Integrated ion, hormone, and proteomic analyses reveal the mechanisms of drought resistance in alfalfa (Medicago sativa)

TL;DR

This study explores how alfalfa resists drought by analyzing ions, hormones, and proteins, revealing key mechanisms and potential targets for improving drought tolerance in crops.

Contribution

The study integrates ion, hormone, and proteomic data to uncover novel molecular mechanisms and candidate proteins involved in alfalfa's drought resistance.

Findings

Dynamic changes in Na+, K+, and Ca2+ ions are crucial for maintaining cellular homeostasis under drought.

Key proteins like MMK1, BSK3, carbonic anhydrase, and MYB11 contribute to drought resistance in alfalfa.

Phenylpropanoid biosynthesis and glutathione metabolism are important pathways in drought response.

Abstract

Drought stress is a major environmental limiting factor limiting forage yield. Alfalfa (Medicago sativa) is a crucial perennial leguminous forage, yet its adaptive mechanisms to drought remain insufficiently understood.

We compared ion homeostasis, endogenous hormone levels, and protein expression in a drought-resistant (‘WL168’) and a drought-sensitive (‘Gannong No. 3’) alfalfa variety under drought stress and rehydration. The results showed that that dynamic changes in ions, particularly Na+, K+, and Ca2+, were closely associated with the maintenance of cellular homeostasis and water balance under drought. Rehydration effectively restored ion balance, especially for K+ and Ca2+. Hormonal analysis suggested that ZT, GA3, IAA, ABA, and SA in roots participated in the response to drought and subsequent recovery. Integrated proteomic and WGCNA revealed key pathways, including phenylpropanoid biosynthesis, starch and sucrose metabolism, and glutathione metabolism, responding to drought stress. Critical proteins such as MMK1, BSK3, carbonic anhydrase, and MYB11 were identified as contributors to alfalfa’s drought resistance.

This study provides new insights into the molecular mechanisms of drought resistance in alfalfa. The identified candidate genes and proteins offer valuable resources for future molecular-assisted breeding aimed at improving drought tolerance.

The online version contains supplementary material available at 10.1186/s12870-026-08198-2.

Figures

Morphological changes of G3 and WL168 under drought stress and rehydration treatments. CK: control, DS: drought stress, R1: rehydration treatment for 1 day, R5: rehydration treatment for 5 days. Each bar represents the mean value ± standard deviation (SD) of three replicates (*n* = 3). Adapted with permission from Chen et al. [[18](#CR18)]. Copyright 2025 American Chemical Society. (A) Morphological phenotypes of G3 and WL168 under drought stress conditions; (B-E) Quantitative analysis of growth parameters. Different lowercase letters indicate significant differences among different treatments

Ion content changes in roots, stems and leaves of alfalfa under drought stress and rehydration treatment. (A-H) Ion contents in leaves; (I-P) Ion contents in stems; (Q-X) Ion contents in roots

Changes in endogenous hormone content in leaf and root sites between G3 and WL168 under drought stress. (A-E) Endogenous hormone contents in leaves; (F-J) Endogenous hormone contents in roots

Venn diagram and KEGG enrichment analysis of DEPs of G3 and WL168 under different treatments. (A) Statistics of number of DEPs in different comparison groups; (B-E) Venn diagram showing the DEGs of different comparison groups; (F) KEGG enrichment of DEPs of G3 down-regulated in DS/CK and up-regulated in R1/DS and R2/DS; (G) KEGG enrichment of DEPs of G3 up-regulated in DS/CK and down-regulated in R1/DS and R2/DS; (H) KEGG enrichment of DEPs of WL168 down-regulated in DS/CK and up-regulated in R1/DS and R2/DS; (I) KEGG enrichment of DEPs of WL168 up-regulated in DS/CK and down-regulated in R1/D

KEGG enrichment analysis of DEPs in two alfalfa varieties under drought stress and rehydration treatment

Heat map analysis of DEPs related to phenylpropanoid biosynthesis, starch and sucrose metabolism in alfalfa under drought stress

Protein Cluster Analysis and Correlation Analysis between Traits and Modules. (A) Hierarchical cluster analysis of co-expressed proteins; (B) Distribution of the number of proteins in each module; (C) Heatmap of expression patterns between modules and samples; (D) Protein function analysis within the black module. (E) Protein-protein interaction network analysis within the black module, red letters represent ion and hormone-related proteins

Analysis of the expression of ion and hormone-related proteins. (A) Heatmap analysis of proteins involved in hormone signal transduction; (B) Heatmap analysis of proteins associated with inorganic ion transport and metabolism; (C-D) Expression profiles of representative proteins from the brown module; (E-I) Expression profiles of candidate proteins from the black module

Relative expression levels of the genes encoding six proteins analyzed by qRT-PCR

Full text

Background

With the intensification of global climate change, drought has become one of the important environmental stresses threatening agricultural production. Alfalfa (Medicago sativa) is the most widely cultivated perennial leguminous forage and nitrogen-fixing crop globally. It has a high biomass and crude protein content, is rich in digestible nutrients and mineral elements, and enjoys the reputation of “the king of forages” [1, 2]. It is also one of the most economically valuable crops in the world. In the United States, it ranks as the fourth most-planted crop, only after wheat (Triticum aestivum), maize (Zea mays), and soybeans (Glycine max) [2, 3]. However, the arid environment has a significant impact on the growth and yield of alfalfa, severely restricting its productivity [4]. Therefore, revealing the physiological and molecular mechanisms of alfalfa under drought stress and breeding drought-resistant alfalfa varieties are of great significance for improving its drought resistance and improving crop varieties.

The response mechanism of alfalfa to drought is complex and diverse. Among them, the regulation of ion content, endogenous hormones, and changes in protein expression are key links in its adaptation to drought stress. Firstly, ionic balance is of vital importance to the physiological stability of plants. Drought stress usually leads to a reduction in water content within plant cells, affecting the absorption, transportation, and accumulation of ions [5]. Plants maintain cell osmotic pressure by regulating the concentrations of ions such as sodium (Na^+^), potassium (K^+^), and calcium (Ca^2+^), ensuring the water balance inside and outside the cells [6]. Ions serve as crucial signaling molecules that trigger plant signal transduction pathways. Under drought stress, both mineral ions and their channel proteins undergo dynamic changes, thereby enabling plants to actively adapt to drought conditions [7]. K^+^ is the primary cationic inorganic nutrient in plants for osmotic regulation, cell growth, and enzyme activation. Previous studies had found that the over-expression of HvAKT1 promoted the absorption of K^+^ as a way for barley (Hordeum vulgare) to cope with drought stress, indicating the crucial role of K^+^ in plants’ adaptation to environmental stresses including drought [8]. Ca^2+^ not only plays a vital role in the osmotic regulation of plants but also participates in the regulation of cell signal transduction and antioxidant responses under drought stress [7]. Recent studies have reported that gene-edited Paghyprp1 poplar (Populus sp.) lines enhance drought and salt tolerance by maintaining root ion homeostasis (Na^+^, K^+^, hydrogen (H^+^), and Ca^2+^) and stabilizing osmotic balance [9]. These findings demonstrated that changes in mineral ion concentrations directly modulated plant osmotic equilibrium, highlighting osmotic regulation as a crucial physiological mechanism in plant stress responses. Secondly, plant hormones, as key regulators of plant growth and stress responses, play a non-negligible role in drought responses. The mechanisms of action of endogenous hormones such as abscisic acid (ABA), auxin (IAA), cytokinin (CTK), gibberellin (GA), and salicylic acid (SA) under drought stress have gradually been revealed [10]. For example, as a dominant hormone in the drought response, ABA can not only enhance the drought resistance of plants by regulating the opening and closing of stomata to reduce water evaporation, but also increase plant drought tolerance by inducing the expression of drought-resistant genes [11, 12]. Other hormones related to this, such as IAA and CTK, also provide adaptive regulation for plants under drought conditions by modulating root growth, leaf development, and the activity of the antioxidant system [13, 14].

