Alfalfa (Medicago sativa) is a highly valuable perennial forage legume that suffers from autotoxicity, which decreases plant resistance, reduces soil fertility, causes serious soil-borne diseases, and promotes ecological imbalance. We evaluated the effects of autotoxicity on the seed germination of 22 alfalfa varieties, and then elucidated the oxidative damage and lipid peroxidation in two alfalfa varieties with contrasting autotoxicity tolerances. The technique for order of preference by similarity to ideal solution (TOPSIS) method was used to rank the germination of the 22 alfalfa varieties when exposed to six autotoxic concentrations (0, 0.025, 0.075, 0.125, 0.175, and 0.225 g∙mL⁻¹). We found WL656HQ and 3105C to be autotoxicity-tolerant and autotoxicity-sensitive varieties, respectively. The germination index mainly affects the comprehensive allelopathic index of WL656HQ and 3105C, which were the simple vigor index and radicle length according to the random forest model, respectively. 3105C eliminates reactive oxygen species (ROS) via antioxidant enzymes and antioxidants under T1 (0.025 g∙mL⁻¹), but the oxidative stress system and the oxidative scavenging system cannot maintain the balance under T2 (0.125 g∙mL⁻¹), causing oxidative bursts. In comparison, WL656HQ used its oxidative scavenging system (peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)) to maintain its redox dynamic balance by removing excess ROS at all concentrations. In conclusion, the positive and negative indicators of autotoxicity for the two varieties were ascorbate (ASA) and hydroxyl free radicals (OH^•), and proline (Pro) and dehydroascrobate (DHA), respectively. The most sensitive autotoxic concentrations of 3105C and WL656HQ were T2 (0.125 g∙mL⁻¹) and T1 (0.025 g∙mL⁻¹), respectively.

Introduction: Alfalfa (Medicago sativa L.) is a globally important legume crop with high nutritional and ecological value. Drought poses a serious threat to alfalfa acreage and yields. Spermine (Spm) has been shown to protect plants from drought damage. The aim of this study was to clarify the mechanism of exogenous Spm to improve drought resistance of alfalfa. Methods: In this study, we root applied 0.1, 0.5, and 1 mM Spm to Gannong No. 3 (G3) alfalfa under drought stress, and then determined their physiological and metabolic changes. Results: The results showed that exogenous Spm increased chlorophyll content, chlorophyll fluorescence parameters and gas exchange parameters, enhanced antioxidant enzymes activity, improved ascorbic acid-glutathione (AsA-GSH) cycle, increased osmoregulatory substances content, reduced hydrogen peroxide and superoxide anion levels, and inhibited malondialdehyde accumulation in alfalfa under drought stress, thereby increasing plant height and leaf relative water content and enhancing drought tolerance of alfalfa. The redundancy analysis of the above physiological indicators showed that the addition of the optimal Spm to improve drought tolerance of alfalfa under drought stress was mainly achieved by increasing catalase activity and improving the ASA-GSH cycle. In addition, metabolomics analysis revealed that exogenous Spm increased the content of oxobutanedioic acid, citric acid, fumaric acid and malic acid to enhance the tricarboxylic acid cycle. Meanwhile, exogenous Spm increased endogenous Spm and proline (Pro) content to resist drought stress by enhancing Spm and Pro metabolism. Moreover, exogenous Spm increased the accumulation of the signaling substance abscisic acid. Discussion: In conclusion, exogenous Spm enhanced drought resistance of alfalfa leaves under drought stress.

Plant height plays an important role in crop yield, product quality, and cultivation management. However, the physiological mechanisms that regulate the establishment of plant height in alfalfa plants remain unclear. Herein, we measured plant height traits, leaf characteristics, photosynthetic physiology, cell wall composition, and endogenous hormone contents of tall- and short-stalked alfalfa materials at different reproductive periods. We analyzed the physiology responsible for differences in plant height. The results demonstrated that the number of internodes in tall- and short-stalked alfalfa materials tended to converge with the advancement of the fertility period. Meanwhile, the average internode length (IL) of tall-stalked materials was significantly higher than that of short-stalked materials at different fertility periods, with internode length identified as the main trait determining the differences in alfalfa plant height. Leaf characteristics, which are closely related to photosynthetic capacity, are crucial energy sources supporting the expression of plant height traits, and we found that an increase in the number of leaves contributed to a proportional increase in plant height. Additionally, a significant positive correlation was observed between plant height and leaf dry weight per plant during the branching and early flowering stages of alfalfa. The leaves of alfalfa affect plant height through photosynthesis, with the budding stage identified as the key period for efficient light energy utilization. Plant height at the budding stage showed a significant positive correlation with soluble sugar (SS) content and a significant negative correlation with intercellular CO₂ concentration. Moreover, we found that alfalfa plant height was significantly correlated with the contents of indole-3-acetic acid in stem tips (SIAA), gibberellin A₃ in leaves (LGA₃), zeatin in stem tips (SZT), and abscisic acid in leaves (LABA). Further investigation revealed that SS, SIAA, and LGA₃ contents were important physiological indicators affecting alfalfa plant height. This study provides a theoretical basis for understanding the formation of alfalfa plant height traits and for genetic improvement studies.

