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Medicago sativa is a globally cultivated forage crop, and improving its digestibility and stress tolerance remains a central breeding goal. The Cellulose synthase ( CESA ) superfamily is central to cell wall biosynthesis and influences plant development, stress responses, and forage quality; however, its evolutionary trajectory in Medicago is poorly resolved. Here, we identified 431 CESA superfamily genes from five Medicago species and four outgroup taxa. Phylogenetic analyses classified these genes into eight clades, including CesA and CslA / B / C / D / E / G / M subfamilies. Gene duplication analyses revealed that both tandem duplication and whole genome duplication (WGD) drove superfamily expansion, with a WGD event occurring in the common ancestor of Papilionooideae (PWGD). Tandem duplication plays a more prominent role than the PWGD-derived duplication. Transcriptomic analyses demonstrated that CESA genes in M. sativa subsp. sativa are broadly involved in development and respond strongly to salt and drought stresses. Notably, loss of CesA gene copies and biased retention of duplicated genes were observed in the MsCslB , MsCslA , MsCslE , and MsCslD subfamilies. Among tandem duplicates, MsCslB5.1 and MsCslB10.1 exhibited pronounced stress-responsive expression. These patterns may reflect domestication-related selection for improved forage quality and stress tolerance. Together, our findings establish a comprehensive evolutionary framework for the CESA superfamily and provide candidate genes with potential value for molecular breeding in Medicago forage crops.

Key message All ten dehydrin genes from three Medicago species are responsive to different kinds of abiotic stress, and CAS31 confers transgenic plants salt tolerance by down-regulating HKT1 expression. Dehydrins are protective proteins playing crucial roles in the tolerance of plants to abiotic stresses. However, a full-scale and systemic analysis of total dehydrin genes in Medicago at the genome level is still lacking. In this study, we identified ten dehydrin genes from three Medicago species ( M . truncatula , M . ruthenica , and M . sativa ), categorizing the coding proteins into four types. Genome collinearity analysis among the three Medicago species revealed six orthologous gene pairs. Promoter regions of dehydrin genes contained various phytohormone- and stress-related cis -elements, and transcriptome analysis showed up-regulation of all ten dehydrin genes under different stress conditions. Transformation of dehydrin gene CAS31 increased the tolerance of transgenic seedlings compared with wild-type seedlings under salt stress. Our study demonstrated that transgenic seedlings maintained the more chlorophyll, accumulated more proline and less hydrogen peroxide and malondialdehyde than wild-type seedlings under salt stress. Further study revealed that CAS31 reduced Na + accumulation by down-regulating HKT1 expression under salt stress. These findings enhance our understanding of the dehydrin gene family in three Medicago species and provide insights into their mechanisms of tolerance.

Understanding the cellular mechanisms of plant tolerance to mercury (Hg) is important for developing phytoremediation strategies of Hg-contaminated soils. The early responses of alfalfa (Medicago sativa) seedlings to Hg were studied using transcriptomics analysis. A Medicago truncatula microarray was hybridized with high-quality root RNA from M. sativa treated with 3 μM Hg for 3, 6 and 24 h. The transcriptional pattern data were complementary to the measurements of root growth inhibition, lipid peroxidation, hydrogen peroxide (H2O2) accumulation and NADPH-oxidase activity as stress indexes. Of 559 differentially expressed genes (DEGs), 91% were up-regulated. The majority of DEGs were shared between the 3 and 6 h (60%) time points, including the ‘stress’, ‘secondary metabolism’ and ‘hormone metabolism’ functional categories. Genes from ethylene metabolism and signalling were highly represented, suggesting that this phytohormone may be relevant for metal perception and homeostasis. Ethylene-insensitive alfalfa seedlings preincubated with the ethylene signalling inhibitor 1-methylcyclopronene and Arabidopsis thaliana ein2-5 mutants confirmed that ethylene participates in the early perception of Hg stress. It modulates root growth inhibition, NADPH-oxidase activity and Hg-induced apoplastic H2O2 accumulation. Therefore, ethylene signalling attenuation could be useful in future phytotechnological applications to ameliorate stress symptoms in Hg-polluted plants.