In recent years, the advancement of high-throughput omics technologies has established comparative proteomics as a key approach for investigating plant molecular responses to drought stress. Proteomic analyses enable in-depth exploration of drought response mechanisms by examining protein alterations across different stress phases and varieties [15]. In alfalfa, drought-responsive proteins primarily include antioxidant enzymes, heat-shock proteins, and ion-channel proteins, which contribute to osmotic adjustment, oxidative damage protection, ion transport regulation, and cellular metabolic stability [16]. Although numerous studies have explored the response mechanisms of alfalfa to drought stress from different perspectives, there are still many unknowns regarding the interrelationships among ion regulation, hormonal signaling, and proteome regulation. Through comprehensive research on ion content, endogenous hormones, and proteomics, we can gain a more comprehensive understanding of the mechanisms by which alfalfa responds to drought stress. In our previous study, we conducted a comprehensive evaluation of drought resistance in 23 alfalfa materials by measuring morphological parameters and chlorophyll content, and based on these assessments, we identified 6 highly drought-tolerant materials and 5 drought-sensitive materials [17]. Therefore, in this study, the ion content and endogenous hormone content in the seedling roots of drought-sensitive alfalfa ‘Gannong No. 3’ (G3) and drought-resistant alfalfa ‘WL168’ (WL168) under drought stress and subsequent rehydration treatment were determined. Through data-Independent Acquisition (DIA) protein sequencing and WGCNA (weighted gene co-expression network analysis) analysis, we identified major drought-responsive pathways including phenylpropanoid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, glutathione metabolism, and amino acid biosynthesis. Additionally, we screened several candidate proteins involved in hormone signaling and inorganic ion transport/metabolism. These findings provide a theoretical framework for elucidating the molecular regulatory network underlying alfalfa drought tolerance.

Results

Morphological changes of alfalfa under drought stress

To explore the impact of drought stress on the seedlings of two alfalfa varieties, we analyzed the phenotypes of the two varieties under four different treatments. The results showed that under drought stress, G3 suffered more severe drought stress than WL168, with a greater degree of leaf curling. After rehydration treatments R1 and R2, both varieties resumed normal growth levels (Fig. 1A). In addition, the plant height of G3 was significantly lower than that of the control under drought stress (P < 0.05), and the plant height of G3 was still significantly lower than that of the control after rehydration treatment, while the plant height of WL168 did not change significantly under drought stress and rehydration treatment (P > 0.05) (Fig. 1B). Under drought stress, the total root length of both varieties decreased, but the total root length of G3 was significantly shorter than that of the control group. And after rehydration treatment, the total root length of both varieties gradually increased, with WL showing a more significant increase (Fig. 1C). The aboveground fresh weight and underground fresh weight of the two varieties decreased significantly under drought stress. With the rehydration treatment, the aboveground fresh weight and underground fresh weight showed an upward trend, but G3 decreased more under drought stress and increased less after rehydration treatment (Fig. 1D and E).

Fig. 1. Morphological changes of G3 and WL168 under drought stress and rehydration treatments. CK: control, DS: drought stress, R1: rehydration treatment for 1 day, R5: rehydration treatment for 5 days. Each bar represents the mean value ± standard deviation (SD) of three replicates (n = 3). Adapted with permission from Chen et al. [18]. Copyright 2025 American Chemical Society. (A) Morphological phenotypes of G3 and WL168 under drought stress conditions; (B-E) Quantitative analysis of growth parameters. Different lowercase letters indicate significant differences among different treatments of each variety at the P < 0.05 level, analyzed by one-way ANOVA. The same below

Changes of ion content under drought stress

In order to explore the ion distribution and transportation of alfalfa seedlings under drought stress, we analyzed the content changes of eight ions (K^+^, Na^+^, Ca^2+^, magnesium (Mg^2+^), iron (Fe^3+^), copper (Cu^2+^), manganese (Mn^2+^) and inc (Zn^2+^)) in leaves, stems and roots of two varieties under four different treatments. In leaves, the results showed that, compared with CK, K^+^ decreased significantly under drought stress and rehydration treatment in G3, while it decreased significantly under drought stress in WL168, returned to the control level under rehydration treatment R1, and exceeded the control level under R2 treatment (P < 0.05) (Fig. 2A). Compared with the control, Na^+^ and Ca^2+^ in G3 showed no significant changes under drought stress and rehydration treatment (P > 0.05), while in WL168 they increased significantly under both conditions (Fig. 2B and C). Fe^3+^ increased significantly under drought stress in both G3 and WL168, and returned to control levels after rehydration treatment (Fig. 2D). Compared with the control, Mg^2+^ and Cu^2+^ did not change significantly under drought stress and rehydration treatment R1 in G3 and WL168 (Fig. 2E and F). Mn^2+^ and Zn^2+^ decreased under drought stress and rehydration treatment in G3 and WL168, but compared with CK, Zn^2+^ had no significant difference under drought stress and R1 treatment in WL168.

Fig. 2. Ion content changes in roots, stems and leaves of alfalfa under drought stress and rehydration treatment. (A-H) Ion contents in leaves; (I-P) Ion contents in stems; (Q-X) Ion contents in roots

In the stems, compared with CK, K^+^, Na^+^ and Ca^2+^ all were no significant changes under drought stress and rehydration treatment R1 in G3 and WL168 (P > 0.05) (Fig. 2I-K), and Fe^3+^ in WL168 stems increased significantly under rehydration treatment (P < 0.05) (Fig. 2L). Compared with CK, Mg^2+^ in stems of G3 and WL168 increased under drought stress and rehydration treatment, but the increasing trend was not significant under drought stress in WL168 (Fig. 2M). Cu^2+^ and Mn^2+^ in stems increased significantly under drought stress in G3, but there was no significant difference under drought stress in WL168 (Fig. 2N-O). Zn^2+^ increased significantly under rehydration treatment R2 in WL168, but no significant change was found in G3 (Fig. 2P).

K^+^ changes were differently in different materials. In G3, the trends of change in leaves, stems, and roots were similar, all decreasing sequentially under drought and rehydration treatments. In WL168, however, K^+^ levels in leaves and roots decreased under drought treatment but rebounded after rehydration, with leaves in the R2 treatment even exceeding the control levels. The changes in stems were the same as in G3. The rebound of K^+^ in WL168 after rehydration was observed, with K^+^ levels under the R2 treatment surpassing the control (Fig. 2Q). (Fig. 2Q); Na^+^ and Ca^2+^ in roots of both G3 and WL168 decreased significantly under drought stress and returned to control levels after rehydration treatment (Fig. 2R-S); Fe^3+^ in roots increased significantly under drought stress in both varieties and returned to control levels after rehydration (Fig. 2T); Mg^2+^ content in roots increased under both drought stress and rehydration treatment in both varieties (Fig. 2U). Cu^2+^ in roots increased significantly under drought stress in G3, remained basically unchanged under subsequent rehydration treatment, but decreased significantly under drought stress in WL168, and then increased significantly under rehydration treatment (Fig. 2V). Under drought stress, Mn^2+^ in roots decreased significantly in G3 and returned to the control level after rehydration, while in WL168, it decreased significantly under both drought stress and rehydration treatment (Fig. 2W). Zn^2+^ in roots decreased significantly under drought stress in WL168 and increased significantly after rehydration; in G3, it increased significantly after rehydration treatment (Fig. 2X).

The ion content in different tissues of alfalfa seedlings showed dynamic changes under drought stress. Compared with G3, WL168 exhibited more pronounced changes in K^+^ in leaves and roots, as well as in Na^+^, Ca^2+^, Mn^2+^ in leaves and stems, and Fe^3+^ in leaves and stems under both drought stress and rehydration treatment. The K^+^/Na^+^ ratios in different tissues of both materials under drought and rewatering treatments are presented in Figure S1. In aboveground organs (leaves and stems), both materials generally showed decreased K^+^/Na^+^ ratios after drought and rewatering treatments (Figure S1A-B). In the roots of WL168, the K^+^/Na^+^ ratio increased after rewatering, with no significant difference observed between the control and R2 treatment (Figure S1C).

