Different rhizobial strains can lead to distinct symbiotic phenotypes in alfalfa, yet molecular differences at the mature nodule stage remain unclear. Here, we analyzed 21-day post-inoculation (dpi) nodules induced by strains WE2 and WWL2. We measured nitrogenase activity (acetylene reduction assay, ARA) and performed dual RNA-seq to compare gene expression in both the alfalfa host and the rhizobia. On the host side, WE2-induced nodules showed higher expression of mature nodule marker genes (ENOD93 and leghemoglobin (Lb) genes) and higher expression of genes encoding SWEET transporters and amino acid and peptide transporters. Host differentially expressed genes were enriched in pathways related to transmembrane transport, redox and heme-related functions, and processes linked to maintaining microaerobic conditions. On the rhizobial side, WE2 nodules showed higher expression of genes involved in microaerobic respiration and nitrogen fixation (e.g., nif/fix and key respiratory chain genes), whereas WWL2 nodules showed higher expression of genes linked to transport, chemotaxis/motility, and environmental information processing. Together, these host and rhizobia expression patterns suggest coordinated differences between host pathways related to resource supply and microaerobic conditions and rhizobial expression programs for respiration and nitrogen fixation. Based on these associations, we propose a working model and provide candidate genes and pathways for functional validation and inoculant screening.

This study investigated the flavor differences among summer pasture-grazed (SM), winter pasture-grazed (WM) and winter stall-fed (HM) yaks using metabolomics, lipidomics and headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The SM group showed n-3 polyunsaturated fatty acids (n-3 PUFAs) were 225% and 209% higher than those in the WM and HM groups, respectively, with higher thiobarbituric acid reactive substances (TBARS) and antioxidant enzyme activities. These changes promoted the formation of (E)-2-decenal and 1-octen-3-ol, which impart green and floral aromas. Conversely, the HM group's high-energy diet increased monounsaturated fatty acids and essential amino acids (EAAs). This promoted the production of Maillard-derived furfural and maintained the oxidative-antioxidant balance, preserving nutty and tallowy flavors. The WM group exhibited off-flavors due to oxidative imbalance, lower PUFAs content, and benzaldehyde accumulation. Overall, feeding regimes shape yak flavor by modulating fatty acid and amino acid compositions and the oxidative-antioxidant balance. This study provides a scientific basis for precision nutrition strategies to optimize flavor in yaks.

Abstract Background Improved symbiotic nitrogen fixation efficiency between alfalfa (Medicago sativa L.) and rhizobia represents a green development strategy that addresses the demand for high‑quality protein, while also serving as a critical measure for safeguarding China’s food security. Currently, there is limited research on how rhizobium inoculation influences alfalfa growth and development through photosynthesis and respiratory metabolism. Furthermore, studies examining the impact of rhizobium strains with differing symbiotic effectiveness on these metabolic pathways remain scarce. Results The number of effective nodules per plant (7), nitrogenase activity (0.29 µmol·g− 1·h− 1), and leghemoglobin content (0.76 mg·g− 1) of the LL2 inoculation group were significantly higher than those of the QL5 group. The aboveground dry weight (0.59 g·10 plants− 1) of LL2 was also significantly greater than that of both the QL5 inoculation treatment and the uninoculated control. These results demonstrate that rhizobium strain LL2 is an efficient symbiotic match for ' Gannong No.9 ' alfalfa, whereas strain QL5 is an inefficient match. Metabolomic analysis revealed that, in leaves, seven differential metabolites were up-regulated in both photosynthetic and respiratory metabolism. Among these, Adenosine 5’-Diphosphate (ADP) was significantly higher in LL2 than in CK (Control) and QL5. In roots, nine differential metabolites were up-regulated. Among these, four metabolites—3-Phosphoglyceric acid, Uridine-5’-diphosphate-glucose, (2 S)-2-Isopropylmalate, and L-Glutamic acid—were present at significantly higher levels in LL2 than in both CK and QL5. Compared to the QL5 group, the LL2 inoculation group resulted in significantly higher contents of ADP in leaves and elevated levels of the root metabolites such as the photosynthetic carbon fixation intermediate 3-Phosphoglyceric acid, the glycosyl donor Uridine-5’-diphosphate-glucose, the respiration and nitrogen metabolism-related compounds (2S)-2-Isopropylmalate and L-Glutamic acid. Additionally, in nodules, the key metabolites trehalose-6-phosphate and alpha-D-glucose-6-phosphate (involved in sugar metabolism and the pentose phosphate pathway) were also significantly elevated Among these, ADP and alpha-D-glucose-6-phosphate participate simultaneously in photosynthetic, respiratory, and symbiotic metabolic pathways; 3-Phosphoglyceric acid is involved in both photosynthetic and symbiotic pathways; while (2S)-2-Isopropylmalate and L-Glutamic acid take part in respiratory and symbiotic pathways. Conclusions Following inoculation with LL2, the levels of key metabolites associated with photosynthesis and respiration underwent systematic changes in the leaves, roots, and nodules of the plants. The enhanced symbiotic nitrogen fixation and plant growth were associated with synergistic changes in the host plant’s photosynthetic carbon metabolism, respiratory energy metabolism, and nitrogen assimilation pathways. The findings of this study suggest potential strategies for enhancing nitrogen accumulation, possibly through modulating the energy balance of the symbiotic system, which could improve nitrogen fixation efficiency and ultimately increase legume yield and quality.

