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Figure 1. 

Motif and gene structure analysis of the M. crystallinum bHLH gene family. (a) The phylogenetic tree of 91 M. crystallinum bHLH. (b) Conserved motif distribution of bHLH protein in M. crystallinum. (c) The genetic structure of the bHLH gene in M. crystallinum. The information of each gene structure is provided in Supplementary Table S4. The red rectangle and the yellow rectangle represent the untranslated region (UTR), and the exon respectively. Introns are indicated by black lines. The scale is shown at the bottom of the chart.

Figure 2. 

Phylogenetic analysis of the bHLH gene family from M. crystallinum and A. thaliana. Phylogenetic trees were constructed using the bHLH protein sequences and different colors indicate different clades.

Figure 3. 

Analysis of the gain and loss of the bHLH gene and evolutionary time in 13 species. Schematic diagram of the gain and loss of the bHLH gene in M. crystallinum and their closely related species. The green rectangle and the numbers in yellow font on the right side of the species picture represent the quantity of bHLH genes in ancestors and existing species. The + sign and the - sign respectively indicate the gain and loss of genes.

Figure 4. 

Chromosome localization of the bHLH gene family in M. crystallinum, A. thaliana, H. undatus, and S. oleracea. The left scale indicates the chromosome length (Mb), with bHLH gene markers on the right side of each chromosome. Among them, blue represents M. crystallinum, green represents A. thaliana, purple represents H. undatus, and orange represents S. oleracea.

Figure 6. 

Co-expression network of the bHLH gene family in M. crystallinum. Based on the correlation of expression levels among the genes of M. crystallinum, a co-expression network is formed. The light purple circle indicates that the genes M. crystallinum do not belong to the bHLH family, and the genes belonging to the bHLH family are marked with different colors. The correlations in the figure are all greater than 0.95.

Figure 7. 

Statistics of cis-acting elements in the bHLH promoter of M. crystallinum. Red represents light response, blue represents stress response, green represents hormone response, and purple represents others.

Figure 8. 

Protein interaction network of the bHLH gene family in M. crystallinum. PPI network based on the bHLH homolog in A. thaliana.

Figure 9. 

Expression pattern analysis of bHLH genes in M. crystallinum. Expression levels of the bHLH gene in the (a) roots, stems and leaves. (b) M. crystallinum grown under different salt concentrations (0, 25, 50, 75, 100, and 125 mM NaCl). (c) M. crystallinum with control and salt treatment (500 mM NaCl) collected at 8:00 and 20:00 from 5 to 7 d after treatment. (d) M. crystallinum under different salt treatments (0, 140, 250, and 500 mM NaCl). (e) M. crystallinum treated with 500 mM NaCl salt concentration and the control group on the first and second days. (f) M. crystallinum treated with CK, LS (100 mM), MS (300 mM), and HS (500 mM) NaCl. The expression levels increase from blue to white and then to red.

Figure 10. 

qRT-PCR analysis of eight differential expression bHLH gene in leaf under different concentrations of salt irrigation. Different letter designations for the same gene indicate significant differences (p < 0.05).