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.

Single-Molecule Real-Time and Illumina Sequencing to Analyze Transcriptional Regulation of Flavonoid Synthesis in Blueberry

“…It was found that only VcMYB1 contained the sg6-motif among the genes up-regulated in blueberries ( Figure 3B ). Studies have shown that the sg6 motif can promote the synthesis of plant anthocyanins ( Stracke et al, 2001 ; Muñoz et al, 2021 ). For further verification, Phylogenetic analysis of all the excavated VcMYB protein sequences and homologous sequences of other 138 species of plants showed that only the VcMYB1 gene has extremely high homology with the Arabidopsis SG-6 R2R3 MYB protein ( Figure 3C ).…”

Genome‐wide analysis of MYB family genes in Tripterygium wilfordii and their potential roles in terpenoid biosynthesis

“…Two species‐specific subgroups (S6 and S24) only contain AtMYBs. Subgroup 6 (AtMYB75, AtMYB90, AtMYB113, and AtMYB114) and Subgroup 24 (AtMYB53, AtMYB92, and AtMYB93) have been shown to be involved in the regulation of anthocyanin and suberin biosynthesis, respectively (Legay et al, 2015 ; Munoz‐Gomez et al, 2021 ; To et al, 2020 ). These species‐specific genes suggest that these proteins might have specialized roles that have been either lost or gained after divergence from their last common ancestor.…”

“…For instance, subgroup 5 is wellknown to play crucial roles in the positive regulation of proanthocyanidin biosynthesis so that the TwMYB in this group might also play similar roles(Wang et al, 2019). There were three species-specific R2R3-MYB subgroups (S27, S28, and S29) in Tripterygium wilfordii.Two species-specific subgroups (S6 and S24) only contain AtMYBs.Subgroup 6 (AtMYB75, AtMYB90, AtMYB113, and AtMYB114) and Subgroup 24 (AtMYB53, AtMYB92, and AtMYB93) have been shown to be involved in the regulation of anthocyanin and suberin biosynthesis, respectively(Legay et al, 2015;Munoz-Gomez et al, 2021;To et al, 2020). These species-specific genes suggest that these proteins might have specialized roles that have been either lost or gained after divergence from their last common ancestor.…”

Genome-wide Analysis of R2R3-MYB Transcription Factors in Boehmeria nivea (L.) Gaudich Revealed Potential Cadmium Tolerance and Anthocyanin Biosynthesis Genes

“…While CaMYB31 is reported to involved in regulation of biosynthesis of capsaicinoids in Capsicum annum [12], DkMyb4 regulated proanthocyanidin biosynthesis in persimmon [13]. A number of MYBs linked to anthocyanin biosynthesis were identi ed in many species, such Arabidopsis (AtMYB75, AtMYB90, AtMYB113 and AtMYB114) [14][15][16], citrus (CsRuby1 and CsMYB3) [17] and tomato (SlMYB12) [18] whereas others involved in cellular morphogenesis, secondary cell wall biosynthesis, meristem formation and cell cycle regulation [19][20][21][22]. A rice R2R3-MYB gene (OsCSA) directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development [23].…”