Origin and diversification of basic-helix-loop-helix proteins in plants

Abstract

Basic helix-loop-helix (bHLH) proteins are a class of transcription factors found throughout eukaryotic organisms. Classification of the complete sets of bHLH proteins in the sequenced genomes of Arabidopsis thaliana and Oryza sativa (rice) has defined the diversity of these proteins among flowering plants. However, the evolutionary relationships of different plant bHLH groups and the diversity of bHLH proteins in more ancestral groups of plants are currently unknown. In this study, we use whole-genome sequences from nine species of land plants and algae to define the relationships between these proteins in plants. We show that few (less than 5) bHLH proteins are encoded in the genomes of chlorophytes and red algae. In contrast, many bHLH proteins (100-170) are encoded in the genomes of land plants (embryophytes). Phylogenetic analyses suggest that plant bHLH proteins are monophyletic and constitute 26 subfamilies. Twenty of these subfamilies existed in the common ancestors of extant mosses and vascular plants, whereas six further subfamilies evolved among the vascular plants. In addition to the conserved bHLH domains, most subfamilies are characterized by the presence of highly conserved short amino acid motifs. We conclude that much of the diversity of plant bHLH proteins was established in early land plants, over 440 million years ago.

FIG. 1.

Phylogenetic relationships of the species used in this study. The total number of bHLH proteins found in the genome of each species is indicated. The cladogram is based on the current view of plant and eukaryotic phylogeny (Baldauf 2003; Lewis and McCourt 2004; Rodríguez-Ezpeleta et al. 2005); ^aRobinson and Lopes (2000); ^bSimionato et al. (2007).

FIG. 2.

Alignment of the bHLH domain of representative plant proteins. A representative of each of the 26 subfamilies of plant bHLH is shown, together with the human protein Max, a well-characterized bHLH protein. The shaded boxes indicate the position of the DNA-binding basic region, the two α-helixes, and the variable loop region (Ferré-D'Amaré et al. 1993). The numbering of the amino acids follows (Atchley and Fitch 1997). This is a subset of the full alignment with all the proteins used in this study (supplementary fig. 1, Supplementary Material online).

FIG. 3.

Twenty subfamilies of bHLH were already established in the common ancestral of vascular plants and mosses. Maximum likelihood analysis of 544 plant bHLH, shown as an unrooted cladogram. The blue balloons delineate the 26 subfamilies of plant bHLH proteins. Colored dots symbolize the species to which the proteins in each group belong (yellow: Oryza sativa [monocot]; red: Arabidopsis thaliana [eudicot]; green: Selaginella moellendorffii [lycophyte]; blue: Physcomitrella patens [moss]; purple: Volvox carteri, Chlamydomonas reinhardtii, Chlorella vulgaris, Ostreococcus tauri, and Cyanidioschyzon merolae (chlorophytes and red algae). A full tree with protein names, proportional branch lengths, and clade support values is given in supplementary fig. S2 (Supplementary Material online).

FIG. 4.

Plant bHLH do not group with other eukaryote bHLH. A Bayesian analysis was performed on an alignment of the bHLH sequence of one representative of each of the 26 subfamilies of plant bHLH, all the chlorophyte and red algae proteins, 5 proteins found in diatom Thalassiosira pseudonana, 8 Saccharomyces cerevisiae proteins, and representatives of 5 major groups of metazoan bHLH in the sponge Amphimedon queenslandica and Homo sapiens. The tree is unrooted. The numbers in the clades are posterior probability values; clades with less than 50% support were collapsed.

FIG. 5.

Non-bHLH amino acid motifs are highly conserved in each bHLH subfamily. An idealized representation of a typical member of each bHLH subfamily is shown, with the bHLH domain and other conserved motifs drawn as shaded boxes. The diagrams are not drawn to scale. The sequences of each motif in individual proteins are given in supplementary table S2 (Supplementary Material online).