Genome-wide identification and characterization of the bHLH gene family and analysis of their potential relevance to chlorophyll metabolism in Raphanus sativus L

Abstract

Background: Green-fleshed radish (Raphanus sativus L.) is an economically important root vegetable of the Brassicaceae family, and chlorophyll accumulates in its root tissues. It was reported that the basic helix-loop-helix (bHLH) transcription factors play vital roles in the process of chlorophyll metabolism. Nevertheless, a comprehensive study on the bHLH gene family has not been performed in Raphanus sativus L.

Results: In this study, a total of 213 Raphanus sativus L. bHLH (RsbHLH) genes were screened in the radish genome, which were grouped into 22 subfamilies. 204 RsbHLH genes were unevenly distributed on nine chromosomes, and nine RsbHLH genes were located on the scaffolds. Gene structure analysis showed that 25 RsbHLH genes were intron-less. Collineation analysis revealed the syntenic orthologous bHLH gene pairs between radish and Arabidopsis thaliana/Brassica rapa/Brassica oleracea. 162 RsbHLH genes were duplicated and retained from the whole genome duplication event, indicating that the whole genome duplication contributed to the expansion of the RsbHLH gene family. RNA-seq results revealed that RsbHLH genes had a variety of expression patterns at five development stages of green-fleshed radish and white-fleshed radish. In addition, the weighted gene co-expression network analysis confirmed four RsbHLH genes closely related to chlorophyll content.

Conclusions: A total of 213 RsbHLH genes were identified, and we systematically analyzed their gene structure, evolutionary and collineation relationships, conserved motifs, gene duplication, cis-regulatory elements and expression patterns. Finally, four bHLH genes closely involved in chlorophyll content were identified, which may be associated with the photosynthesis of the green-fleshed radish. The current study would provide valuable information for further functional exploration of RsbHLH genes, and facilitate clarifying the molecular mechanism underlying photosynthesis process in green-fleshed radish.

Keywords: Basic helix-loop-helix transcription factor; Chlorophyll content; Photosynthesis; Raphanus sativus L.

Fig. 1

Chromosomal locations of RsbHLH genes

Fig. 2

The exon–intron organization of RsbHLH genes. The yellow box represents the exon, the green box represents the untranslated regions (UTR), and the black line represents the intron. The sizes of exons and introns are estimated by the scale at the bottom

Fig. 3

An un-rooted phylogenetic tree of the RsbHLH proteins and AtbHLH proteins. 22 subfamilies are marked with different colors

Fig. 4

The conserved motif distribution of RsbHLH proteins. Different motifs are displayed by different colored boxes. The motif size is estimated by the scale at the bottom

Fig. 5

Partial display of multiple sequence alignment for RsbHLH proteins. The basic helix-loop-helix domain was displayed. The height of a letter indicates its relative frequency at the given position. This image shows only a small part of the alignment

Fig. 6

a The collinear analysis of bHLH genes between Raphanus sativus L. and Arabidopsis thaliana. The curves of brown, orange, blue, purple, and red link the bHLH genes on the At01, At02, At03, At04, and At05 chromosomes of A. thaliana and their syntenic orthologous genes in Raphanus sativus L., respectively. b The collinear analysis of bHLH genes between Raphanus sativus L. and Brassica rapa/Brassica oleracea. The curves of orange and brown link the syntenic orthologous bHLH genes between Raphanus sativus L. and Brassica rapa/Brassica oleracea, respectively

Fig. 7

Cis-regulatory elements in the promoter regions of the RsbHLH genes. Different cis-regulatory elements are represented with different colored boxes, which are placed at the top on the right. The element size is estimated by the scale at the bottom

Fig. 8

The GO annotation of RsbHLH genes. All annotated GO terms include cellular component, molecular function and biological process. The y axis indicates the number of genes

Fig. 9

Expression patterns of RsbHLH genes at five development stages of GF and WF. GF indicates the green-fleshed radish ‘Cuishuai’; WF indicates the white-fleshed radish ‘Zhedachang’. The colour scale is shown at the right top. Higher expression level is in red, while lower expression level is in blue

Fig. 10

a Hierarchical cluster tree revealing gene co-expression modules identified by WGCNA. The branches contain 15 modules labeled in different colors. Except the gray module, the modules are named ME1 to ME15. b Module-trait associations. Columns correspond to chlorophyll content, and rows correspond to the characteristic genes of the modules. The correlation between two is shown in cell by Pearson correlation coefficient, and p-value is in parentheses. Cell color ranges from red (high positive correlation) to blue (high negative correlation)

Fig. 11

DEGs co-expressed with RsbHLH52 in the blue module (a) and RsbHLH140 in the turquoise module (b)