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Light is a vital regulator of photosynthesis, energy production, plant growth, and morphogenesis. Although these key physiological processes are well understood, the effects of light quality on the pigment content, oxidative stress, reactive oxygen species (ROS) production, antioxidant defense systems, and biomass yield of plants remain largely unexplored. In this study, we applied different light-emitting diode (LED) treatments, including white light, red light, blue light, and a red+blue (1:1) light combination, to evaluate the traits mentioned above in alfalfa (Medicago sativa L.). Fluorescence staining showed that red light significantly triggered the oxidative stress indicators compared to blue and white light, while the combined red and blue light treatment significantly reduced the ROS (O2•−, H2O2) intensity in alfalfa seedlings. Interestingly, the combined light treatment significantly boosted the seed germination rate (%), maximum photochemical quantum yield of PSII (Fv/Fm), leaf greenness (SPAD score), photosynthetic pigment levels (chlorophyll a, chlorophyll b, and carotenoids), and plant biomass yield in alfalfa seedlings. The red and/or combined (red+blue) light treatments significantly regulated antioxidant enzymes (SOD, CAT, APX, and GR) and the expression of genes related to the ascorbate–glutathione (AsA-GSH) pathway, including monodehydroascorbate reductase (MsMDHAR), dehydroascorbate reductase (MsDHAR), ascorbate peroxidase (MsAPX), and glutathione reductase (MsGR). These results indicate that light quality is crucial for regulating the morphological, physiological, and molecular traits linked to alfalfa improvement. These findings suggest a new approach to enhancing the adaptation, as well as the morphological and agronomic yield, of alfalfa and forage legumes through light-quality-mediated improvement.

Background Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop’s sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. Results To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13 , and DIHYDROFLAVONOL-4-REDUCTASE ( DFR ) regulating WD40–1 . Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40–1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40–1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. Conclusions Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40–1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Anthers consist of various specialised cell types and play a significant role in plant reproduction. Although the molecular mechanisms underlying anther development and regulation have been extensively studied, the single‐cell transcriptional landscape and dynamic regulation during anther development in M. sativa remain largely unexplored. In the present study, we constructed the first single‐cell transcriptome atlas of M. sativa anthers to provide a comprehensive view of cell type‐specific gene expression and epigenetic modifications. The reconstruction of the developmental trajectories of tapetum and microspores led to the identification of novel genes and elucidated the regulatory networks involved in tapetum formation. Our findings revealed rapid functional transitions in tapetum during the tetrad stage, including cell formation, specialisation, and programmed cell death (PCD). Additionally, we analysed the stages at which tapetal degradation and microspore shrinkage occurred in the sterile line. Overall, this study offers valuable insights into the molecular mechanisms underlying anther development in M. sativa. Specifically, MsKIN14P was identified as a key regulator of microtubules and the cytoskeleton during mitosis, and the transcription factors ERF3 and ERF025 were shown to influence anther development through the ethylene response pathways. These findings provide an essential theoretical foundation for the development of novel male‐sterile lines and enhance the breeding capabilities of M. sativa.

Background Calcium-dependent protein kinases (CDPKs), play multiple roles in plant development, growth and response to bio- or abiotic stresses. Calmodulin-like domains typically contain four EF-hand motifs for Ca²⁺ binding. The CDPK gene family can be divided into four subgroups in Arabidopsis , and it has been identified in many plants, such as rice, tomato, but has not been investigated in alfalfa ( Medicago sativa subsp. sativa ) yet. Results In our study, 38 non-redundant MsCDPK genes were identified from the “XinJiangDaYe” alfalfa genome. They can be divided into four subgroups which is the same as in Arabidopsis and Medicago truncatula , and there were 15, 12,10 and 1 in CDPK I, II, III and IV, respectively. RNA-seq analysis revealed tissue-specificity of 38 MsCDPK genes. After researching the transcriptome data, we found these 38 MsCDPK members responsive to drought, salt, and cold stress treatments. Further analysis showed that the expression of almost all the MsCDPKs is regulated by abiotic stresses. In addition, we chose MsCDPK03 , MsCDPK26 , MsCDPK31 and MsCDPK36 for RT-qPCR validation which was from CDPK I-IV subgroups respectively. The result showed that the expression of these four genes was significantly induced by drought, salt and cold treatments. The subcellular location experiment showed that these four proteins were all located in nucleus. Conclusion In our study, we identified 38 distinct MsCDPK genes within the alfalfa genome, which were classified into four groups. We conducted a comprehensive analysis of various gene features, including physicochemical properties, phylogenetic relationships, exon-intron structures, conserved motifs, chromosomal locations, gene duplication events, cis -regulatory elements, 3D structures, and tissue-specific expression patterns, as well as responses to drought, salt, and cold stresses. These results also provide a solid foundation for further investigations into the functions of MsCDPKs aimed at improving drought tolerance in autotetraploid cultivated alfalfa through genetic engineering.