Analysis of ion transport capacity under drought stress

We analyzed the selective transport coefficients SK, Na, SCa, Na, SMg, Na, SCu, Na, SFe, Na, SZn, Na and SMn, Na in leaves, stems, and roots of the two alfalfa varieties under drought stress and rehydration treatments (Table 1). Compared with the control, the roots of G3 accumulated more Na⁺ under drought stress, with increased transport capacity of Ca^2+^ and Mn^2+^ to stems. Under rehydration treatment, the transport capacities of K^+^, Mg^2+^, Cu^2+^, Fe^3+^ and Zn^2+^ increased compared to drought stress. Under drought stress, the roots of WL168 accumulated Na^+^, with increased transport capacity of K^+^ and Ca^2+^ to stems compared to the control. Under rehydration treatment, the transport capacities of Fe^3+^ and Mn^2+^ increased compared to drought stress levels. Analysis of ion transport from stems to leaves showed that the Mn²^+^ transport capacity in G3 increased significantly under drought stress (P < 0.05), while other ion transport capacities showed no significant changes. The transport capacity of K^+^, Ca^2+^, Mg^2+^, Fe^3+^, Zn^2+^ and Mn^2+^ in WL168 stems was significantly reduced under drought stress, and the transport capacity of Fe^3+^ to leaves continued to decrease under R1 and R2 treatments, while the transport capacity of other ions under R2 treatment was significantly lower than that under drought stress. In general, drought stress significantly affected the ion selective transport capacity in different organs of alfalfa seedlings. Different alfalfa varieties exhibited distinct patterns of ion accumulation and transport under drought stress. Rehydration treatment promoted the recovery of ion transport capacity, particularly for certain mineral ions.

Table 1. Ion transport of different organs of alfalfa under different treatmentsTreatmentS_K, Na_S_Ca, Na_S_Mg, Na_S_Cu, Na_S_Fe, Na_S_Zn, Na_S_Mn, Na_Root-stemG3-CK2.04 ± 0.24b2.06 ± 0.10bc1.74 ± 0.29a2.53 ± 0.30a1.85 ± 0.04a1.37 ± 0.15a1.19 ± 0.14bcG3-DS1.90 ± 0.18b2.65 ± 0.35a0.85 ± 0.10bc0.86 ± 0.12bc0.22 ± 0.02d0.61 ± 0.10c1.34 ± 0.16bG3-R12.52 ± 0.11a2.37 ± 0.07abc1.15 ± 0.04b1.09 ± 0.09b0.34 ± 0.02 cd0.90 ± 0.07b0.54 ± 0.06eG3-R22.25 ± 0.10ab2.51 ± 0.16ab0.82 ± 0.04bc0.78 ± 0.05bc0.36 ± 0.05 cd0.60 ± 0.07c0.75 ± 0.08deWL-CK1.08 ± 0.02 cd1.47 ± 0.04de0.90 ± 0.03bc0.74 ± 0.04bc0.23 ± 0.02d0.42 ± 0.06 cd0.64 ± 0.05eWL-DS1.47 ± 0.08c2.20 ± 0.12abc0.65 ± 0.05c0.66 ± 0.04c0.28 ± 0.03d0.40 ± 0.02 cd0.44 ± 0.02eWL-R11.40 ± 0.05c1.90 ± 0.08 cd0.67 ± 0.05c0.65 ± 0.03c0.50 ± 0.10bc0.39 ± 0.05 cd2.22 ± 0.17aWL-R20.82 ± 0.04d1.38 ± 0.18e0.54 ± 0.03c0.69 ± 0.05bc0.61 ± 0.11b0.29 ± 0.03d1.00 ± 0.01 cdStem-leafG3-CK1.75 ± 0.11b2.01 ± 0.15ab1.80 ± 0.09b1.28 ± 0.17bc0.77 ± 0.06de1.86 ± 0.09bc2.06 ± 0.10bcG3-DS1.61 ± 0.15bc2.08 ± 0.17ab1.67 ± 0.13b1.22 ± 0.08bc1.13 ± 0.18 cd1.71 ± 0.23c1.19 ± 0.05deG3-R11.99 ± 0.22ab2.48 ± 0.31a2.15 ± 0.31ab1.78 ± 0.30ab1.70 ± 0.33bc2.26 ± 0.34bc3.46 ± 0.60aG3-R21.85 ± 0.07ab2.39 ± 0.10a1.88 ± 0.21b1.34 ± 0.26b0.85 ± 0.09de1.62 ± 0.32c1.93 ± 0.18bcdWL-CK2.18 ± 0.11a2.36 ± 0.04a2.46 ± 0.22a2.24 ± 0.32a2.49 ± 0.32a3.80 ± 0.20a2.37 ± 0.14bWL-DS1.69 ± 0.05b1.72 ± 0.02bc1.93 ± 0.12ab1.64 ± 0.07ab1.86 ± 0.16b2.45 ± 0.23b1.51 ± 0.10cdeWL-R11.60 ± 0.07bc1.88 ± 0.03bc1.62 ± 0.05b1.16 ± 0.02bc0.77 ± 0.14de2.29 ± 0.12bc1.17 ± 0.08eWL-R21.25 ± 0.08c1.51 ± 0.09c0.86 ± 0.07c0.65 ± 0.11c0.30 ± 0.04e0.79 ± 0.08d0.43 ± 0.04fS_X, Na_ = sink organ [X/Na^+^]/source organ [X/Na^+^], X represents the content of any one of the ions such as K^+^, Ca^2+^, Mg^2+^, Fe^3+^, Cu^2+^, Mn^2+^ and Zn^2+^ The value is the mean ± standard error (n=3). Different lowercase letters indicate significance of the same column at P<0.05, one-way ANOVA

Changes in endogenous hormone content under drought stress

The contents of zeatin (ZT), GA_3_, IAA, ABA and SA in leaves and roots were measured. In leaves, ZT content significantly decreased in G3 under rehydration treatment R1, and significantly decreased under drought stress and rehydration treatment in WL168 (Fig. 3A) (P < 0.05). Compared with the control, GA_3_ content significantly increased under drought stress and rehydration treatment in both G3 and WL168, but began to decrease at R1 treatment in WL168 (Fig. 3B). IAA content increased significantly under drought stress in both G3 and WL168, but decreased to control levels under rehydration treatment R1 (Fig. 3C). The ABA content was significantly increased under drought stress and R1 treatment in G3 and WL168, but decreased under rehydration treatment R2 (Fig. 3D). The SA content increased significantly in G3 under drought stress and R1 treatment, as well as in WL168 under drought stress. However, it decreased significantly compared to the control in G3 under R2 treatment, and in WL168 under R1 and R2 treatments (Fig. 3E).

Fig. 3. Changes in endogenous hormone content in leaf and root sites between G3 and WL168 under drought stress. (A-E) Endogenous hormone contents in leaves; (F-J) Endogenous hormone contents in roots

In the roots, the ZT content showed a trend of first decreasing and then increasing in G3 and WL168. However, the ZT content increased significantly as early as under the R1 treatment and was higher than the control level in WL168 (P < 0.05) (Fig. 3F). The contents of GA_3_, IAA, ABA, and SA in G3 and WL168 increased significantly under drought stress and then decreased under rehydration treatment. Specifically, the IAA content returned to the control level under rehydration treatment, while the contents of GA_3_ and ABA were lower than the control level in G3 and WL168 under rehydration treatment R2, and under R1 treatment, the ABA content in both G3 and WL168 remained higher than that in the control (Fig. 3G-I). Although the SA content decreased under rehydration treatment compared with drought stress, it was still significantly higher than the control (Fig. 3J).

