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Figure 1. 

Phylogenetic analysis of CYP genes from alfalfa, thistle alfalfa, and Arabidopsis. Different colors represent different groups.

Figure 2. 

Phylogeny, conserved motifs, and exon–intron structure of MsCYP genes.

Figure 3. 

Chromosomal locations of MsCYP genes. Tandem duplicates are marked with curved lines.

Figure 4. 

Synteny analysis of alfalfa CYP genes. Different segmental duplicate pairs are linked with different colors.

Figure 5. 

Expression pattern of nine key MsCYP genes. The column indicates RNA-seq data, the red line indicates RT-qPCR data, and the error bar indicates the standard error of the mean (three biological replicates and three technical replicates). **, p < 0.01. ns, not significant. N0 refers to gene expression before salt treatment; N2 refers to gene expression after salt treatment for 2 h; and NH refers to gene expression after rehydration for 3 d.

Figure 6. 

Analysis of the MsCYP273 protein. (a) RT-PCR results of five MsCYP273 overexpressing lines. (b) Subcellular localization of MsCYP273 protein. Bar = 20 μm. Bright: Bright field; GFP: green fluorescent protein of 495-545 nm; CHF: chloroplast autofluorescence of 590–670 nm. (c) Phenotype of MsCYP273 overexpressing lines (MsCYP273-line2 and MsCYP273-line10) under salt stress. (d) Root length analysis of MsCYP273 overexpressing lines (MsCYP273-line2 and MsCYP273-line10) under salt stress. Bar = 1 cm. ***, p < 0.001.