Sorghum, the fifth-largest cereal crop globally and a C4 crop, mainly grows in arid and semiarid areas. In 2021 to 2023, a new foliar disease of sorghum occurred in China. The diseased leaves showed water-soaked symptoms in the leaf tip and margins, resulting in half- and full-leaf desiccation and necrosis, thus affecting plant photosynthesis. A total of 24 Eutiarosporella strains were isolated from symptomatic leaves. Based on morphological characteristics, multilocus phylogenetic analysis involving ITS, LSU, and EF1-alpha sequences, and the pathogenicity test, the pathogen of sorghum causing leaf blight in China was identified as Eutiarosporella dactylidis. The virulence of all E. dactylidis strains was evaluated using the spray-mycelium method. Different strains showed significantly different pathogenicities toward a susceptible cultivar, Longza 10, with disease indexes ranging from 23.76 to 60.37. This study first reported leaf blight of sorghum caused by E. dactylidis and named it "Eutiarosporella leaf blight," which provides a theoretical basis for farmers in disease management.

BACKGROUND:Alfalfa (Medicago sativa) is a perennial high-quality legume forage widely cultivated worldwide, but drought stress severely restrict its growth and development. Plant homeodomain finger (PHD) family genes are a type of zinc finger transcription factors widely distributed in eukaryotes. They act as histone code identifier to regulate the expression of downstream genes and play important biological roles in plant growth, development, and stress response. However, information about the PHD family genes in alfalfa remains limited at present. RESULTS:We identified 56 MsPHD genes containing 67 PHD domains in the alfalfa, and these genes were unevenly distributed on 8 chromosomes of alfalfa. There are 9 pairs of gene fragment duplicates in the MsPHD family. The 56 MsPHD genes are evolutionarily divided into 14 subgroups. There are differences in the conservative motifs and gene structure composition patterns of MsPHD among different subgroups. In addition, we identified 11 MsPHD genes related to drought stress, and their promoter regions all contained elements related to stress response. Quantitative Real-time PCR (QRT-PCR) further verified that the expression levels of these 11 MsPHD genes were significantly up-regulated in both roots and leaves, with MsPHD9 showing the highest upregulation. 57.14% of MsPHD proteins were predicted to be located in the nucleus, and the co-localization analysis of MsPHD9 protein confirmed that it was primarily located in the nucleus. CONCLUSIONS:The identification of alfalfa PHD family genes not only provides important genetic resources for in-depth analysis of the molecular mechanism of alfalfa drought resistance, but also lays a foundation for creating new germplasm of alfalfa with strong drought resistance through genetic engineering technology.