Proteomic analysis of drought stress and rehydration response

The samples of G3 and WL168 under drought stress and rehydration treatment (R1 and R2) were further analyzed with proteomics using DIA based on screening of differentially expressed proteins (DEPs) when FC ≥ 1.5 and P-value ≤ 0.05 (Table S1). DEP analysis revealed that in the DS/CK comparison, which directly reflects the drought stress response, the more drought-tolerant WL168 identified only 452 DEPs, while the less drought-tolerant G3 identified 1,064 DEPs (Fig. 4A). The Venn diagram showed that there were 58 (3.87%) DEPs of DS/CK down-regulated proteins and R1/DS and R2/DS up-regulated proteins in the comparison group of G3 (Fig. 4B), while there were 92 (8.16%) DEPs of DS/CK up-regulated proteins and R1/DS and R2/DS down-regulated proteins in the comparison group (Fig. 4C). In contrast, the more drought-tolerant WL168 had significantly fewer DEPs showing the same expression trends, with only 5 (0.38%) and 37 (2.50%) DEPs in the corresponding categories (Fig. 4D, E). Notably, two of the five DEPs were peroxidase and fatty acid desaturase. However, there were 37 (2.50%) DEPs in DS/CK up-regulated proteins and R1/DS and R2/DS down-regulated proteins in the comparison group (Fig. 4E). These proteins, which reverse their expression patterns during stress and recovery, may serve as potential candidates for regulating drought adaptation.

Fig. 4. Venn diagram and KEGG enrichment analysis of DEPs of G3 and WL168 under different treatments. (A) Statistics of number of DEPs in different comparison groups; (B-E) Venn diagram showing the DEGs of different comparison groups; (F) KEGG enrichment of DEPs of G3 down-regulated in DS/CK and up-regulated in R1/DS and R2/DS; (G) KEGG enrichment of DEPs of G3 up-regulated in DS/CK and down-regulated in R1/DS and R2/DS; (H) KEGG enrichment of DEPs of WL168 down-regulated in DS/CK and up-regulated in R1/DS and R2/DS; (I) KEGG enrichment of DEPs of WL168 up-regulated in DS/CK and down-regulated in R1/DS and R2/DS

Functional analysis of deps under drought stress and rehydration treatment

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed distinct molecular strategies between G3 and WL168 in response to drought stress and rewatering recovery (Fig. 4F-I, Table S2). This difference was primarily reflected in two types of pathways with reversed expression patterns: In the less drought-tolerant G3, drought stress suppressed “growth and repair” pathways, including ribosome biogenesis, carotenoid and isoflavonoid biosynthesis (Fig. 4F), while activating “acute stress protection” pathways represented by proline metabolism, fatty acid degradation, and ascorbate metabolism (Fig. 4G). In contrast, the highly drought-tolerant WL168 exhibited a more anticipatory and regulated response pattern. Its “stress-downregulated, recovery-upregulated” pathways were concentrated in fatty acid metabolism (Fig. 4H), while its “stress-upregulated, recovery-downregulated” pathways were enriched in flavonoid biosynthesis, MAPK signaling, and starch and sucrose metabolism (Fig. 4I). These pathways collectively coordinated proactive signal transduction, antioxidant protection, and carbon resource allocation. Additionally, both cultivars were commonly enriched in fundamental metabolic pathways such as phenylpropanoid biosynthesis.

Functional enrichment analysis of deps in different comparison groups

GO enrichment analysis revealed differences in strategies between the drought-tolerant WL168 and the drought-sensitive G3 during drought stress and recovery (Figure S2, Table S2). At the DS stage, DEPs in the less drought-tolerant G3 were primarily enriched in regulatory functions such as “intracellular signal transduction (GO:0035556)” and “hormone metabolic process (GO:0009800).” In contrast, the drought-tolerant WL168 was enriched in fundamental metabolic and structural maintenance functions like “cellular respiration (GO:0016042)” and “microtubule organization (GO:0030388).” During the subsequent recovery phase (R1), the two cultivars converged on shared pathways such as “chitin metabolic process (GO:0006032),” but differed in molecular functions: G3 was enriched in “glycoside hydrolase activity (GO:0004568),” while WL168 was enriched in “metal ion binding (GO:0005507)” and “protein folding chaperone activity (GO:0051087).” By the late recovery stage (R2), WL168 demonstrated a more comprehensive reconstruction capacity in cellular components, with its DEPs enriched in structural-related functions such as “ribosomal protein synthesis (GO:0017087)” and “cytoskeleton (GO:0030312),” whereas G3 exhibited a more limited response.

To reveal the biological mechanisms of DEPs under drought stress and rehydration treatment, we performed a clustering analysis of pathway enrichment based on the KEGG database. As shown in Fig. 5, the pathways significantly enriched between the up-and downregulated DEPs were different in the six controls. Under drought stress, in both G3 and WL168, the up-regulated DEPs were most enriched in phenylpropanoid biosynthesis, glycolysis/gluconeogenesis, and starch and sucrose metabolism. The down-regulated DEPs were most enriched in phenylpropanoid biosynthesis, carbon metabolism, and biosynthesis of amino acids. Compared with drought stress, under R1 treatment, the up-regulated DEPs in both G3 and WL168 were most enriched in phenylpropanoid biosynthesis, carbon metabolism, and biosynthesis of amino acids, and the down-regulated DEPs were most enriched in carbon metabolism and biosynthesis of amino acids. Compared with drought stress, under R2 treatment, the up-regulated DEPs in both G3 and WL168 were most enriched in phenylpropanoid biosynthesis, carbon metabolism, biosynthesis of amino acids, and starch and sucrose metabolis, and the down-regulated DEPs were most enriched in glycolysis/gluconeogenesis, carbon metabolism, and biosynthesis of amino acids.

Fig. 5KEGG enrichment analysis of DEPs in two alfalfa varieties under drought stress and rehydration treatment

Compared with G3, under drought stress, WL168 had more up-regulated DEPs enriched in arginine and proline metabolism, glycerolipid metabolism, and glutathione metabolism, and more down-regulated DEPs enriched in glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and MAPK signaling pathway-plant. Under R1 treatment, WL168 had more up-regulated DEPs enriched in glycolysis/gluconeogenesis, and more down-regulated DEPs enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism. Under R2 treatment, WL168 had more up-regulated DEPs enriched in cysteine and methionine metabolism and endocytosis, and more down-regulated DEPs enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism.

Analysis of pathways related to drought stress

Although phenylpropanoid biosynthesis and starch and sucrose metabolism play important roles in the drought response of both alfalfa cultivars, their molecular strategies differ (Fig. 6). In the phenylpropanoid pathway, the drought-tolerant WL168 exhibited more precise regulation: its peroxidase (POD) was significantly activated during drought stress and the early recovery (R1) stage, promoting rapid lignin synthesis for “early reinforcement.” In contrast, POD activity in G3 was not substantially induced until the late recovery (R2) stage, displaying a “delayed repair” characteristic. In starch and sucrose metabolism, WL168 actively regulated osmotic balance and carbon source supply by upregulating enzymes such as β-fructofuranosidase (INV) and trehalose synthase (TPS), whereas G3 primarily upregulated fructokinase (FK) and phosphoglucomutase (PGM), indicating different carbon flow directions. These differences may be the main reasons for the variation in drought tolerance between the two cultivars.

Fig. 6. Heat map analysis of DEPs related to phenylpropanoid biosynthesis, starch and sucrose metabolism in alfalfa under drought stress

Protein co-expression network analysis

To identify the co-expression patterns among proteins in alfalfa under drought stress and rehydration treatments, we employed WGCNA, which had the function of clustering proteins with similar expression patterns into modules. Thirteen co-expression modules were constructed from the protein expression data of 24 alfalfa root samples (Fig. 7A). Further analysis of the distribution of proteins in the module showed that the number of proteins in the turquoise module was the largest, followed by blue module, brown module and yellow module (Fig. 7B). Through the heat-map analysis of the pearson correlation coefficients between module eigenvalue and trait data, we found that the module most significantly positively correlated with ZT, GA_3_, IAA, and ABA was the pink module. The module most significantly positively correlated with GA_3_, IAA, ABA, SA, and Mn^2+^ was the brown module. The module most significantly positively correlated with K^+^, Ca^2+^, Na^+^, and SA was the black module. Therefore, we conducted a further analysis of these three modules that were significantly associated with traits (Fig. 7C).

