Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon

Abstract

Background: As a superfamily of transcription factors (TFs), the basic helix-loop-helix (bHLH) proteins have been characterized functionally in many plants with a vital role in the regulation of diverse biological processes including growth, development, response to various stresses, and so on. However, no systemic analysis of the bHLH TFs has been reported in Brachypodium distachyon, an emerging model plant in Poaceae.

Results: A total of 146 bHLH TFs were identified in the Brachypodium distachyon genome and classified into 24 subfamilies. BdbHLHs in the same subfamily share similar protein motifs and gene structures. Gene duplication events showed a close relationship to rice, maize and sorghum, and segment duplications might play a key role in the expansion of this gene family. The amino acid sequence of the bHLH domains were quite conservative, especially Leu-27 and Leu-54. Based on the predicted binding activities, the BdbHLHs were divided into DNA binding and non-DNA binding types. According to the gene ontology (GO) analysis, BdbHLHs were speculated to function in homodimer or heterodimer manner. By integrating the available high throughput data in public database and results of quantitative RT-PCR, we found the expression profiles of BdbHLHs were different, implying their differentiated functions.

Conclusion: One hundred fourty-six BdbHLHs were identified and their conserved domains, sequence features, phylogenetic relationship, chromosomal distribution, GO annotations, gene structures, gene duplication and expression profiles were investigated. Our findings lay a foundation for further evolutionary and functional elucidation of BdbHLH genes.

Keywords: Brachypodium distachyon; Expression profile; Genome-wide; Transcription factor; bHLH.

Fig. 1

Genomic distribution of bHLH genes and the gene duplications in the Brachypodium distachyon (a), rice (b), maize (c), and sorghum (d) genome

Fig. 2

Multiple Sequence Alignment of the bHLH Domains. The amino acids with identity more than 50% are labeled with colored boxes

Fig. 3

The interaction network of bHLHs in Brachypodium distachyon according to the orthologs in Arabidopsis. The BdbHLHs are in yellow blocks, the MYBs are in green blocks while other genes are in blue blocks

Fig. 4

Phylogenetic tree constructed with the BdbHLH domains based on the neighbor-joining method. The tree shows the 24 phylogenetic groups (numbered within blue circles). The circles of different colors represent the predicted DNA-binding activity of each protein: G-box in brown, Non-G-box in gray, Non-E-box in red and No-DNA binding in purple

Fig. 5

Intron distribution patterns in the coding sequence of the bHLH domain of BdbHLHs. The intron distribution patterns are marked with different colors, and position of introns is indicated by triangles. BRADI1G58230, as an example, is shown at top

Fig. 6

The expression profiles of BdbHLH genes in different tissues. The color scale is shown at the top. Higher expression levels are shown in red

Fig. 7

The expression profiles of BdbHLHs treated with high and low concentration of various phytohormones. The heatmap was generated with log2 based values. The color scale is shown at the top. Higher expression levels are shown in red while lower expression levels are shown in green. H stands for high concentration of phytohormone treatment while L stands for low concentration treatment

Fig. 8

Quantitative RT-PCR analysis of 29 selected BdbHLH genes. The relative expression levels of the 29 genes in (a) different organs namely root, stem, leaf and inflorescence; (b and c) root with different treatments including 20% PEG6000, 200 mM NaCl, 100 μM MeJA, 100 μM ABA, 20 μM 6-BA and 1 mM SA; (d) leaf with treatments of high temperature (45 C) and low temperature (4 C)