Systematic analysis of the basic/helix-loop-helix (bHLH) transcription factor family in pummelo (Citrus grandis) and identification of the key members involved in the response to iron deficiency

Abstract

Background: Iron (Fe) deficiency is a common problem in citrus production. As the second largest superfamily of transcription factors (TFs), the basic/helix-loop-helix (bHLH) proteins have been shown to participate in the regulation of Fe homeostasis and a series of other biological and developmental processes in plants. However, this family of members in citrus and their functions in citrus Fe deficiency are still largely unknown.

Results: In this study, we identified a total of 128 CgbHLHs from pummelo (Citrus grandis) genome that were classified into 18 subfamilies by phylogenetic comparison with Arabidopsis thaliana bHLH proteins. All of these CgbHLHs were randomly distributed on nine known (125 genes) and one unknown (3 genes) chromosomes, and 12 and 47 of them were identified to be tandem and segmental duplicated genes, respectively. Sequence analysis showed detailed characteristics of their intron-exon structures, bHLH domain and conserved motifs. Gene ontology (GO) analysis suggested that most of CgbHLHs were annotated to the nucleus, DNA-binding transcription factor activity, response to abiotic stimulus, reproduction, post-embryonic development, flower development and photosynthesis. In addition, 27 CgbHLH proteins were predicted to have direct or indirect protein-protein interactions. Based on GO annotation, RNA sequencing data in public database and qRT-PCR results, several of CgbHLHs were identified as the key candidates that respond to iron deficiency.

Conclusions: In total, 128 CgbHLH proteins were identified from pummelo, and their detailed sequence and structure characteristics and putative functions were analyzed. This study provides comprehensive information for further functional elucidation of CgbHLH genes in citrus.

Keywords: Citrus; Gene expression; Motif; Stress; bHLH.

Fig. 1

Phylogenetic tree and subfamily classifications of CgbHLH and AtbHLH proteins. The conserved protein sequences of a total of 128 CgbHLHs and 136 AtbHLHs were used to construct neighbor-joining phylogenetic tree. Arabic numerals 1 to 18 represent 18 of subfamilies. All CgbHLH proteins are highlighted with red color. The G-box, non-G-box, non-E-box and non-DNA binding of CgbHLHs are marked with different colored symbols

Fig. 2

Conserved amino acids in CgbHLH domains. The height of each amino acid represents its conservation at that position. The amino acids conserved with more than 50% consensus ratio among the 128 CgbHLH domains are indicated in black letters

Fig. 3

Gene structures and protein motifs of CgbHLHs. The intron-exon structures, predicted motifs 1 to 20, and conserved domains of all CgbHLHs are shown according to their phylogenic classifications

Fig. 4

Chromosomal localizations and gene duplications of CgbHLH genes. The green and red colors represent tandem duplicated and segmental duplicated genes, respectively. The CgbHLH genes followed by the same number (highlighted with blue circles) are collinear genes

Fig. 5

Functional annotation and tissue expression of CgbHLH genes. a All annotated GO terms including biological process (BP), cellular component (CC) and molecular function (MF) of 126 CgbHLHs (two other CgbHLHs were not annotated a GO term). b Expression heatmap of CgbHLHs. The TPM values were generated from the RNAseq data of pummelo roots in a public database. c Venn diagram shows the number of the genes that respond to abiotic stimulus and expresssed in roots

Fig. 6

Predicted protein-protein interactions of CgbHLHs according to their orthologs in A. thaliana. In the network, only the pairs with higher than 40% sequence identity between CgbHLHs and AtbHLHs and with an interaction score > 0.7 are shown. Line and node colors indicate the different kinds and degrees of interactions, respectively. The filled or empty nodes represent known or unknown 3D structures, respectively. The abbreviated names are the genes that have been reported in A. thaliana

Fig. 7

qRT-PCR analysis of 34 CgbHLH genes in pummelo roots. Relative expression levels of 34 CgbHLHs were determined in 0.5 d, 1.5 d, 2 d, 7 d and 12 d of Fe-deficient (−Fe) roots and normal (CK) roots sampled at corresponding time points. Data are means ± SE of three biological replicates. Asterisks on the error bars indicate significant differences (t-test, P < 0.05) between -Fe and CK