Fig. 7. Protein Cluster Analysis and Correlation Analysis between Traits and Modules. (A) Hierarchical cluster analysis of co-expressed proteins; (B) Distribution of the number of proteins in each module; (C) Heatmap of expression patterns between modules and samples; (D) Protein function analysis within the black module. (E) Protein-protein interaction network analysis within the black module, red letters represent ion and hormone-related proteins

Functional analysis and network interaction analysis of proteins in related modules

To further analyze the metabolic pathways of proteins in these modules in response to drought stress, we performed KEGG enrichment analysis of proteins within the brown, pink and black modules. The proteins of the brown module were mainly enriched in metabolic pathways, secondary metabolic biosynthesis, carbon metabolism, citric acid cycle (TCA cycle), and amino acid biosynthesis (Figure S3A). The hub proteins within this module mainly include probable fructokinase-4 (MS.gene032177), fructose-bisphosphate aldolase 6 (MS.gene47343), pyruvate dehydrogenase E2 component (MS.gene052712), general regulatory factor 2 (A0A072VBW0), threonine synthase (A0A072UTS3), glutaredoxin-dependent peroxiredoxin (G7KPG2), and MYB11 (MS.gene005539) (Figure S3B). The proteins in the pink module were mainly enriched in metabolic pathways, biosynthesis of secondary metabolites, carbon metabolism, pentose phosphate pathway, citric acid cycle, glycolysis/gluconeogenesis, and glutathione metabolism (Figure S3C). The proteins in the black module were mainly enriched in aminoacyl-tRNA biosynthesis, nucleocytoplasmic transport, plant hormone signal transduction, MAPK signaling pathway-plant, and ABC transporters (Fig. 7D). The hub proteins of this module mainly include hypothetical protein Lal_00028479 (MS.gene032452), uncharacterized protein LOC25490008 (MS.gene32840), 40 S ribosomal protein S2-4 (MS.gene026524), and RNA helicase (G7IQH3) (Fig. 7E).

Heat map analysis of proteins related to ion transport and hormone signal transduction

To further analyze the roles of hormone signaling and ion transport-related proteins under drought stress and rehydration treatment, we conducted a heatmap analyses on proteins associated with hormonal signal transduction, as well as inorganic ion transport and metabolic processes. We found that the proteins related to hormone signal transduction could be divided into three distinct subclasses (Table S3 and Fig. 8A). Subclass a proteins exhibited significant up-regulation primarily under rehydration treatment R2 in G3. Subclass b proteins showed marked up-regulation during WL168 rehydration treatment R2. Subclass c proteins were predominantly down-regulated under drought stress conditions in G3, whereas MS.gene008714 and MS.gene006134 demonstrated significant up-regulation under drought stress in WL168, and their change patterns were not obvious during rehydration R1 and R2 treatments in both materials. In addition, the proteins related to inorganic ion transport and metabolism could be divided into four distinct subclasses (Table S4 and Fig. 8B). Subclass a proteins were down-regulated in G3 under drought stress, showed no significant difference during rehydration treatments, but exhibited marked up-regulation in WL168 under R2 treatment. Subclass b proteins demonstrated significant up-regulation primarily in G3 under R2 treatment. Subclass c proteins were predominantly down-regulated in G3 under R2 treatment, with no obvious changes in WL168 under various treatments. Subclass d proteins were mainly significantly up-regulated in WL168 under the R1 treatment, while there were no obvious changes in WL168 under other treatments. Interestingly, within the brown module, we also identified one protein related to plant hormone signal transduction and one protein related to inorganic ion transport and metabolism, namely mitogen-activated protein kinase homolog MMK1 (MS.gene009099) and carbonic anhydrase 2 (MS.gene027637) (Figure S3B and Fig. 8C-D). Within the black module, we identified two proteins related to plant hormone signal transduction and three proteins related to inorganic ion transport and metabolism, which were probable inactive receptor kinase (MS.gene037415), serine/threonine-protein kinase BSK3 (MS.gene27729), glutathione gamma-glutamylcysteinyltransferase 3 isoform X1 (MS.gene008714), carbonic anhydrase (MS.gene018673), and plasma membrane ATPase 4 (MS.gene037008) (Figs. 7E and 8E-I).

Fig. 8. Analysis of the expression of ion and hormone-related proteins. (A) Heatmap analysis of proteins involved in hormone signal transduction; (B) Heatmap analysis of proteins associated with inorganic ion transport and metabolism; (C-D) Expression profiles of representative proteins from the brown module; (E-I) Expression profiles of candidate proteins from the black module

Transcriptional analysis of genes encoding relevant proteins

To evaluate the correlation between mRNA and protein levels, we randomly selected six proteins from the brown module and performed qPCR-based transcriptional analysis on their encoding genes. As shown in Fig. 9, the changing trends of the relative expression levels of six genes (probable fructokinase-4, mannosyl glycoprotein endo-beta-mannosidase, 20 kDa chaperonin, fructose-bisphosphate aldolase 6, probable alpha-mannosidase, MYB11) were consistent with the trends of protein expression abundance under drought stress and rehydration treatments in G3. The changing trends of the relative expression levels of probable fructokinase-4, 20 kDa chaperonin, and MYB11 were opposite to the trends of protein expression abundance under drought stress and R2 treatment in WL168. The differences between the transcriptional levels of these three genes and the abundances of their corresponding proteins indicated that their encoding genes may be influenced by other factors such as post-transcriptional regulation.

Fig. 9. Relative expression levels of the genes encoding six proteins analyzed by qRT-PCR

Discussion

Effect of drought stress on ion content

Dynamic ions and morphological differences of alfalfa under drought stress and rehydration treatment

Drought stress triggers morphological changes across plant tissues, typically leading to poor growth, shorter stature, reduced leaf water content, leaf curling, and enhanced root development. As the primary organ for soil anchoring and water uptake, roots are among the first to adjust under drought conditions [19]. This study found that the plant height, aboveground fresh weight, and underground fresh weight of drought-sensitive materials decreased under drought stress and rebounded after rehydration, but still remained lower than the control. However, the total root length of WL after rehydration was higher than the control. For drought-resistant materials (WL), plant height stagnated under drought and rehydration conditions, while the total root length and underground fresh weight after rehydration were higher than the control. The results indicate that the root system is significantly affected by water stress. Strong drought-resistant materials primarily respond to drought through their roots, while the growth of aboveground parts is less impacted under short-term drought. Overall, drought causes stagnation in plant growth, alters morphological structures, and significantly reduces yield, but rehydration can restore this growth stagnation.

