Evolution and diversification of the ACT-like domain associated with plant basic helix-loop-helix transcription factors

Abstract

Basic helix-loop-helix (bHLH) proteins are one of the largest families of transcription factor (TF) in eukaryotes, and ~30% of all flowering plants' bHLH TFs contain the aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain at variable distances C-terminal from the bHLH. However, the evolutionary history and functional consequences of the bHLH/ACT-like domain association remain unknown. Here, we show that this domain association is unique to the plantae kingdom with green algae (chlorophytes) harboring a small number of bHLH genes with variable frequency of ACT-like domain's presence. bHLH-associated ACT-like domains form a monophyletic group, indicating a common origin. Indeed, phylogenetic analysis results suggest that the association of ACT-like and bHLH domains occurred early in Plantae by recruitment of an ACT-like domain in a common ancestor with widely distributed ACT DOMAIN REPEAT (ACR) genes by an ancestral bHLH gene. We determined the functional significance of this association by showing that Chlamydomonas reinhardtii ACT-like domains mediate homodimer formation and negatively affect DNA binding of the associated bHLH domains. We show that, while ACT-like domains have experienced faster selection than the associated bHLH domain, their rates of evolution are strongly and positively correlated, suggesting that the evolution of the ACT-like domains was constrained by the bHLH domains. This study proposes an evolutionary trajectory for the association of ACT-like and bHLH domains with the experimental characterization of the functional consequence in the regulation of plant-specific processes, highlighting the impacts of functional domain coevolution.

Keywords: evolution; gene regulation; green algae; protein–protein interaction.

Fig. 1.

Association of ACT-like and bHLH domains. (A) Schematic representation of bHLH and ACT-like domains. α and β represent the α-helices and β-sheets, respectively. The L within the gray box denotes the loop region. (B) Presence of bHLH-associated ACT-like domains in various species. Green and yellow circles indicate bHLH proteins with or without the ACT-like domains, respectively. The eukaryote phylogenetic tree was modified from the literature (35, 36). Species information and data source for the bHLH sequences used in this study are presented in Dataset S1, and Pfam analysis result is in Dataset S2. AlphaFold search result is in SI Appendix, Fig. S1B and Dataset S2. Numbers of the ACT-like domains in each species are described in Dataset S1.

Fig. 2.

bHLH domains associated with ACT-like domains form distinct clades in their phylogeny. ML phylogenetic tree was reconstructed with bHLH domains of 797 bHLH proteins retrieved from 37 chlorophytes, five streptophyte algae, and seven embryophytes, with an outgroup of three bHLH proteins from haptophytes. Colored rectangles on outer rings denote the species from which the bHLH proteins derived. Arcs of a circle in black indicate the bHLH proteins containing ACT-like domain. Shades of gray in the tree indicate clades corresponding to the described 30 bHLH subfamilies. Thick lines indicate branches with SH-aLRT support >80. Branches in yellow color highlight the chlorophyte bHLH^ACT+. Branches in red and blue colors indicate two respective chlorophyte bHLH^ACT− clades. Asterisks indicates the streptophyte algae bHLH^ACT+ proteins belonging to subfamilies Vb and XIII. The phylogeny was calculated using RAxML-NG, 1,000 iterations, and Jones–Taylor–Thornton model with a gamma distribution (JTT+G). Full details of the phylogenetic tree are provided in Dataset S5.

Fig. 3.

bHLH-associated ACT-like domains form an independent clade distinct from ACRs and GlnDs. ML phylogeny was inferred from ACT-like domains of 279 bHLH proteins in 48 chlorophytes, two streptophyte algae, and 229 embryophytes; from 18 ACRs in chlorophytes, and 67 GlnDs in bacteria, using a JTT model implemented in RAxML-NG with 2,000 bootstraps. Shades in brown, gray, and green indicate monophyletic groups that consist of ACT-like domains of ACRs (Group III) and GlnDs, ACRs (Group I and II), chlorophytes and subfamily Vb, and 12 subfamilies (Ia, Ib1, Ib2, II, IIIa+c, IIIb, IIId+e, IIIf, IVa, IVd(1), XVI, and XVII). Blackish lines indicate branches with bootstrap support >70%. A detailed phylogenetic tree is presented in Dataset S8.

Fig. 4.