Salinity is a major abiotic stress limiting plant productivity. The Salt Overly Sensitive (SOS) pathway, comprising SOS1, SOS2, and SOS3, is central to ion homeostasis under salt stress. While the pathway has been extensively studied in model plants, its components remain uncharacterized in Medicago sativa (alfalfa), a moderately salt-tolerant forage crop of global economic importance. Here, we report the functional characterization of MsSOS2 , a CIPK family gene and putative ortholog of AtSOS2 . Sequence analysis revealed that MsSOS2 possesses conserved domains essential for kinase activity, including a kinase domain, activation loop, the phosphatidic acid (PA)-binding site, and NAF motif. Phylogenetic analysis placed MsSOS2 with AtSOS2 and OsSOS2. Yeast-two-hybrid assays confirmed the interaction between MsSOS2 and MsSOS3, mimicking the AtSOS2 – AtSOS3 complex. Transgenic expression of MsSOS2 in the Arabidopsis sos2 mutant restored salinity tolerance during seed germination, seedling development, and late vegetative growth. Ion profiling showed reduced Na accumulation and enhanced K retention in MsSOS2 -expressing lines under salinity. We propose that MsSOS2 activates AtSOS1 to facilitate Na⁺ efflux and disrupts AtCBL10-mediated inhibition of AKT1 to promote K⁺ uptake. This study provides the first functional validation of an SOS pathway component in alfalfa and highlights MsSOS2 as a promising candidate for improving salinity tolerance in legumes.

Background Alfalfa ( Medicago sativa L.) has the benefits of high yield and nutritional value as a sustainable forage. However, the water deficit significantly limits its growth and yield performance. Nitric oxide (NO) is a signal molecule that can enhance plant tolerance. The majority of previous studies focus on the role of exogenous NO in plant tolerance. However, the underlying mechanism of endogenous NO in alfalfa drought tolerance remains largely unexplored. Results To explore the mechanism of the endogenous NO-mediated water deficit resistance in the alfalfa, seedlings were exposed to polyethylene glycol 6000 (PEG) and NO scavenger (cPTIO). Results showed that PEG treatment significantly augmented alfalfa endogenous NO, MDA, O 2 ·− , and H 2 O 2 levels. In parallel, eliminating endogenous NO under PEG stress (PEG-NO) significantly diminished NO level, exacerbated MDA and reactive oxygen species accumulation, and decreased the activities of key enzymes involved in carbon fixation and TCA cycle, such as Rubisco, FBA, PDH, α-KGDH, and SDH, as well as reduced ABA and IAA content in alfalfa leaves. RNA-Seq and bioinformatics analysis suggested that endogenous NO-responsive DEGs primarily relate to carbon metabolism and hormone signal transduction. In further studies of these DEGs, we speculated that GH3 , SAUR , SnRK2 , and ABF genes and FBA, GAPDH, SBP, and CS are critical genes in response to endogenous NO under PEG stress. Conclusions In summary, our study innovatively proposes a mechanism model of how endogenous NO enhances alfalfa tolerance to water deficiency at the physiological and molecular levels. The novel candidate genes can give genetic resources for the subsequent molecular-assisted breeding of drought-resistant alfalfa crops.