Inorganic ions play a key role in osmotic regulation during plant drought response. Their accumulation enhances plant resistance to water stress through improved ion uptake and related mechanisms [20]. Wang et al. [21] found that compared with the osmolytes or organic solutes accumulated in the cytoplasm. Na^+^ was more effective in alleviating or mitigating the damage caused to plants under high-salt stress and water deficit. In maintaining water balance, its effect was even more obvious than that of K^+^. In this study, variations in K^+^ content and transport capacity across organs and materials suggest that the stem is not the primary organ for drought resistance in alfalfa. The rise in K^+^ levels in WL168 leaves and roots after rehydration indicates a robust post-drought recovery ability, consistent with the findings of Zhang et al. [22]. Under drought stress, Ca^2+^ helps maintain cell membrane integrity by binding to phospholipids, phosphates, and protein carboxyl groups, thereby reducing membrane permeability and enhancing leaf water retention. Additionally, Ca^2+^ participates in drought signal transduction and induces the synthesis of ABA and proline [23]. We analyzed the variation patterns of mineral ions in alfalfa under drought stress and rehydration treatments and their potential mechanisms. The results showed that drought stress significantly affected the contents of mineral ions in different parts (leaves, stems, roots) of alfalfa, while rehydration treatment promoted the recovery of mineral ions to a certain extent, demonstrating the dynamic changes of different mineral ions. These findings align with Bao et al. [6], who reported that transgenic alfalfa accumulated more Na^+^, K^+^, and Ca^2+^ under stress, aiding in the maintenance of water status and ion homeostasis. In addition, the contents of Mn^2+^ and Zn^2+^ in leaves showed a decreasing trend under drought stress and rehydration treatments in both G3 and WL168, indicating that they might be inhibited during the drought-tolerance process of alfalfa. The interaction between drought stress and rehydration may regulate the water and nutrient balance of plants by altering the transmembrane transport and redistribution of ions. In the roots of G3 and WL168, the content of K^+^ decreased significantly under drought stress and the rehydration treatment R1. The contents of Na^+^ and Ca^2+^ decreased significantly under drought stress and gradually recovered to the control level with rehydration. Interestingly, the trend of K^+^/Na^+^ ratios was consistent with that of K^+^. These findings demonstrate that K^+^/Na^+^ can serve as a reliable indicator of plant drought resistance, while also distinguishing plant organs with rapid responses to water changes, making it a valuable marker for monitoring drought-induced physiological alterations in plants. Under mannitol-induced osmotic stress, both K^+^ and Na^+^ efflux/absorption rates changed significantly in all plants. Transgenic lines exhibited higher K^+^ uptake compared to controls. While Na^+^ efflux decreased in all plants under osmotic stress, only transgenic plants achieved net Na^+^ absorption, indicating that WRKY29 overexpression enhances osmotic regulation and drought tolerance in Ammopiptanthus nanus [20]. Fe^3+^ content increased under drought and recovered to control levels after rehydration, suggesting that rehydration effectively restores nutrient absorption and transport systems. Additionally, the increase in Mg^2+^ content in stems and roots suggests its important role in drought stress response, potentially related to antioxidant activity and cellular stability. Changes in Cu^2+^ and Zn^2+^ levels in roots may reflect plant demand for trace elements and regulation of drought resistance mechanisms. This study reveals dynamic ion changes in alfalfa under drought and rehydration conditions, demonstrating regulated ion absorption and redistribution. Variations in Na^+^, K^+^, and Ca^2+^ appear closely associated with mechanisms maintaining cellular homeostasis and water balance during drought stress. Rehydration facilitated recovery of most ion concentrations, particularly K^+^ and Ca^2+^, supporting theoretical understanding of alfalfa regrowth under drought conditions. Future research should further explore specific roles of ions in drought resistance physiology.

Effects of drought stress on ion transport capacity in different tissues of alfalfa seedlings

Drought severely affects the nutritional relationships of plants. Under drought conditions, the reduced water availability usually leads to decreased nutrient uptake by plants and lower tissue concentrations. Ions such as Ca^2+^ and Mg^2+^ are absorbed by plant roots along with water and transported to the above-ground parts. However, under drought conditions, the transpiration of plants is weakened, and the diffusion and movement of water and the ions within it are restricted, thus hindering plant growth [24, 25]. While mineral ion uptake varies by species and genotype, drought generally reduces the absorption of essential ions including K^+^, Ca^2+^, Cu^+^, Zn^+^, Mg^+^, and Mn^+^, thereby disrupting numerous physiological processes [5]. This study demonstrates that drought significantly alters ion accumulation and transport capabilities across root, stem, and leaf tissues in alfalfa seedlings, with distinct varietal response patterns. Under drought stress, the roots of G3 accumulated Na^+^, and the root’s transport capabilities for Ca^2+^ and Mn^2+^ were enhanced. The roots of WL168 also accumulated Na^+^, promoting an increase in the transport capabilities of K^+^ and Ca^2+^ to the stems. This may be a way for alfalfa to cope with drought conditions by regulating ion flow in the roots, reducing water loss and the loss of mineral ions. Rehydration treatment has a certain promoting effect on the restoration of the ion transport capabilities of alfalfa varieties with weak drought resistance. The transport capabilities of ions such as K^+^, Mg^2+^, Cu^2+^, Fe^2+^, and Zn^2+^ were significantly improved in G3, this phenomenon may be due to the fact that the rehydration process promoted the re-absorption and transportation of water and mineral ions, helping plants to restore normal physiological functions, especially for the transport of some key ions. In addition, the differences in ion transport capacity among different varieties under drought stress may be closely related to their genotypes and adaptive strategies. The G3 variety may enhance its drought tolerance to some extent by regulating the absorption and transportation of Ca^2+^ and Mn^2+^ in the roots, while the WL168 variety may do so through the absorption and transportation of K^+^ and Ca^2+^. These findings indicate that ion transport capacity serves as an important indicator of water use efficiency and overall stress resistance in alfalfa.

Response of endogenous hormones to drought stress

This study investigated dynamic changes in endogenous hormones (ZT, GA_3_, IAA, ABA, and SA) in alfalfa leaves and roots under drought stress and rehydration. These hormones displayed significant fluctuations, indicating their crucial regulatory roles in drought adaptation and recovery processes. CTKs can delay senescence, over-expressing the ipt gene involved in its biosynthesis can lead to an increase in endogenous CTK content, thereby enhancing drought tolerance by delaying drought-induced senescence [13]. At the same time, CTKs are negative regulators of root growth and branching, and their specific degradation in roots can promote the growth and branching of the main root, thus improving drought tolerance [26]. Chen et al. [27] reported that the increase in cytokinin levels promoted the growth of cotton roots, thereby enhancing its drought tolerance. In the leaves of WL168, the content of ZT decreased significantly under drought stress and rehydration treatments, which may imply that it played a negative regulatory role in regulating the drought recovery process. GA_3_, as a hormone that promotes plant growth and development, also plays a central role in regulating drought stress [28]. GA_3_ content increased significantly under drought stress in both G3 and WL168, particularly in WL168 roots, aligning with its recognized role in promoting plant stress responses [29]. However, after rehydration, the content of GA_3_ decreased significantly, indicating that plants may regulate GA_3_ to restore normal physiological states during the recovery stage. IAA is a key hormone for plant cell growth, leaf expansion, organogenesis and other life activities [14]. Transcriptome data indicated that some drought-responsive genes in Arabidopsis thaliana were regulated by IAA, and studies in plants such as rice (Oryza sativa) and potato (Solanum tuberosum) also found that changes in auxin balance affected the drought tolerance of plants [30, 31]. Under drought stress, the content of IAA in both G3 and WL168 showed a significant increasing trend, which was consistent with the characteristic of IAA as a growth hormone and may help plants maintain growth under drought conditions. However, after rehydration, IAA levels rapidly returned to control values, indicating its involvement in both drought adaptation and recovery processes. ABA, as the primary hormone mediating plant responses to water stress, helps reduce water loss through regulating stomatal closure and root water uptake. Drought stress triggers ABA synthesis and accumulation, leading to stomatal closure, growth inhibition, and activation of transcription factors that induce downstream drought-responsive genes, thereby enhancing plant adaptation to water deficit [11, 12]. ABA also participates in expression pathways regulated by transcription factors including MYB (AtMYB2), MYC (AtMYC2), and NAC (AtRD26) [32, 33]. Under drought stress, ABA content significantly increased in both G3 and WL168. During rehydration, tissue-specific responses were observed: in leaves, ABA levels continued to rise under R1 treatment but decreased under R2 treatment, remaining higher than control in G3 while falling below control in WL168. In roots, ABA concentrations declined after rewatering, with both varieties showing lower levels under R2 treatment compared to the control. These differential responses between roots and leaves indicate distinct temporal patterns in water stress perception and recovery. ABA enhances plant hydraulic conductivity and promotes root cell elongation, improving survival under severe water deficit [34]. Overall, ABA played a complex regulatory role in the drought recovery process, which may be related to the plant’s gradual adaptation to water restoration. SA regulates growth and stress responses, including drought tolerance. Exogenous SA application increases biomass, leaf water content, and solute accumulation in drought-stressed wheat [35]. During drought stress, the content of SA increased significantly in both the roots and leaves of G3 and WL168. During the rehydration process, the content of SA gradually decreased, indicating that it may play a key regulatory role in both the drought stress and rehydration recovery stages. In addition, in the roots, the response patterns of these hormones were similar to those in the leaves. The content of ZT in the roots increased after drought stress, suggesting that ZT may promote plant adaptation during the drought recovery process by regulating root growth. GA_3_, IAA, and ABA all showed initial increases followed by decreases during drought and rehydration, reflecting root sensitivity to water status changes. These hormonal interactions appear crucial for both drought adaptation and rehydration recovery, warranting further investigation into their specific roles and crosstalk in alfalfa drought tolerance.