Background/Objectives: Alfalfa is a widely cultivated high-quality forage crop, and salinity tolerance is one of the most important breeding goals. Glycine max SALT INDUCED NAC 1 (GmSIN1) was found to enhance salinity tolerance in soybean plants. The phylogenetic analysis showed there were two homologs of GmSIN1 in Medicago truncatula, MtSIN1a and MtSIN1b. This raised questions regarding the roles of MtSIN1s in alfalfa under salinity stress. Methods: From a Tnt1 mutant collection, we identified the mutants of MtSIN1a. We recorded the survival rate and plant height of mtsin1a-1 and mtsin1a-2 after 100 mM NaCl treatment. Subsequently, we generated 35S:MtSIN1a-GFP transgenic alfalfa lines via genetic transformation. Two lines with relatively high MtSIN1a expression, 35S:MtSIN1a-GFP#3 and 35S:MtSIN1a-GFP#4, were selected for gradient NaCl treatments. In addition, DAB and NBT staining were performed, and the H2O2 content and catalase (CAT) activity were determined. Then, we used RNA-seq analysis and RT-qPCR to study the mechanism of its tolerance. Results: This study found that after salt treatment, the survival rate and plant height of mtsin1a-1 and mtsin1a-2 were significantly lower than those of the WT. The mutants of MtSIN1a were sensitive to salinity stress. The transgenic alfalfa plants exhibited higher plant height, weaker DAB staining, stronger NBT staining, less H2O2 content, and enhanced CAT activity. The transgenic alfalfa constructed by transforming MtSIN1a showed enhanced salinity tolerance with elevated ROS scavenging. We identified MsSOD1 showing elevated expression levels in transcriptomic analysis. Conclusions: MtSIN1a is a positive regulator for enhancing salinity tolerance in alfalfa with activated ROS scavenging.

Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula . CO perception is a function of the receptor-like kinases Mt CERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment. Polysaccharide molecules chitooligosaccharides (COs) and peptidoglycan not only activate plant immunity but also trigger plant symbiosis signalling. Here the authors show that a combination of COs and lipochitooligosaccharides (LCOs) act synergistically to suppress immunity and promote symbiosis to facilitate beneficial fungal associations.

Medicago falcata is a legume species that exhibits great capacity of tolerance to abiotic stresses. To elucidate the mechanism underlying tolerance of M. falcata to freezing, we compared the characteristics of M. falcata in response to cold acclimation and freezing with those of the legume model plant Medicago truncatula. M. falcata seedlings were more tolerant to freezing than M. truncatula, as evidenced by a lower value of EL50 (temperature at which 50% electrolyte leakage after freezing) and greater survival rate for M. falcata than M. truncatula. Cold acclimation led to greater reduction in EL50 for M. falcata than M. truncatula. Sucrose was the most abundant sugar in both M. falcta and M. truncatula, and a greater accumulation of sucrose and Pro in M. falcata than in M. truncatula during cold acclimation was observed. Cold acclimation induced small amounts of raffinose and stachyose in M. falcata, but not in M. truncatula. The activities of sucrose phosphate synthase and sucrose synthase were greater in M. falcata than in M. truncatula. In contrast, the activity of acid invertase was higher in M. truncatula than in M. falcata. There was an increase in transcript of CRT binding factor (CBF) upon exposure to low temperature in the two species. The low temperature-induced increase in transcript of CBF2 was much higher in M. truncatula than in M. falcata, while transcript of CBF3 in M. falcata was greater than that in M. truncatula. There were sustained increases in transcripts of cold acclimation specific (CAS), a downstream target of CBF, during cold acclimation and the increases were greater in M. falcata than in M. truncatula. These results demonstrate that accumulation of greater amounts of soluble sugars coupled with higher CBF3 and CAS transcript levels in M. falcata may play a role in conferring greater tolerance of M. falcata to freezing than that of M. truncatula.

