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. 2023 Feb 9;24(4):3498.
doi: 10.3390/ijms24043498.

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cinnamomum camphora ('Gantong 1')

Xue Gong  1   2 , Tengfei Shen  3 , Xiuqi Li  1   2 , Hanbin Lin  1   2 , Caihui Chen  2 , Huihu Li  2 , Zhaoxiang Wu  2 , Qiaoli Liu  2 , Meng Xu  3 , Bo Zhang  4 , Yongda Zhong  1   2
Affiliations

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cinnamomum camphora ('Gantong 1')

Xue Gong et al. Int J Mol Sci. .

Abstract

Cinnamomum camphora is one of the most commonly used tree species in landscaping. Improving its ornamental traits, particularly bark and leaf colors, is one of the key breeding goals. The basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial in controlling anthocyanin biosynthesis in many plants. However, their role in C. camphora remains largely unknown. In this study, we identified 150 bHLH TFs (CcbHLHs) using natural mutant C. camphora 'Gantong 1', which has unusual bark and leaf colors. Phylogenetic analysis revealed that 150 CcbHLHs were divided into 26 subfamilies which shared similar gene structures and conserved motifs. According to the protein homology analysis, we identified four candidate CcbHLHs that were highly conserved compared to the TT8 protein in A. thaliana. These TFs are potentially involved in anthocyanin biosynthesis in C. camphora. RNA-seq analysis revealed specific expression patterns of CcbHLHs in different tissue types. Furthermore, we verified expression patterns of seven CcbHLHs (CcbHLH001, CcbHLH015, CcbHLH017, CcbHLH022, CcbHLH101, CcbHLH118, and CcbHLH134) in various tissue types at different growth stages using qRT-PCR. This study opens a new avenue for subsequent research on anthocyanin biosynthesis regulated by CcbHLH TFs in C. camphora.

Keywords: Cinnamomum camphora; RNA sequencing; anthocyanin; basic helix–loop–helix (bHLH) transcription factors; collinearity analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phylogenetic relationships of bHLH proteins from C. camphora and Arabidopsis. Each subgroup is shown in a different color. The subgroup names and ID numbers are indicated in the outer circle of the phylogenetic tree.
Figure 2
Figure 2
CcbHLH gene family segment duplication events. Segmental repeats formed CcbHLH genes linked by red lines.
Figure 3
Figure 3
Collinearity analysis of the bHLH TF family in C. camphora, A. thaliana, and P. trichocarpa. Red lines link identified collinear genes.
Figure 4
Figure 4
Protein interaction network prediction for CcbHLH001, CcbHLH022, CcbHLH118, and CcbHLH134 (shown in red) based on their orthologs in Arabidopsis.
Figure 5
Figure 5
The heatmap of CcbHLH genes in different tissues of C. camphora (‘Gantong 1’). The scale bars represent the log2(FPKM) transformations of the FPKM values.
Figure 6
Figure 6
Relative expression levels of CcbHLH genes determined by qRT-PCR in the leaves (A) and bark (B) of C. camphora (‘Gantong 1’) and control plants collected year-round. Black and grey colors represent the relative expression levels in C. camphora ‘Gantong 1’ and the control plants, respectively. Figures with different numbers of asterisks represent significant differences on different levels. ***, p < 0.001; **, p < 0.01; *, p < 0.05.
Figure 6
Figure 6
Relative expression levels of CcbHLH genes determined by qRT-PCR in the leaves (A) and bark (B) of C. camphora (‘Gantong 1’) and control plants collected year-round. Black and grey colors represent the relative expression levels in C. camphora ‘Gantong 1’ and the control plants, respectively. Figures with different numbers of asterisks represent significant differences on different levels. ***, p < 0.001; **, p < 0.01; *, p < 0.05.
Figure 7
Figure 7
Phenotypic comparison between C. camphora ‘Gantong 1’ and the control group in different months.
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