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Figure 1. 

The distribution of the MsPAP gene family on alfalfa chromosomes.

Figure 2. 

Structures of MsPAP genes in alfalfa. (a) Phylogenetic tree of MsPAP proteins. (b) Schematic diagram of gene structure. Green boxes represent exons (CDS), black lines represent introns, and yellow boxes represent untranslated regions (UTRs). The lengths of exons and introns of each gene are displayed proportionally.

Figure 3. 

Conserved protein motifs of the MsPAP gene family in alfalfa.

Figure 4. 

Phylogenetic analysis of PAP proteins from A. thaliana, M. truncatula, and alfalfa.

Figure 5. 

Syntenic relationships of the PAP gene family in A. thaliana, M. truncatula, and alfalfa. (a) Syntenic relationships of MsPAP and AtPAP; (b) syntenic relationships of MsPAP and MtPAP.

Figure 6. 

Analysis of MsPAP genes' expression in different tissues of alfalfa. Hierarchical clustering heatmap of the transcriptional expression levels of the MsPAP gene family in the tissues of (a) elongated stems, (b) nodules, (c) leaves, (d) roots, (e) unelongated stems, and (f) flowers in alfalfa. The genes are arranged according to the results of the cluster analysis; the color gradient represents the relative expression levels (green indicates high expression, white indicates low expression), and the color scale on the right shows the range of expression values using FPKM-normalized values.

Figure 7. 

Expression of MsPAP genes in alfalfa under cold, drought, alkali, and salt stress. Hierarchical clustering heatmap of the transcriptional expression levels of the MsPAP gene family in the stress under (a) drought, (b) salt, (c) cold, and (d) alkali. The color gradient represents the level of gene expression, where red typically indicates high expression and blue typically indicates low expression. The color scale represents log₂(FPKM + 1).

Figure 8. 

Analysis of the relative expression levels of MsPAP family members under low-P stress. The abscissa represents different sampling time points: 0 and 1 h, and 1, 3, 5, and 7 d; the ordinate represents the relative expression level of each gene. Pink bars represent the 50-μmol·L⁻¹ low-P treatment group (50 μmol·L⁻¹ KH₂PO₄), and green bars represent the control group. The data are presented as the mean ± SD, n = 3. Significance of differences: *, 0.01 < p ≤ 0.05; **, 0.001 < p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001, extremely significant. The y-axis shows relative expression (2^−ΔΔCᴛ). Data are mean ± SD (n = 3), with the control (0 h) set to 1.