AIMS:Fertilizers can significantly influence leaf senescence and hormonal regulation, which in turn impacts crop yield. Despite significant advancements in understanding fertilizer effects on plant growth, the specific molecular mechanisms through which fertilizers influence hormonal regulation and leaf senescence, and subsequent impact on yield, remain underexplored. This study addresses this critical gap by examining transcriptional, physiological, and molecular mechanisms in the semiarid regions of rainfed spring maize under long-term fertilizers. METHODS:Fertilizer treatments include no amendment (NA), inorganic fertilizer (CF), combined inorganic and organic fertilizer (SC), organic fertilizer (SM), and maize straw (MS) replicated three times. RESULTS:The highest number of differentially expressed genes (DEGs) were observed under CF (3972) followed by SC (1949) in comparison to NA, showing a strong effect of inorganic fertilizer on gene expressions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that numerous genes involved in the biosynthesis of secondary metabolites, plant hormone signaling, photosynthesis pathways, and metabolic pathways showed varied expressions of up- and downregulation. Genes involved in the ethylene, abscisic acid, jasmonic acid, salicylic acid, and brassinosteroid pathways indicated their interaction and promoted leaf senescence, whereas those related to auxin and gibberellin pathways had minimal impact. In the ethylene pathway known to influence senescence, two ethylene receptor (ETR) genes (Zm00001d013486 and Zm00001d021687) were downregulated, whereas, two ethylene-insensitive proteins 3 (EIN2) genes (Zm00001d053594 and Zm00001d033625) showed upregulation in the CF, SC and SM treatments. Furthermore, 86 highly up-regulated genes involved in the photosynthesis pathway encompassing components such as photosynthesis antenna, photosynthesis complexes II, cytochrome complexes, photosynthesis electron transport, and ATP complex in SC and CF compared to SM and MS. CONCLUSION:In summary, the study finds that DEGs showed stronger responses to inorganic fertilizers, likely due to organic fertilizers decomposing at a slower rate. Nevertheless, transcriptional and physiological analyses indicate that the SC treatment sustainably enhances maize productivity without causing adverse environmental effects, outperforming the other treatments (NA, CF, SM, MS). These results provide new perspectives on genetic regulation and pathway discovery in rainfed maize cultivation in semiarid areas.

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. 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). 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.

Endophytic rhizobia have the functions of dissolving organic phosphorus, secreting auxin, fixing nitrogen, and promoting growth. The proliferation of endophytic rhizobia in alfalfa and their symbiotic nodulation with alfalfa seedlings are regulated by various plant hormones. In this study, the alfalfa seeds (Medicago sativa L.) containing CFP-labeled rhizobium R.gn5f (isolated from the seeds of Gannong No.5 alfalfa) were used as materials, and the concentrations of 3-indoleacetic acid (3-IAA), 6-benzylaminopurine (6-BA) and homobrassinolide (HBR) suitable for the growth of R.gn5f were used for seed soaking treatment, and distilled water was used as the control. The proliferation of endophytic rhizobium, plant nodulation, nitrogen fixation performance and plant growth ability of alfalfa at different growth stages were determined. The effects of hormone types and concentrations on the proliferation and nitrogen fixation of endophytic rhizobia were analyzed to provide a theoretical basis for accurately promoting the nodulation, nitrogen fixation and growth-promoting ability of endophytic rhizobia in seeds. The results showed that the optimal concentrations of 3-IAA, 6-BA and HBR were 12 mg·L−1, 16 mg·L−1 and 2.47 mg·L−1, respectively. The nitrogen fixation performance of endophytic rhizobium plants containing three hormones was higher at the branching stage and budding stage. The growth ability of the plant was better at the flowering stage. The hormone 2.47 mg·L−1 of HBR was beneficial to the proliferation, nodulation, nitrogen fixation and plant growth of endophytic rhizobia in alfalfa at the vegetative and reproductive growth stages, and the number of R.gn5 f in the seeds of HBR plants at the mature stage was the largest (281.25 CFU·g−1). Therefore, the hormone 2.47 mg·L−1 of HBR was better for the proliferation of endophytic rhizobia R.gn5 f and plant growth in alfalfa. These findings provide a theoretical basis for precisely leveraging the nodulation and nitrogen-fixing capabilities of seed-borne endophytic rhizobia, thereby laying a foundation for the symbiotic breeding of alfalfa and rhizobia.