Drought is considered a primary factor constraining alfalfa ( Medicago sativa L.) yield and acreage. To understand the internal drought resistance mechanisms of alfalfa is essential for breeding drought-resistant alfalfa varieties. Here, we compared the phenotypic characteristics, metabolic pathways and metabolites of drought-resistant (Longzhong, LZ) and drought-sensitive (Gannong No. 3, G3) varieties under drought stress. Phenotypic analysis revealed that drought stress reduced plant height, single plant fresh weight, single plant dry weight and leaf RWC in alfalfa, with a greater effect observed in G3. The root length of LZ increased under drought stress, whereas there was no significant change in the root length of G3. Widely targeted metabolomics revealed that LZ could maintain higher glycolysis/gluconeogenesis and tricarboxylic acid cycle under drought stress, which provided more ATP and substrates for amino acids biosynthesis, arginine and proline metabolism and phenylpropanoid metabolism. This allowed LZ to accumulate more amino acids, spermidine, spermine, 4-aminobutyric acid, naringenin, isoliquiritigenin, glycitein, glycitin, calycosin, ferulate, scopoline, scopoletin, sinapyl alcohol and coniferin, which favor the enhancement of drought resistance in alfalfa. Moreover, widely targeted metabolomics showed salicylic acid and trans-zeatin were key hormones involved in drought resistance in alfalfa. The results of this study provide useful insights into the enhancement of drought resistance in alfalfa through metabolic regulatory mechanisms, which provides a theoretical basis for the breeding drought-resistant alfalfa varieties.

The symbiotic nitrogen-fixing system formed between alfalfa (Medicago sativa L.) and rhizobia requires precise regulation of carbohydrate and lipid metabolism to sustain their high-energy-demand system. However, metabolic divergence between roots and nodules remains poorly characterized. Using comparative transcriptomics, we analyzed gene expression profiles in pink nodules (PN), white nodules (WN), Pink nodule roots (PNR), white nodule roots (WNR), non-nodule roots (NNR) and control roots (CKR) from rhizobia-inoculated plants at 35 days post-inoculation. Key findings revealed metabolic specialization between tissues: PN exhibited elevated expression of lipid catabolism genes (MsECHIA, MsACX) and key genes of the TCA cycle regulators, driving direct energy supply for nitrogenase activity. PNR, WNR preferentially expressed glycolysis (MsPKP2) and pentose phosphate pathway (MsG6PD5) genes to convert photoassimilates into dicarboxylic acids via a directional transport system to nodules. WN showed enriched fatty acid elongation genes (MsKCR1, MsHACD2), suggesting compensatory synthesis of structural lipid to maintain symbiotic interfaces under carbon limitation. NNR, CKR retained starch metabolism dominance. Weighted geneco-expression network analysis revealed that symbiotic signaling synchronizes nodule lipid degradation with root carbon repartitioning to prioritize photoassimilate allocation to nodules. Nodulated roots may supplement nodule energetics through lipid precursor synthesis or storage lipid hydrolysis, thereby forming a “root-nodule metabolic relay” mechanism. Our results demonstrate that the alfalfa-rhizobia symbiosis establishes a hierarchical energy distribution network through tissue-specific regulation of metabolic genes, coordinating nitrogen fixation efficiency with energy supply homeostasis. This study elucidates metabolic coordination mechanisms underlying legume-rhizobial symbiosis, providing a theoretical framework for optimizing symbiotic energy economics through targeted gene editing approaches.

To investigate the precise and efficient symbiosis between Sinorhizobium meliloti LL2 and different alfalfa varieties, we conducted experiments using eight alfalfa varieties along with the S. meliloti LL2. Our objective was to identify highly effective symbiotic combinations by analyzing differences in nodulation, nitrogen fixation, and biomass accumulation. The results revealed that Gannong NO.9 had higher values for single effective root nodule weight (1.30 mg) and the number of infected cells in root nodules (2795) compared to other varieties (p < 0.05). Additionally, Gannong NO.9 exhibited the highest nitrogenase activity (0.91 μmol·g⁻¹·h⁻¹), nitrogen fixation percentage (67.16%), and amount of nitrogen fixation (18.80 mg/pot). Moreover, there was a significant 26.50% increase in aboveground tissue nitrogen accumulation compared to the control check (CK) (p < 0.05). Furthermore, underground tissue showed excellent values for nitrogen accumulation (35.68 mg/plant) and crude protein content (17.75%) when compared with other treatments. The growth of plants was demonstrated by the combined impact of nodulation and nitrogen fixation. The distribution of biomass after nitrogen fixation was compared to the control group (p < 0.05) to investigate accumulation. The eight combinations of symbiotic nitrogen fixation (SNF) were classified into six distinct types based on their significantly different biomass growth rates compared to CK. ① Aboveground accumulation type: Gannong NO.9 (there was a 24.31% increase in aboveground dry weight); ② aboveground and underground accumulation type: Qingshui (the aboveground dry weight increased by 135.94%, while the underground dry weight grew by 35.26%); ③ aboveground accumulation, underground depletion type: Gannong NO.5 ( ); ④ zero-growth type (there was no significant difference in dry weights, both above and below ground, compared to CK): WL168HQ, WL319HQ and Longzhong; ⑤ aboveground and underground depletion type: WL298HQ (the aboveground dry weight decreased by 29.29%, while the underground dry weight decreased by 20.23%); ⑥ underground depletion type: Gannong NO.3 (the underground dry weight showed a decrease of 34.49%); no type with aboveground consumption and underground accumulation was found. The study clarified the optimal combination of LL2 and Gannong NO.9, finding that biomass accumulation after symbiotic nitrogen fixation is variety-dependent.