Effect of drought stress on protein function

Analysis of the metabolic pathways under drought stress

Through biological pathway enrichment analysis of DEPs under drought and rehydration treatments, this study revealed distinct metabolic regulation patterns during stress and recovery phases. The results showed that drought stress mainly responds to drought stress by regulating metabolic pathways such as phenylpropanoid biosynthesis, glycolysis/gluconeogenesis, and starch and sucrose metabolism. In contrast, the rehydration treatment was more involved in the regulation of pathways such as carbon metabolism, amino acid biosynthesis, and phenylpropanoid biosynthesis to restore normal growth. These adjustments reflect optimized energy allocation and defense mechanisms during drought, followed by metabolic rebalancing during recovery. As key plant defense compounds, phenylpropanoids-including hormones, lignin, flavonoids, and phenolic acids-likely provide crucial antioxidant protection against drought-induced oxidative damage [36].

The phenylpropanoid pathway often mediates the biosynthesis of various drought-resistant secondary metabolites by maintaining high levels of POD activity, thereby conferring drought resistance to the root system and enabling it to withstand drought stress [37]. Notably, phenylpropanoid biosynthesis exhibited significant alterations under both drought and rehydration conditions. Previous studies have identified differential gene expression in phenylpropanoid and flavonoid biosynthesis as a key mechanism for enhancing plant drought tolerance. For instance, MYB family transcription factors can significantly induce the synthesis of phenylpropanoid metabolites, thereby improving plant drought tolerance [38]. The activation of glycolysis/gluconeogenesis pathways indicates plants utilize carbohydrate metabolism to maintain energy supply under water deficit conditions [39, 40]. Carbohydrate metabolism plays a central role in plant metabolism, providing energy for the normal growth and development of plants and serving as a bridge in the communication among proteins, lipids, and metabolism [41]. The regulation of metabolites in the amino acid biosynthesis pathway also plays a crucial role in the survival of plants under drought stress [39]. Amino acid metabolism is essential for various cellular processes, such as defense and signal transduction [42]. Metabolic patterns during rehydration differed markedly from those under drought stress, with carbon metabolism and amino acid biosynthesis being significantly activated. Following rewatering, plants require rapid restoration of physiological functions. The reactivation of these pathways demonstrates that plants reestablish growth and repair mechanisms through amino acid synthesis and carbon network reconstruction, indicating a precisely regulated recovery process beyond simple water replenishment. In addition, the enrichment of cysteine and methionine metabolism and endocytosis in WL168 under R2 treatment may be closely related to the antioxidant protection of plant cells and the activation of signal transduction pathways, contributing to the rapid restoration of cell functions and the regulation of stress responses. Particularly, in the comparison between WL168 and G3, it was found that WL168 showed significant up-regulation in pathways such as arginine and proline metabolism, glycerolipid metabolism, and glutathione metabolism. The activation of these pathways was closely related to the water-protection mechanisms of plants under drought stress. Research has shown that proline and arginine are key metabolites for plants to cope with water stress, regulating the osmotic potential of cells and protecting cells from the effects of water deficiency [43, 44]. The involvement of glycerolipids and glutathione helps to enhance the plant’s tolerance and protection against oxidative stress [45, 46]. During the rehydration process, the restoration of these metabolic pathways in WL168 further emphasizes the metabolic adaptability of plants in response to drought and rehydration.

WGCNA analysis of the protein expression profiles under drought stress

Plant response to drought at the molecular level is a complex process involving stress perception, signal transduction, and transcriptional regulation [47]. Through gene module enrichment analysis under drought stress, this study revealed characteristic metabolic pathway responses. KEGG analysis showed that proteins in the brown module were primarily enriched in metabolic pathways, secondary metabolite biosynthesis, carbon metabolism, TCA cycle, and amino acid synthesis. As key energy metabolic pathways, the regulation of glycolysis and TCA cycle may help plants optimize energy synthesis and utilization under water deficit [48]. This study also found that the core proteins in the brown module mainly include probable fructokinase-4, fructose-bisphosphate aldolase 6, pyruvate dehydrogenase E2 component, General regulatory factor 2, threonine synthase, glutaredoxin-dependent peroxiredoxin, and MYB11. Interestingly, our qPCR analysis revealed distinct expression patterns of probable fructokinase-4, fructose-bisphosphate aldolase 6, and MYB11 during drought-rehydration cycles compared to controls, suggesting their involvement in alfalfa’s drought recovery process. Notably, the differential expression of probable fructokinase-4 and fructose-bisphosphate aldolase 6 between the two accessions may partially explain WL168’s superior drought tolerance. The enrichment of these proteins reflects plants’ strategy to maintain cellular functions through metabolic adjustments under drought stress. For instance, MYB-type transcription factors play crucial roles in regulating plant responses to environmental stresses, potentially enhancing stress resistance by activating specific response proteins [49, 50]. Proteins in the pink module were mainly enriched in metabolic pathways, secondary metabolite biosynthesis, carbon metabolism, the pentose phosphate pathway, the citric acid cycle (TCA cycle), glycolysis/gluconeogenesis, and glutathione metabolism. The enrichment of the glutathione metabolism pathway indicated that the proteins in this module may be involved in enhancing the plant’s antioxidant defense mechanism, which helped to alleviate oxidative damage caused by drought [51]. Proteins in the black module were associated with aminoacyl-tRNA biosynthesis, nucleocytoplasmic transport, plant hormone signal transduction, phagosome, MAPK signaling pathway in plants, and ABC transporters. These pathways indicate this module may optimize plant physiological states during drought through regulating protein synthesis, hormone signaling, and cellular signal transduction. The MAPK signaling pathway particularly contributes to drought adaptation by regulating cellular adaptive responses [52].

Ion transport and hormone-related proteins are involved in drought regulation in alfalfa

The MAPK cascade is a crucial cellular signal transduction mechanism that plays a significant role in plant growth, development, metabolism, and stress responses [53]. For instance, the silencing of GhMAPK3 can reduce the tolerance of cotton (Gossypium hirsutum) to salt and drought stresses [54]. When exogenous ABA was used to simulate drought stress, the activity of carbonic anhydrase (CA) in barley plants increased by more than two-fold [55]. GST, a key enzyme in glutathione metabolism, functions as a multifunctional antioxidant and plays important roles in plant development and stress resistance [51]. Previous studies have found that compared with drought-sensitive varieties, drought-resistant varieties can maintain greater expansin activity and cell-wall extension, which helps them grow more rapidly under water stress [56]. In Arabidopsis, BSK proteins interact directly with BRI1 to regulate brassinosteroid signaling and plant development [57]. Based on these findings, we propose that several identified proteins-including mitogen-activated protein kinase MMK1, carbonic anhydrase 2, probable inactive receptor kinase, serine/threonine-protein kinase BSK3, glutathione transferase, and plasma membrane ATPase 4-may participate in alfalfa’s drought response and rehydration recovery. These represent important candidate genes for further investigation into alfalfa’s adaptation to drought stress.