Introduction:Whole-plant corn silage (WPCS) is an important roughage source in ruminant nutrition, and its nutritional value can vary significantly with corn variety. Understanding how different WPCS varieties influence gastrointestinal microbiota and metabolic profiles is essential for optimizing feed efficiency and animal health. Methods:This study examined the effects of three corn varieties (2 introduced - Tunyu 168: TY; Yu silage 23: YQZ, and 1 local Longsheng 1: LS) in WPCS on gastrointestinal bacteria and metabolites in lambs. Thirty 4-month-old female Hu lambs (19.6 ± 0.26 kg) were assigned randomly to three groups (n = 10 per groups). After 90 days, 6 random lambs from each group were slaughtered, and contents from the rumen, ileum and cecum were collected. Results:The LS silage had the highest crude protein (CP) content, the TY silage had the lowest neutral detergent fiber (NDF) content, and the YQZ silage had the highest ammonia nitrogen (NH3-N) content. Dry matter intake (DMI) was greater in lambs fed the YQZ and TY silages than the LS silage, while average daily gain (ADG) was greater in lambs fed the TY silage than the YQZ and LS silages. The greatest concentration of total volatile fatty acids (TVFAs) in the rumen was measured in lambs fed the YQZ silage, and in the ileum and cecum was measured in lambs fed the TY silage. Lambs fed the YQZ silage increased the relative abundances of bacteria that degrade carbohydrates and synthesize volatile fatty acids (VFAs) in the gastrointestinal tract, and decreased the relative abundances of pathogenic bacteria in the rumen; while lambs fed the TY silage increased the relative abundances of bacteria in the cecum that degrade carbohydrate, protein and starch, and decreased the relative abundances of pathogenic bacteria in the rumen. The pathways of nicotinate and nicotinamide metabolism and folate biosynthesis were upgraded with the TY silage; whereas, pentose phosphate metabolism, histidine metabolism and folate biosynthesis were upgraded with the YQZ silage. Conclusion:These findings suggests that the YQZ and TY silages mediate rumen fermentation by altering rumen bacterial populations and metabolic activities, thereby maintaining rumen health and improving lamb growth performance. Lambs fed the TY silage had the greatest ADG and best feed conversion ratio (FCR: DMI/ADG), but the YQZ silage may have greater potential in sheep as it mediates a wider range of metabolic pathways.

Coumarins are the key autotoxins in alfalfa (Medicago sativa), which inhibit seed germination and seedling growth of offspring through autotoxicity, causing continuous cropping obstacles and reduced productivity in alfalfa cultivation systems. However, the biosynthetic regulation mechanism of coumarins in alfalfa is still unclear. Here, cinnamate 4-hydroxylase (MsC4H) positively regulates the biosynthesis of scopoletin, the key coumarins in alfalfa, was first elucidated by combined physiological, transcriptomic, metabolomics and functional verification analysis. We identified 17 differential coumarins in "Longzhong" and "WL656HQ", along with 89 DEGs related to coumarins biosynthesis. These DEGs encoded key enzymes, including PAL, 4CL, C4H, HCT, CSE, COMT, CCoAOMT, BGLU, F6H, TOGT1, and S8H. The differential regulation of scopoletin biosynthesis and its conversion with esculetin may represents a key factor underlying the distinct autotoxic substance profiles observed between "Longzhong" and "WL656HQ". Scopoletin accumulation was strongly correlated with the upregulated expression of MsC4H. The MsC4H protein was localized in the endoplasmic reticulum, and consisted of 506 amino acids with a molecular weight of 58,180.43 kDa. Overexpression of MsC4H significantly increased both C4H activity and scopoletin content in alfalfa leaves, whereas MsC4H RNAi alfalfa exhibited significantly reduced C4H activity and scopoletin accumulation. Correlation analysis of structural genes and TFs showed that bHLH (MS.geng025894) or MYB-related (MS.gene22755) may regulate scopoletin biosynthesis together with MsC4H.

