolochiaceae; Asarum; Saruma; anthocyanins; flavonoids; floral color; petaloid sepals; subgroup 6 R2R3-MYB genes.

FIGURE 1

Summarized phylogeny of the Aristolochiaceae with representative photographs for the main lineages. (A) Saruma henryi. (B) Asarum canadense. (C) Thottea siliquosa. (D) Aristolochia macrophylla. (E) A. manshuriensis. (F) A. arborea. (G) A. deltantha. (H) A. praevenosa. (I) A. clematitis. (J) A. fimbriata. (K) A. lindneri. (L) A. ringens.

FIGURE 2

Maximum likelihood analysis of the SG6 R2R3-MYB genes with expanded view of the non-core eudicot gene homologs. Yellow star indicates large-scale duplication events in the Aristolochiaceae prior to the diversification of Asarum and Saruma. Red stars represent species-specific duplication events. Color clades follow the conventions in the top left.

FIGURE 3

Maximum likelihood analysis of the SG6 R2R3-MYB genes with expanded view of the core-eudicot gene homologs. Yellow stars indicate large-scale duplication events in Solanum and Brassicales. Red stars represent species-specific duplication events. Color clades follow the conventions in the top left.

FIGURE 4

MEME analysis of selected SG6 R2R3-MYB proteins from core and non-core eudicots. Motifs found are indicated by color boxes. The sequence corresponds to the consensus, that is the amino acids occurring at higher rates in the motif. The R2 domain corresponds to motifs 1 and 3; R3 domain corresponds to motif 2. For gene codes, see Figures 2, 3 and Supplementary Table 1.

FIGURE 5

Protein sequence alignment of the R2 and R3 MYB domains from Subgroup 6 R2R3MYB proteins of selected basal angiosperms, monocots, basal eudicots and core eudicots. Asterisks represent conserved tryptophan residues. Left box indicates bHLH interacting motif. Right box indicates specific anthocyanin promoting MYB motif. For gene codes, see Figures 2, 3 and Supplementary Table 1.

FIGURE 6

Expression of SG6 R2R3-MYB genes in (A) Saruma henryi. (B) Asarum canadense. (C) Aristolochia manshuriensis. (D) A. fimbriata. ACTIN was used as a positive control. l, limb; lf, leaf; p, petal; pe, perianth; s, sepal; t, tube; u, utricle; -c, amplification reaction without cDNA (negative control).

FIGURE 7

Expression analysis of genes involved in flavonoid production in Aristolochia fimbriata. The heatmap was generated based on normalized RNA-seq data. Ten genes were analyzed, six structural genes and four regulatory genes involved in the flavonoid biosynthetic pathway. Two different developmental stages (S6 and S9) and three portions of the perianth (limb, tube, and utricle) were compared. The color codes indicate upregulated (red) and downregulated (green).

FIGURE 8

KEGG analysis of selected Aristolochiaceae species using predicted peptides. Summary flavonoid/anthocyanin biosynthetic pathway based on KEGG results. Colored enzyme names (in red and purple) represent those enzymes present in the peptide data from the mixed reference transcriptome in most analyzed species. For exceptions, see results. In black are the enzymes that were not found. 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; UFGT, Anthocyanidin 3-O-glucosyltransferase; UGT79B1, Anthocyanidin 3-O-glucoside 2^″′-O-xylosyltransferase.