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Figure 1. 

The phylogenetic tree shows the relationships between CBL protein sequences from N. tangutorum and 12 other species. Phylogenetic analysis was performed using IQ-TREE software with maximum likelihood (ML) method and subjected to 1,000 bootstrap replicates. The modules are color coded to represent the five subclades of CBLs. NtCBLs are indicated by dark red stars. Green dots of different sizes indicate Bootstraps confidence levels above 80.

Figure 2. 

Genome-wide synteny analysis of CBL gene family among N. tangutorum and three other species. (a) inter-chromosomal relationships of NtCBLs (the links on the green curve indicate synteny relationships between genes). (b) Synteny analyses between the CBLs of N. tangutorum, Arabidopsis, Vitis vinifera, and Oryza sativa, the links between species indicate homologous relationships between genes.

Figure 3. 

Gene structure of the CBL gene family in N. tangutorum. The dark green boxes represent the UTR (Untranslated Region), the light green boxes represent the CDS (gene coding region), and the black lines represent the intron region.

Figure 4. 

Conservative motifs distribution in N. tangutorum. (a) Distribution of different motifs on NtCBL genes. (b) Specific sequence information of different motifs, with larger letters indicating higher conservation.

Figure 5. 

The gene expression characteristics of N. tangutorum CBL genes in root, stem, and leaf tissues were analyzed by fluorescence quantitative PCR. **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

Figure 6. 

Expression patterns of NtCBL genes during drought (upper panels) and salt stress (lower panels) in N. tangutorum. 'ck' stands for untreated control, while '1h', '4h', '8h', and '24h' respectively represent different time points of salt or stress treatment, which are 1, 4, 8, and 24 h. **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

Figure 7. 

Heterologous expression of NtCBL1-1 increases Arabidopsis germination rates under salt and drought stress. (a)−(c) Phenotypic charts of germination rates under normal growth conditions, 150 mM NaCl treatment, and 300 mM mannitol treatment, with about 70 seeds per dish and three replicates for each experiment. (d) Germination rate statistics of Arabidopsis overexpressing NtCBL1-1 under normal growth conditions, 150 mM NaCl treatment, and 300 mM mannitol treatment. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

Figure 8. 

Heterologous overexpression of NtCBL1-1 increased salt tolerance in Arabidopsis. (a) Wild-type Arabidopsis phenotypes and overexpression lines exposed to 200 mM NaCl for 0–7 d. (b) Chlorophyll content, soluble protein content, and MDA content after 0–5 d of exposure to 200 mM NaCl. Statistical significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

Figure 9. 

Heterologous overexpression of NtCBL1-1 in Arabidopsis increased drought stress tolerance. (a) Phenotypic comparison between wild-type Arabidopsis and overexpression lines under 300 mM mannitol exposure for 0–7 d. (b) Chlorophyll content, soluble protein content, and MDA content of wild-type Arabidopsis and overexpression lines exposed to 300 mM mannitol for 0–5 d. Statistical significance denoted as *p < 0.05, **p < 0.01 (ANOVA followed by Tukey’s HSD).