Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar “XinjiangDaye”) reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I–EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa (Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf–stem ratio (LSR) and repression of several lignin-synthesis genes (MsNST, MsPAL1, MsC4H, and Ms4CL). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage.

Alfalfa (M. sativa), a perennial legume forage, is known for its high yield and good quality. As a long-day plant, it is sensitive to changes in the day length, which affects the flowering time and plant growth, and limits alfalfa yield. Photoperiod-mediated delayed flowering in alfalfa helps to extend the vegetative growth period and increase the yield. We isolated a blue-light phytohormone gene from the alfalfa genome that is an ortholog of soybean FKF1 and named it MsFKF1. Gene expression analyses showed that MsFKF1 responds to blue light and the circadian clock in alfalfa. We found that MsFKF1 regulates the flowering time through the plant circadian clock pathway by inhibiting the transcription of E1 and COL, thus suppressing FLOWERING LOCUS T a1 (FTa1) transcription. In addition, transgenic lines exhibited higher plant height and accumulated more biomass in comparison to wild-type plants. However, the increased fiber (NDF and ADF) and lignin content also led to a reduction in the digestibility of the forage. The key genes related to GA biosynthesis, GA20OX1, increased in the transgenic lines, while GA2OX1 decreased for the inactive GA transformation. These findings offer novel insights on the function of MsFKF1 in the regulation of the flowering time and plant height in cultivated M. sativa. These insights into MsFKF1’s roles in alfalfa offer potential strategies for molecular breeding aimed at optimizing flowering time and biomass yield.

JASMONATE ZIM domain (JAZ) proteins, inhibitors of the jasmonic acid (JA) signaling pathway, are identified in different plants, such as rice and Arabidopsis. These proteins are crucial for growth, development, and abiotic stress responses. However, limited information is available regarding the JAZ family in alfalfa. This study identified 11 JAZ genes (MsJAZs) in the “Zhongmu No.1” reference genome of alfalfa. The physical and chemical properties, chromosome localization, phylogenetic relationships, gene structure, cis-acting elements, and collinearity of the 11 MsJAZ genes were subsequently analyzed. Tissue-specific analysis revealed distinct functions of different MsJAZ genes in growth and development. The expression patterns of MsJAZ genes under salt stress conditions were validated using qRT-PCR. All MsJAZ genes responded to salt stress, with varying levels of upregulation over time, highlighting their role in stress responses. Furthermore, heterogeneous expression of MsJAZ1 in Arabidopsis resulted in significantly lower seed germination and survival rates in OE-2 and OE-4 compared to the WT under 150 mM NaCl treatment. This study establishes a foundation for further exploration of the function of the JAZ family and provides significant insights into the genetic improvement of alfalfa.