Coevolution of ACT-like and bHLH domains. (A) Proportion of amino acid sequence similarity of ACT-like and bHLH domains in chlorophytes and streptophytes. The pairwise comparison analysis was performed with ACT-like and bHLH domains of 54 chlorophytes and 231 streptophytes. The proportion of the number of sequence pairs within the same range of similarity (number of pairs within same ranges of similarity/total number of pairs of bHLH domains and ACT-like domains * 100) is displayed. (B) Nonsynonymous (dN)-to-synonymous substitutions (dS) rates (dN/dS; ω) in bHLH and ACT-like domains. The box plot shows the range of the global ω values of the bHLH and ACT-like domains in bHLH orthologs in Brassicaceae. The ω values of individual subfamilies are indicated as dots, and the data are provided in SI Appendix, Table S2. The horizontal lines in the boxes represent median values, box heights interquartile range, and the vertical lines data point within 1.5× interquartile range. Asterisks denote statistically significant difference (P < 0.001) in Wilcoxon signed-rank test. The species and genes used in this analysis are provided in SI Appendix, Table S1 and Dataset S10. (C) Correlated evolutionary rates between ACT-like domain and bHLH domains. Global ω rates were estimated from nucleotide sequences encoding the ACT-like and bHLH domains of bHLH orthologs in Brassicaceae. The line represents the linear regression, and the coefficient correlation value (R²) is indicated in the graph. The ω values, species, and genes used in this analysis are provided in SI Appendix, Tables S1 and S2 and Dataset S10. (D) Tanglegram of bHLH and ACT-like domains in 231 streptophytes. ML phylogenetic trees of bHLH domains and ACT-like domains in streptophytes were individually reconstructed by RAxML-NG (43), with 1,000 bootstraps, based on “JTT+G” model, and plotted face to face with links between same proteins in the two trees. Line and tip colors indicate the various bHLH subfamilies. The x-axis represents branch lengths. Detailed phylogenetic trees with tip labels and bootstrap values are provided in Dataset S11.

Fig. 5.

Dimerization and influence of C. reinhardtii ACT-like domains on the DNA-binding activity of bHLH domains. (A) Schematic representation of the domain architecture of seven C. reinhardtii bHLH^ACT+ and one bHLH^ACT− proteins showing the fragments expressed in E. coli as N-terminal fusions to N₆His-SUMO or N₆His and GST and affinity-purified. Yellow rectangles depict the bHLH domain, with gray indicating the loop regions, green the β-sheets, and yellowish green the α-helices on the ACT-like domain. (B) Phylogenetic relationships of the bHLH domains of the nine C. reinhardtii bHLH factors biochemically analyzed. The ML phylogenetic tree was constructed with the bHLH domains from the nine C. reinhardtii species using Le and Gascuel model with gamma distribution (LG+G) and 1,000 Felsenstein bootstraps in MEGA. Bootstrap values >50 are shown as percentages at the respective branch points. The dark green shades highlight proteins harboring ACT-like domains, and light green shades indicate those with ACT-like domain lacking the β₁ sheet. Gray shades indicate proteins without ACT-like domains. (C) Dissociation constants (K_d) for dimerization of ACT-like domains and G-box binding of bHLH domains and C-terminal regions (both bHLH and ACT-like domains). K_d values were determined by saturation binding assays using ALPHA. The ACT-like domains lacking the first β-sheet formed inclusion bodies when expressed in E. coli (Cre01.g011150, Cre07.g353555, Cre07.g332250, and Cre04.g224600). For the DNA-binding assays, various concentrations (0 to 4 µM) of the GST-tagged proteins (Cre07.g349152, Cre02.g109683, and Cre04.g216200) were incubated with 500 nM of the same protein fused to N₆His-SUMO and subjected to fluorescence signal measurement. The G-box-binding strengths were determined by competition binding assays with N₆His-SUMO-tagged bHLH domains or His₆-SUMO-tagged C-terminal regions (SI Appendix, Materials and Methods). K_d values were calculated by one-site fit model in GraphPad Prism v6.0. For each interaction, K_d values (mean ± SD) of two biological repeats are shown, and the SD reflects the variation between three technical replicates.

Fig. 6.

Proposed evolutionary model of ACT-like domain in plant bHLH TFs. Possible parsimonious evolutionary model of ACT-like domain in bHLH TFs is proposed based on the relationships inferred from the phylogenies presented in this study. Ancestor bHLH protein acquired the ACT-like domain from type I or II ACRs. Then, subfamilies XIII and Vb diverged early before the division of streptophyte algae and embryophytes, while other bHLH^ACT+ subfamilies radiated in the evolution of embryophytes. Subfamilies IVb and IVc might descend from a common ancestor with most bHLH^ACT+ subfamilies but might have lost the ACT-like domain later. Most bHLH^ACT− subfamilies, except for IVb and IVc, evolved along different lineages. The arrow in gray represents the association event between the ACT-like and bHLH domains. The blue line indicates the lineages of the bHLH^ACT+. Dashed lines indicate branches whose positions are not clearly resolved, based on the available data.