Background Salt-alkali stress is an abiotic stress that inhibits crop growth and reduces yield. It significantly affects various physiological processes in plants, including photosynthesis, osmotic regulation, and antioxidant defense. However, studies on the transcriptional response mechanisms of Medicago falcata L. under salt-alkali stress are limited. In this study, RNA-seq technology was used to analyze differentially expressed genes (DEGs) in salt-alkali tolerant M.falcata breeding lines (LM18) and the salt-alkali sensitive Hulunbeier (HL) under salt-alkali stress. Furthermore, physiological indicators such as chlorophyll content, proline accumulation, and superoxide dismutase (SOD) activity were assessed to compare the responses of LM18 and HL to salt-alkali stress. By integrating transcriptomic and physiological analyses, this study provides new insights into the physiological and molecular regulatory mechanisms of M. falcata in response to salt-alkali stress. Results The results showed that compared to the untreated controls, 10,289 and 2,478 DEGs were detected in LM18 and HL M.falcata seedlings, with 788 shared DEGs detected in both. GO functional analysis classified these DEGs into three categories: Biological Process, Cellular Components, and Molecular Functions, with significant enrichment in GO terms such as “response to osmotic stress”, “intramolecular oxidoreductase activity” and “antioxidant activity”. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the involvement of these DEGs in key metabolic pathways, including “Phenylpropanoid biosynthesis”, “Plant hormone signal transduction”, “Plant-pathogen interaction”, “Isoflavonoid biosynthesis”, “Circadian rhythm-plant” and “Photosynthesis—antenna proteins”. Physiological indicators and membership function analysis confirmed that LM18 has greater salt-alkali tolerance than HL. Transcription factor analysis identified 42 transcription factor families, with the ERF family being the most abundant, followed by MYB-related, WRKY, bHLH, and MYB families. Weighted Gene Co-expression Network Analysis (WGCNA) showed that the MEturquoise module exhibited a significant positive correlation with salt-alkali stress and several physiological indicators. Module gene network analysis and GO enrichment revealed that MS.gene64536 (MYBP), MS.gene76249 (SRM1) and MS.gene049843 (MPK3) have functions related to “response to salt stress” and “positive regulation of response to salt stress”, suggesting their key roles in salt-alkali tolerance in M.falcata . All three genes were upregulated in the salt-alkali tolerant LM18. Conclusions The GO terms and KEGG pathways significantly enriched in LM18 involved a significantly higher number of DEGs compared to HL , suggesting a more robust and effective mechanism in LM18. These findings highlight the robust molecular and physiological adaptations of LM18 in response to salt-alkali stress.

Low temperature triggers Ca 2+ signalling and reprogramming of gene expression and metabolism in plants. However, how the Ca 2+ signal is transduced to the downstream metabolic pathways remains unknown. The involvement of a cold‐induced calmodulin‐like protein, MfCML50, from Medicago falcata in regulation of cold tolerance was examined in the present study. The interaction of MfCML50 with isopiperitenol/carveol dehydrogenase (MfCDH) was identified, which was dependent upon Ca 2+ and led to activated activity of MfCDH. MfCDH catalysed the production of carvone from carveol, with K m value of 10.25 μM. Overexpression of MfCML50 or MfCDH in Medicago truncatula led to enhanced cold tolerance with increased accumulation of carvone under cold conditions. The knockout mutation of the ortholog MtCML50 and MtCDH in M. truncatula led to reduced cold tolerance with decreased accumulation of carvone in the mtcml50 , mtcdh , or mtcml50 mtcdh mutants under cold conditions, which could be recovered by expressing MfCML50 or MfCDH or exogenous application of carvone. The diphenyl‐2‐picrylhydrazyl (DPPH) assay showed that carvone exhibited strong antioxidant activity. Less ROS were accumulated in MfCML50 and MfCDH overexpressing lines, but more ROS were accumulated in mtcml50 and mtcdh mutants under cold conditions. The results suggested that the Ca 2+ signal activated the MfCML50‐MfCDH module regulates cold tolerance through promoted production of carvone to maintain ROS homeostasis.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

The ability of root cells to distinguish mutualistic microbes from pathogens is crucial for plants that allow symbiotic microorganisms to infect and colonize their internal root tissues. Here we show that Lotus japonicus and Medicago truncatula possess very similar LysM pattern-recognition receptors, LjLYS6/MtLYK9 and MtLYR4, enabling root cells to separate the perception of chitin oligomeric microbe-associated molecular patterns from the perception of lipochitin oligosaccharide by the LjNFR1/MtLYK3 and LjNFR5/MtNFP receptors triggering symbiosis. Inactivation of chitin-receptor genes in Ljlys6, Mtlyk9, and Mtlyr4 mutants eliminates early reactive oxygen species responses and induction of defense-response genes in roots. Ljlys6, Mtlyk9, and Mtlyr4 mutants were also more susceptible to fungal and bacterial pathogens, while infection and colonization by rhizobia and arbuscular mycorrhizal fungi was maintained. Biochemical binding studies with purified LjLYS6 ectodomains further showed that at least six GlcNAc moieties (CO6) are required for optimal binding efficiency. The 2.3-Å crystal structure of the LjLYS6 ectodomain reveals three LysM βααβ motifs similar to other LysM proteins and a conserved chitin-binding site. These results show that distinct receptor sets in legume roots respond to chitin and lipochitin oligosaccharides found in the heterogeneous mixture of chitinaceous compounds originating from soil microbes. This establishes a foundation for genetic and biochemical dissection of the perception and the downstream responses separating defense from symbiosis in the roots of the 80–90% of land plants able to develop rhizobial and/or mycorrhizal endosymbiosis.