The proteome responses to heat stress have not been well understood. In this study, alfalfa (Medicago sativa L. cv. Huaiyin) seedlings were exposed to 25°C (control) and 40°C (heat stress) in growth chambers, and leaves were collected at 24, 48 and 72 h after treatment, respectively. The morphological, physiological and proteomic processes were negatively affected under heat stress. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and differentially expressed protein spots were identified by mass spectrometry (MS). Totally, 81 differentially expressed proteins were identified successfully by MALDI-TOF/TOF. These proteins were categorized into nine classes: including metabolism, energy, protein synthesis, protein destination/storage, transporters, intracellular traffic, cell structure, signal transduction and disease/defence. Five proteins were further analyzed for mRNA levels. The results of the proteomics analyses provide a better understanding of the molecular basis of heat-stress responses in alfalfa.

Medicago polymorpha is a nutritious and palatable forage and vegetable plant that also fixes nitrogen. Here, we reveal the chromosome-scale genome sequence of M. polymorpha using an integrated approach including Illumina, PacBio and Hi-C technologies. We combined PacBio full-length RNA-seq, metabolomic analysis, structural anatomy analysis and related physiological indexes to elucidate the important agronomic traits of M. polymorpha for forage and vegetable usage. The assembled M. polymorpha genome consisted of 457.53 Mb with a long scaffold N50 of 57.72 Mb, and 92.92% (441.83 Mb) of the assembly was assigned to seven pseudochromosomes. Comparative genomic analysis revealed that expansion and contraction of the photosynthesis and lignin biosynthetic gene families, respectively, led to enhancement of nutritious compounds and reduced lignin biosynthesis in M. polymorpha. In addition, we found that several positively selected nitrogen metabolism-related genes were responsible for crude protein biosynthesis. Notably, the metabolomic results revealed that a large number of flavonoids, vitamins, alkaloids, and terpenoids were enriched in M. polymorpha. These results imply that the decreased lignin content but relatively high nutrient content of M. polymorpha enhance its edibility and nutritional value as a forage and vegetable. Our genomic data provide a genetic basis that will accelerate functional genomic and breeding research on M. polymorpha as well as other Medicago and legume plants.

Plant leaf patterns and shapes are spectacularly diverse. Changing the complexity of leaflet numbers is a valuable approach to increase its nutrition and photosynthesis. Alfalfa (Medicago sativa) is the most important forage legume species and has diversified compound leaf patterns, which makes it a model species for studying compound leaf development. However, transcriptomic information from alfalfa remains limited. In this study, RNA-Seq technology was used to identify 3746 differentially expressed genes (DEGs) between multifoliate and trifoliate alfalfa. Through an analysis of annotation information and expression data, SPL, one of the key regulators in modifiable plant development and abiotic stress response, was further analyzed. Here, thirty MsSPL genes were obtained from the alfalfa genome, of which 16 had the putative miR156 binding site. A tissue expression pattern analysis showed that the miR156-targeted MsSPLs were divided into two classes, namely, either tissue-specific or widely expressed in all tissues. All miR156-targeted SPLs strongly showed diversification and positive roles under drought and salt conditions. Importantly, miR156/MsSPL08 was significantly suppressed in multifoliate alfalfa. Furthermore, in the paralogous mutant of MsSPL08 isolated from Medicago truncatula, the phenotypes of mutant plants reveal that miR156/MsSPL08 is involved not only involved the branches but also especially regulates the number of leaflets. The legume is a typical compound leaf plant; the ratio of the leaflet often affects the quality of the forage. This study sheds light on new functions of SPL genes that regulate leaflet number development.

Breeding improvement in alfalfa (Medicago sativa L.) is often constrained by the complexity of agronomic traits and trade-offs among yield-related characteristics. Conventional single-trait selection rarely captures the full range of phenotypic variation or the interactions among traits. To address this, we developed a principal component analysis (PCA)-based framework for multivariate selection in hybrid breeding. Six yield-related traits—plant height, branch number, fresh/hay yield ratio (FHR), leaf/stem ratio (LSR), multifoliolate leaf frequency, and dry weight per plant—were quantified in two parental lines and their F₁/F₂ generations. PCA identified three principal components (PC1–PC3) with eigenvalues >1, explaining 71.14% of the total phenotypic variance: PC1 (32.43% variance) was predominantly loaded with positive contributions from dry weight per single plant, height, and branches, biologically representing overall plant vigor and biomass accumulation; PC2 (21.77% variance) showed strong negative loadings for LSR, capturing architectural trade-offs between stem dominance and leaf production; PC3 (16.94% variance) had positive loadings on multifoliolate leaf rate and fresh/dry ratio, embodying quality and physiological resilience traits. Based on PCA scores, a composite selection index was constructed, and the top 31.1% of F1 hybrids were selected. Their F₂ progeny showed significant improvements in dry weight (+15.56%, p < 0.01), multifoliolate leaf frequency (+74.78%, p < 0.001), and reduced FHR (–8.2%, p < 0.05), accompanied by lower yield decline (−7.2% versus −14.1% in controls). These results show that PCA-based multivariate selection effectively balances trait trade-offs, enhances intergenerational stability, and improves selection efficiency. This framework offers a practical tool for alfalfa breeding.

Wenling Medic (Medicago polymorpha) is common in southern China and has long been utilized as a vegetable in eastern China, as well as a significant raw ingredient for livestock and pickled meals. As a legume, there is still a research vacuum, and the first problem Wenling Medic faces in production is the problem of seed germination. The germination percentage of Wenling Medic was low, according to production practice and laboratory tests performed in this work. Furthermore, after more than two years of storage, the germination percentage of Wenling Medic dropped sharply, and it lacked a long enough seed life. An attempt was made to restore the viability of the seeds using the polyethylene glycol (PEG) osmoregulation-mediated priming method, after the physiological and biochemical characteristics of the seeds were assessed using the anthrone method, Bradford assay, 3,5-dinitrosalicylic acid (DNS) method, and thiobarbituric acid (TBA) method. The findings showed that it has a relatively short storage age and, with a useable life of only two years under normal aging circumstances, is not deactivated soon after harvesting. In addition, whereas protein content and α-amylase concentration did not significantly correlate (p > 0.05) with storage life, the conductivity of exudate, malondialdehyde content, and carbohydrate content did (p < 0.05). The seed viability was not considerably increased by the saturation initiation mediated by osmoregulation, utilizing PEG. In conclusion, the decrease in Wenling Medic seeds’ germination ability was substantially connected with higher levels of lipid peroxidation and decreased carbohydrate levels, but not with protein concentrations or α-amylase activity. The timing of dehydration may need to be carefully controlled when using PEG osmoregulation to prime Wenling Medic seeds.