Expansion and diversification of the Populus R2R3-MYB family of transcription factors

Abstract

The R2R3-MYB proteins comprise one of the largest families of transcription factors in plants. R2R3-MYB family members regulate plant-specific processes, such as the elaboration of specialized cell types, including xylem, guard cells, trichomes, and root hairs, and the biosynthesis of specialized branches of metabolism, including phenylpropanoid biosynthesis. As such, R2R3-MYB family members are hypothesized to contribute to the emergence of evolutionary innovations that have arisen in specific plant lineages. As a first step in determining the role played by R2R3-MYB family members in the emergence of lineage-specific innovations in the genus Populus, the entire Populus trichocarpa R2R3-MYB family was characterized. The Populus R2R3-MYB complement is much larger than that found in other angiosperms with fully sequenced genomes. Phylogenetic analyses, together with chromosome placement, showed that the expansion of the Populus R2R3-MYB family was not only attributable to whole genome duplication but also involved selective expansion of specific R2R3-MYB clades. Expansion of the Populus R2R3-MYB family prominently involved members with expression patterns that suggested a role in specific components of Populus life history, including wood formation and reproductive development. An expandable compendium of microarray-based expression data (PopGenExpress) and associated Web-based tools were developed to better enable within- and between-species comparisons of Populus R2R3-MYB gene expression. This resource, which includes intuitive graphic visualization of gene expression data across multiple tissues, organs, and treatments, is freely available to, and expandable by, scientists wishing to better understand the genome biology of Populus, an ecologically dominant and economically important forest tree genus.

Figure 1.

The R2 and R3 MYB repeats are highly conserved across all R2R3-MYB proteins in the P. trichocarpa genome. The sequence logos of the R2 (A) and R3 (B) MYB repeats are based on full-length alignments of all PtrR2R3-MYB proteins. The bit score indicates the information content for each position in the sequence. Highly conserved Trp residues required for DNA binding are highlighted in red. PtrMYB proteins in clade 27 have an additional four amino acids directly before the first conserved Trp in R3 (Supplemental Fig. S2A).

Figure 2.

Phylogenetic relationships and subgroup designations in MYB proteins from P. trichocarpa (Ptr), V. vinifera (Vv), and Arabidopsis (At). The neighbor-joining tree includes 192 R2R3-MYB proteins from Populus, 118 from Vitis, 126 from Arabidopsis, and a further 54 from other plant species, in addition to five 3R-MYB proteins from each of Populus, Vitis, and Arabidopsis. The proteins are clustered into 49 subgroups (triangles), designated with a subgroup number (e.g. C1). Eleven proteins did not fit well into clusters (lines). Some landmark R2R3-MYB proteins are listed to the right of the clades for reference. The membership of each subgroup is described in the table at right. Several clades are highlighted that exemplify lineage-specific R2R3-MYB gene expansion. C3 and C25 (green) show dramatic expansion in the P. trichocarpa lineage. C13 and C37 (yellow) do not include any PtrMYB proteins. C14 and C22 (blue) show limited expansion in the P. trichocarpa lineage. Many clades do not include any Arabidopsis R2R3-MYB proteins (orange). The uncompressed tree with full taxa names is available as Supplemental Figure S1.

Figure 3.

R2R3-MYB and 3R-MYB proteins are present on all LG in the P. trichocarpa genome. One hundred seventy-two of the Populus R2R3-MYB genes are mapped to LG according to Joint Genome Institute Poplar Genome version 1.1. Each vertical bar represents one LG drawn to scale. The identities of the MYB genes are indicated to the right of the LG on which they are located, with horizontal lines intersecting with the LG indicating their positions. In cases in which multiple MYB genes are located in close proximity on a LG, lines connecting individual gene names and the LG have been omitted. In these cases, angled lines have been used to bracket the relevant gene names and to display their genomic locations. The colored boxes indicate groups of MYB proteins with paralogous and syntenic genes on two LG. The groups indicated are not exhaustive but serve to indicate the widespread evidence of the salicoid-specific whole genome duplication event in the P. trichocarpa genome.

Figure 4.

Populus R2R3-MYB genes exhibit differential expression across a range of tissues, organs, and treatments. The patterns of relative transcript accumulation of each of 180 R2R3-MYB genes and five 3R-MYB genes as determined by microarray analysis are presented as a heat map, with red indicating higher levels and green indicating lower levels of transcript accumulation. Each column represents a discreet biological sample, and all treatments are presented as biological triplicates. CL, Seedlings grown in continuous light; DL, seedlings grown in continuous darkness and then transferred to light for 3 h; CD, seedlings grown in continuous darkness; YL, young leaf; ML, mature leaf; R, root; DX, differentiating xylem; FC, female catkins; MC, male catkins. Data are normalized within each row.

Figure 5.

PtrR2R3-MYB genes show conserved patterns of transcript accumulation across tissues. A, Clade 10 includes R2R3-MYB genes isolated from the wood-forming tissues of a number of species, including P. glauca (Pg), P. taeda (Pt), E. gunnii (Eg), V. vinifera (Vv), and Arabidopsis (At) as well as four previously uncharacterized R2R3-MYB genes from P. trichocarpa (Ptr). B, The relative transcript accumulation of each of the PtrR2R3-MYB genes in clade 10 as determined by microarray analysis. C, Clade 27 includes several well-known R3R3-MYB genes with demonstrated roles in anthocyanin biosynthesis in a variety of organisms, including AtPAP1/MYB75, AtPAP2/MYB90, and P. hybrida ANTHOCYANIN2 (PhAN2) as well as six previously uncharacterized PtrR2R3-MYB genes. D, The relative transcript accumulation of each of the PtrR2R3-MYB genes in clade 27 as determined by microarray analysis. The scale bars under the trees represent 0.2 substitutions. The heat maps are clustered by Pearson correlation coefficient. Red indicates higher levels and green represents lower levels of transcript accumulation. CL, Seedlings grown in continuous light; DL, seedlings grown in continuous darkness and then transferred to light for 3 h; CD, seedlings grown in continuous darkness; YL, young leaf; ML, mature leaf; R, root; DX, differentiating xylem; FC, female catkins; MC, male catkins.

Figure 6.

eFP display of transcript accumulation patterns across a variety of Populus organs and treatments. Poplar eFP browser presents the transcript accumulation pattern of PtrMYB025 in a variety of tissues and organs. In all cases, red indicates higher levels of transcript accumulation and yellow indicates a lower level of transcript accumulation.