Evolution and functional diversification of R2R3-MYB transcription factors in plants

Abstract

R2R3-MYB genes (R2R3-MYBs) form one of the largest transcription factor gene families in the plant kingdom, with substantial structural and functional diversity. However, the evolutionary processes leading to this amazing functional diversity have not yet been clearly established. Recently developed genomic and classical molecular technologies have provided detailed insights into the evolutionary relationships and functions of plant R2R3-MYBs. Here, we review recent genome-level and functional analyses of plant R2R3-MYBs, with an emphasis on their evolution and functional diversification. In land plants, this gene family underwent a large expansion by whole genome duplications and small-scale duplications. Along with this population explosion, a series of functionally conserved or lineage-specific subfamilies/groups arose with roles in three major plant-specific biological processes: development and cell differentiation, specialized metabolism, and biotic and abiotic stresses. The rapid expansion and functional diversification of plant R2R3-MYBs are highly consistent with the increasing complexity of angiosperms. In particular, recently derived R2R3-MYBs with three highly homologous intron patterns (a, b, and c) are disproportionately related to specialized metabolism and have become the predominant subfamilies in land plant genomes. The evolution of plant R2R3-MYBs is an active area of research, and further studies are expected to improve our understanding of the evolution and functional diversification of this gene family.

Figure 1

Phylogenetic relationships of 75 plant species in which the R2R3-MYB gene family has been comprehensively analyzed. Phylogenetic relationships among these species were obtained from the NCBI taxonomy database (https://www.ncbi.nlm.nih.gov/taxonomy). In total, 10 270 sequences of 75 plant species from Rhodophyta to angiosperms have been reported. Numbers in round brackets indicate the number of R2R3-MYB family members in each species. Detailed information, including corresponding references for each species, is provided in Supplemental Table 1. The end date for these statistics is November 2021.

Figure 2

Domain structure and functional characterization status of R2R3-MYB transcription factors. a. The R2R3-MYB transcription factor is composed of a MYB region and a non-MYB region. The DNA-binding domain, also called the MYB region, contains conserved R2 and R3 repeats. In most activators and some repressors [43], there is a conserved bHLH-interacting motif ([D/E] Lx2[R/K]x3Lx6Lx3R) within the first two helixes of the R3 domain that enables interactions with bHLH proteins [48] to form the MYB-bHLH-WDR transcriptional complex. The protein sequences in the C-terminal region often show divergence, with one or a few typical repressor motif(s) such as the EAR motif (ERF-associated amphiphilic repression), SID motif (Sensitive to ABA and Drought 2 protein interact motif), and TLLLFR. H, Helix; T, turn; W, tryptophan; X, amino acid. ^* indicates that the motif is not included in all R2R3-MYBs, but only in some. b. Number of species for which the functions of one or more R2R3-MYB genes were identified as of 2020. In the last decade (2011–2020), there was explosive growth in the breadth of taxa for which R2R3-MYB data were available. It is noteworthy that most of the increase in species number is ascribed to the vast number of orthologous MYB genes characterized in horticultural plants that have similar functions, especially in phenylpropanoid biosynthesis. c. Number of Arabidopsis R2R3-MYBs for which biological role(s) have been identified. In Arabidopsis, all the increase is due to paralogs with new functions. When a gene had different functions published in different years, we sorted it into the year of the first publication. The dark grey bar represents the number of R2R3-MYBs with unknown (i.e. not experimentally verified) functions.

Figure 3

Major metabolite biosynthetic pathways regulated by R2R3-MYBs and simplified schemes are shown. a. The most intensively investigated regulatory metabolites are phenylpropanoid biosynthesis-derived compounds, including flavonoids and lignins. Typical groups of flavonoids are shown in the yellow box, several representative groups for specific branches of flavonoid biosynthesis are shown in the deep yellow box, and typical groups for lignins are shown in the light blue box. More information regarding the phenylpropanoid pathway has been reviewed recently [190]. b. R2R3-MYB regulation of glucosinolate biosynthesis in S12 has primarily been studied in Brassicaceae. The synthesis pathway involves three phases, with two types of starting amino acids, that are regulated by different R2R3-MYBs. A fine review with more details has recently been published by Mitreiter and Gigolashvili [191]. c. The best-studied terpenoids regulated by R2R3-MYBs are floral volatiles.

Figure 4

Phylogenetic relationships among functionally characterized plant R2R3-MYBs. a. The number and percentage of R2R3-MYBs displaying each intron pattern as shown in Supplemental Fig. 1 in Physcomitrella patens (Pp), Selaginella moellendorfii (Sm), Zea mays (Zm), Oryza sativa (Os), Aquilegia coerulea (Ac), Solanum lycopersicum (Sl), Solanum tuberosum (St), Vitis vinifera (Vv), Arabidopsis thaliana (At), Citrus sinensis (Csi), Medicago truncatula (Mt), Populus trichocarpa (Pt), and Brassica napus (Bna). b. The maximum likelihood (ML) tree was constructed with 100 replications using the JJT + G model. It contains 598 nonredundant R2R3-MYBs, including 126 A. thaliana R2R3-MYBs [9] and 435 functionally characterized R2R3-MYBs from other plant taxa (Supplemental Table 2), as well as representatives of 73 subfamilies from our previous results [10]. The tree was rooted using S29 (the CDC5-like protein) as the outgroup. The scale bar represents 2 substitutions per site. Detailed information on the ML tree is provided in Supplemental Fig. 2. Detailed information on the sequences used for phylogenetic analyses is provided in Supplemental Table 3. The subfamily classification and nomenclature of the 598 proteins were based on those of Arabidopsis [9] and our previous study [10]. The new subfamilies, with the exception of the 25 subfamilies from Arabidopsis [9], are underlined, and their detailed functions and classification information are provided in Supplemental Table 2 and Fig. 2. The colored squares indicate the intron pattern to which each subfamily member belongs (Supplemental Fig. 1). Nodes with bootstrap values ≥70% and ≥50% are shown as black and gray dots, respectively, in the phylogenetic tree.