Allelopathy plays a major role in agricultural production, influencing plant protection, crop yield, and crop rotation systems. This study investigated the effects of root exudates on 3105c alfalfa (Medicago sativa) seeds and seedlings to identify crops with strong and weak allelopathic potential. The results revealed that corn (Zea mays L.) (T1) exhibited the strongest allelopathic effects, whereas soybean (Glycine max (Linn.) Merr.) (T10) exhibited the weakest effects. T1 promoted seed germination by increasing radicle length and the simple vitality index. Both T1 and T10 promoted 3105c seedling growth and enhanced antioxidant capacity, albeit through different mechanisms. T1 primarily increased antioxidant capacity by elevating ascorbate and dehydroascorbate levels while reducing malondialdehyde content. In contrast, T10 enhanced antioxidant capacity by increasing soluble sugar and protein levels via hydroxyl free radical inhibition. These findings demonstrate that the allelopathic properties of corn effectively promote alfalfa growth by enhancing seed germination and improving physiological stress resistance.

BACKGROUND: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. RESULTS: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. CONCLUSIONS:This study elucidates metabolic coordination mechanisms underlying legume-rhizobial symbiosis, providing a theoretical framework for optimizing symbiotic energy economics through targeted gene editing approaches.

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.

This study investigates how seasonal grazing and housed feeding regimes alter the rumen microbiome and metabolite networks in yaks, thereby influencing meat quality. We evaluated the effects of these feeding strategies on Warner-Bratzler shear force (WBSF) and other key meat quality parameters using an integrated approach combining eDNA-metagenomics and metabolomics. This study revealed that warm-season forages, which are abundant in n-3 polyunsaturated fatty acids (n-3 PUFA), promote rumen metabolites associated with PUFA retention. This process resulted in an increased n-3 PUFA content in yak meat. Housed yaks fed high-energy diets exhibited increased Bacillota abundance and acetate biosynthesis, changes that enhanced IMF and crude protein levels, thereby improving meat tenderness and marbling. In contrast, cold-season grazing yaks ingested a higher proportion of Fabaceae and Ranunculaceae species. A high-fibre diet increased the abundance of Prevotella and Fibrobacter, genera that mitigate cold stress through enhanced propionate production and fatty acid catabolism. However, this dietary pattern led to a reduction in key meat quality indicators. In conclusion, the yak rumen microbiome employs distinct adaptive strategies under different feeding systems, thereby exerting differential effects on meat quality.