Peroxidase (POD) is a widespread and highly active enzyme in plants that plays an important role in plant growth and development and stress response. No genome-wide analysis and characterization of the POD gene family in alfalfa has been performed yet. In this study, we used bioinformatics techniques to identify 343 members of this family in alfalfa and performed predictive analyses of their physicochemical properties, subcellular localization, phylogenetic relationships and conserved motifs. Expression analysis showed that 58 of the 343 genes were specifically expressed. Expression pattern analysis under different stresses showed that the MsPOD gene family was responsive to salt stress, cold stress, and drought stress, and there were genes responsive to multiple stresses. Among them, 24 MsPOD genes responded to all three stresses. Understanding the expression patterns of alfalfa MsPOD family members can enhance alfalfa's ability to resist abiotic stresses, thereby providing a theoretical basis for increasing alfalfa yield under adverse conditions.

Background The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa. Results In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs . Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs ( MsMAPK70 and MsMAPK75 ) responds to salt and cold stresses; two MsMAPKs ( MsMAPK60 and MsMAPK73 ) responds to cold and drought stresses; four MsMAPKs ( MsMAPK1 , MsMAPK33 , MsMAPK64 and MsMAPK71 ) responds to salt and drought stresses; and two MsMAPKs ( MsMAPK5 and MsMAPK7) responded to all three stresses. Conclusion This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance.

Background Alfalfa ( Medicago sativa L.) is a high-quality, high-protein forage, and the improvement and breeding of key traits are important for enhancing the productivity of alfalfa. Plant height is an important trait that affects crop yield, and its regulatory network mechanism has been widely reported in model plants, however, there are fewer studies on the developmental regulatory of plant height in alfalfa. Results In this study, we screened tall (WL525HQ) and short (WL343HQ) alfalfa materials through field experiments and analyzed the regulatory mechanism of plant height based on the multidimensional joint analysis of phenotype, cell, physiology, and molecular biology. The results showed that internode length was an important factor determining plant height in alfalfa, and cell size affected the internode elongation to a certain extent, whereas cell size was limited by cell wall. Moreover, changes in cell wall components play an important role in cell wall expansion, especially lignin synthesis. Transcriptome analysis showed that the high expression of hydrolase activity in T1 (initiation growth period) facilitates the expansion of the cell wall, the significant enrichment of the cellular modification process in T3 (rapid growth period) increases the cell size, and the synthesis of cell wall structural constituents and plant-type cell wall organization in T5 (growth stabilization) further improves and modifies the cell wall structure. Differential genes involved in cell wall biosynthesis and expansion were mainly enriched in cellulose synthesis, pectin cleavage, lignin formation, expansion protein (EXP), and xyloglucan endotransglycosidase (XTH). Conclusions These findings elucidated the plant height regulation mechanisms throughout the alfalfa plant and provided a theoretical basis for the generation of ideal alfalfa plant height germplasm.

Sowing date and soil fertility are very important factors in the overwintering and production performance of alfalfa ( Medicago sativa L.), yet there’s a knowledge gap in knowledge on how late-seeded alfalfa responds to phosphorus (P) fertilization. A field study was conducted in Inner Mongolia from 2020 to 2022 using a split-plot design. The main plots consisted of five sowing dates (31 July, 8, 16, and 24 August, and 1 September), while the subplots involved five P application rates (0, 40, 70, 100, and 130 kg P 2 O 5 ha −1 ). Throughout the growing seasons, the overwintering rate, root traits, forage yield, and yield components were measured. The results revealed a consistent decrease in overwintering ability and productivity with the delayed sowing. This reduction in overwintering rate was mainly due to diminished root traits, while the decrease in forage yield was largely associated with a reduction in plants per square meter. However, P fertilizer application to late-seeded alfalfa demonstrated potential in enhancing the diameter of both the crown and taproot, thus strengthening the root system and improving the overwintering rate, the rate of increase ranges from 11.6 to 49%. This adjustment could also improve the shoots per square meter and mass per shoot, increasing by 9.4–31.3% and 15.0–27.1% respectively in 2 years, which can offset the decline in forage yield caused by late sowing and might even increase the forage yield. Regression and path analysis indicated that alfalfa forage yield is primarily affected by mass per shoot rather than shoots per square meter. This study recommended that the sowing of alfalfa in similar regions of Inner Mongolia should not be later than mid-August. Moreover, applying P fertilizer (P 2 O 5 ) at 70.6–85.9 kg ha −1 can enhance the forage yield and persistence of late-seeded alfalfa. Therefore, appropriate late sowing combined with the application of P fertilizer can be used as an efficient cultivation strategy for alfalfa cultivation after a short-season crop harvest in arid and cold regions.