Conclusion

In this study, the ion homeostasis, endogenous hormone levels, and protein expression profiles of two alfalfa varieties with different drought resistances were analyzed under drought stress and rehydration treatments. The results showed that the ability of alfalfa to maintain cell homeostasis under drought stress was mainly related to the dynamic changes of ions such as Na^+^, K^+^, and Ca^2+^, and rehydration treatment helped to restore the normal levels of most ions. There were differences in the expression levels of endogenous hormones ZT, GA_3_, IAA, ABA, and SA in the leaves at different treatment stages of materials with different drought resistances, but the contents of GA_3_, IAA, ABA, and SA increased significantly under drought stress in both materials and showed a decreasing trend under rehydration treatment. KEGG enrichment analysis of DEPs indicated that pathways such as phenylpropanoid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, glutathione metabolism, and amino acid biosynthesis responded to drought stress. Further combined with WGCNA analysis, it was shown that proteins including MMK1, BSK3, carbonic anhydrase, and MYB11 could be potential candidate genes for alfalfa drought resistance.

Materials and methods

Plant varieties and drought treatment

Based on preliminary experimental results, we selected the drought-resistant alfalfa variety ‘WL168’ and drought-sensitive ‘Gannong No. 3’ [17]. Each variety was cultivated in artificial climate chambers with a mixture of field soil/vermiculite (V: V = 2:1). Each treatment was planted with 5 pots (diameter: height = 130 cm: 120 cm) and 10 plants in each pot. The temperature was kept at 25 ± 2 ℃ for 16 h in the daytime and 22 ± 2 ℃ for 8 h at night. After emerging, alfalfa seedlings were watered every three days using the weighing method to maintain a water content in each pot of soil at 70–80%. The experimental treatments were started after 30 days of growth. Alfalfa seedlings were divided into four groups: (1) Control: plants received regular watering (CK); (2) Drought stress: plants grown without watering for 7 d before sampling (DS); (3) Rewater for 1 d: plants grown without irrigation during the first 7 d and rewatering 1 d before sampling (R1); (4) Rewater for 5 d: plants grown without irrigation during the first 7 d and rewatering 5 d before sampling (R2). The roots of alfalfa seedlings from each treatment were sampled, immediately frozen in liquid nitrogen, and stored at −80℃ for further analysis.

Determination of ion content and endogenous hormones

The ion content in the seedling roots, stems and leaves of G3 and WL168 under drought stress and rehydration (R1 and R2) treatments was determined by atomic absorption spectrometry (AAS). The specific steps were as follows: The alfalfa root samples at different time points of drought-stress treatment were first fixed at 105 ℃ and then dried to a constant weight at 75 ℃. After the dried samples were ground into fine powder, digestion was carried out using the H₂SO₄-H₂O₂ method in a graphite digestion instrument (SH220F). First, accurately weigh 0.50 g of the dry weight of each sample was into a digestion tube. Then, added 5 mL of concentrated sulfuric acid and 2 mL of 30% hydrogen peroxide to each digestion tube. Digesting the mixture on a graphite digestion apparatus until a clear liquid was obtained. Transferred the cooled clear liquid into a 50 mL volumetric flask, diluted it with deionized water and made up to the mark for standby use. Finally, using an atomic absorption spectrophotometer (TAS-990Super, Beijing Purkinje General Instrument Co., Ltd., Sichuan) to measure the absorbance values of K^+^, Na^+^, Ca^2+^, Mg^2+^, Fe^3+^, Cu^2+^, Mn^2+^ and Zn^2+^. The calculation of ion selective transport capacity adopted the method of Yang et al. [58]. The calculation formula was: S_X, Na_ = sink organ [X/Na^+^]/source organ [X/Na^+^], X represents the content of any one of the ions such as K^+^, Ca^2+^, Mg^2+^, Fe^3+^, Cu^2+^, Mn^2+^ and Zn^2+^. The larger the value of S_X, Na_, the stronger the ability of the source organ to accumulate Na^+^ and promote the transport of X to the sink organ, that was, the stronger the ability of the sink organ to selectively transport X.

The endogenous hormones (IAA, ABA, CTK, GA_3_ and SA) in the seedling roots and leaves of G3 and WL168 under drought stress and rehydration treatments R1 and R2 were determined by high-performance liquid chromatography (HPLC) [59]. First, 0.20 g root and leaf samples of the alfalfa under each treatment were weighed, cut into small pieces, and then the endogenous hormones were extracted using a methanol-water solution (V: V = 80: 20). The extract was concentrated to dryness by a rotary evaporator, and then an appropriate amount of mobile phase was added to dissolve the sample. The separation was carried out using HPLC, and the wavelength was set at 254 nm or other suitable wavelengths for hormone absorption. The concentrations of hormones in the samples were calculated by comparing with the standard hormone solutions of known concentrations.

Total protein extraction, polypeptide preparation, and library construction

Total proteins of all samples were extracted using the phenolic-based method described by Wang et al. [60]. The extracted protein concentrations were measured by Bradford protein analysis [61]. Peptide preparation and library construction were performed as described by Wang et al. [60]. Briefly, 50 µg of protein was extracted from each sample for reduction. Enzyme release salt, peptide dry powder dissolved in 0.1% FA formic acid water, and iRT standard peptide were added after two incubations. 1 µg of sample was then injected for liquid mass detection using Q Exactive HF-X mass spectrometer with a NanosprayFlex™ (ESI) ion source. Then, GO and InterPro (IPR) were analyzed using the interproscan-5 program, and protein families and pathways were analyzed using COG (homologous group cluster) and KEGG databases. A STRING-db server was used to predict possible interaction partners (http://string.embl.de/) according to related species. Enrichment analysis was performed separately for GO and KEGG using the enrichment pipeline [62]. The spectral map library was built using the DDA and DIA data. Proteomics data were stored in ProteomeXchange database with the accession number of PXD051465. Selected DEPs were considered up-regulated when FC ≥ 1.50 and P-value ≤ 0.05 and down-regulated when FC ≤ 1/1.50 and P-value ≤ 0.05.

Weighted Gene Co-expression Network Analysis (WGCNA)

A gene co-expression network was constructed using the expression data of 24 alfalfa root samples. The thresholds were established as follows: an average gene expression FPKM value ≥ 20, a minimum number of modules of 20, and a maximum number of modules of 50. The gene expression adjacency matrix was constructed and used for the network topology analysis. A module correlation analysis was performed using the module feature values and all physiological data determined in this study, and the correlation coefficients between the physiological data and the gene module features were calculated using the Pearson correlation method. The correlation heatmap was then drawn.

Gene expression analysis by quantitative real-time PCR

To enhance the reliability of Proteomics data validation, the total RNA used for library construction was reverse transcribed and cDNA synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA, Japan). Specific primers for 6 DEGs were designed using Primer-BLAST on NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast) (Table S5). RT-qPCR amplification was performed on LightCycler 96 (Roche, Basel, Switzerland) using super real premix plus (SYBR Green) (Tiangen, Shanghai, China). The amplification process followed the procedure of Chen et al. [63]. The relative transcription levels of the genes encoding the selected proteins were calculated with the 2^−ΔΔCT^ method and normalized to the expression levels of the MsActin gene.

Data statistics and analysis methods

The physiological and biochemical indices and RT-qPCR data of two alfalfa varieties were organized using Microsoft Excel 2016. These data were subjected to one-way ANOVA using IBM SPSS Statistics Software 22.0 (Armonk, USA), with the significance level defined as P < 0.05. Graphs were generated using GraphPad Prism 8 (GraphPad Software, USA).

Supplementary Information

Supplementary Material 1.

Supplementary Material 2.

Supplementary Material 3.

Supplementary Material 4.

Supplementary Material 5.

Supplementary Material 6.