Abstract Background Seed aging, a natural and inevitable process occurring during storage. Oats, an annual herb belonging to the Gramineae family and pooideae. In addition to being a healthy food, oats serve as ecological pastures, combating soil salinization and desertification. They also play a role in promoting grassland agriculture and supplementing winter livestock feed. However, the high lipid and fat derivatives contents of oat seeds make them susceptible to deterioration, as fat derivatives are prone to rancidity, affecting oat seed production, storage, development, and germplasm resource utilization. Comparative studies on the effects of aging on physiology and cytological structure in covered and naked oat seeds are limited. Thus, our study aimed to determine the mechanism underlying seed deterioration in artificially aged ‘LongYan No. 3’ (A. sativa) and ‘BaiYan No. 2’ (A. nuda) seeds, providing a basis for the physiological evaluation of oat seed aging and serving as a reference for scientifically safe storage and efficient utilization of oats. Results In both oat varieties, superoxide dismutase and catalase activities in seeds showed increasing and decreasing trends, respectively. Variance analysis revealed significant differences and interaction in all measured indicators of oat seeds between the two varieties at different aging times. ‘LongYan No. 3’ seeds, aged for 24–96 h, exhibited a germination rate of < 30%, Conductivity, malondialdehyde, soluble sugar, and soluble protein levels increased more significantly than the ‘BaiYan No. 2’. With prolonged aging leading to cell membrane degradation, reactive oxygen species accumulation, disrupted antioxidant enzyme system, evident embryo cell swelling, and disordered cell arrangement, blocking the nutrient supply route. Simultaneously, severely concentrated chromatin in the nucleus, damaged mitochondrial structure, and impaired energy metabolism were noted, resulting in the loss of ‘LongYan No. 3’ seed vitality and value. Conversely, ‘BaiYan No. 2’ seeds showed a germination rate of 73.33% after 96 h of aging, consistently higher antioxidant enzyme activity during aging, normal embryonic cell shape, and existence of the endoplasmic reticulum. Conclusions ROS accumulation and antioxidant enzyme system damage in aged oat seeds, nuclear chromatin condensation, mitochondrial structure damage, nucleic acid metabolism and respiration weakened, oat seed vigor decreased. ‘LongYan No. 3’ seeds were more severely damaged under artificial aging than ‘BaiYan No. 2’ seeds, highlighting their heightened susceptibility to aging effects.

Evaluating key traits of male sterile mutant accessions in rhizomatous alfalfa (Medicago sativa L.) is crucial for selecting plants for artificial hybrid breeding of rhizomatous maternal lines. In this study, branch cuttings from four male sterile mutant accessions of ‘Qingshui’ alfalfa were used as experimental samples. We evaluated phenotypic traits, which included pollen viability and stigma receptivity, as well as nutritional quality, using difference analysis, correlation analysis, and principal component analysis. Prioritizing pollen viability and stigma receptivity, while considering phenotypic traits and nutritional quality as supplementary factors, allowed us to comprehensively evaluate 24 rhizomatous alfalfa individuals. This evaluation led to the identification of four male sterile mutant accessions with superior traits. The pollen from accession 4-4 was found to be partially fertile, whereas the remaining 23 alfalfa individuals were entirely male sterile. All 24 individuals exhibited stigma receptivity levels suitable for effective pollination. Principal component analysis revealed that among the assessed traits, the leaf–stem ratio contributed most significantly, followed by crude protein content, while neutral detergent fiber content had the least impact on overall quality. Additionally, the number of branches showed a strong positive correlation with individual plant yield (p < 0.01). No significant correlations were detected among plant height, stem diameter, forage grading index, crude protein, neutral detergent fiber, acid detergent fiber content, and yield. Overall, our comprehensive evaluation suggests that accessions 1-2, 2-2, 3-1, and 4-3 are most suitable for use as parental lines in artificial hybrid breeding.

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 mu molg(-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.

IntroductionAlfalfa (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. MethodsIn 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. ResultsThe 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. DiscussionIn conclusion, exogenous Spm enhanced drought resistance of alfalfa leaves under drought stress.

Megachile saussurei (Hymenoptera, Megachilidae) is a primary insect pollinator of alfalfa (Medicago sativa L.) in northwestern China. However, the mechanisms underlying the olfactory responses of M. saussurei induced by alfalfa volatiles is still unclear. Here, the interaction between MsauOBP4 and alfalfa floral volatiles was first elucidated. Results suggested that thirty-two alfalfa floral volatiles were identified and MsauOBP4 was successfully expressed with the consistent molecular mass as predicted results. MsauOBP4 displayed a broad binding spectrum to 32 volatiles, among which MsauOBP4 showed the strongest binding ability to (Z)-3-Hexen-1-ol. In the Y-tube olfactometer behavioral bioassay, M. saussurei elicited the most significant behavioral preference (Z)-3-Hexen-1-ol. MsauOBP4 showed an optimal binding feature to (Z)-3-Hexen-1-ol and valine was the key residue in binding the ligands. After silencing the MsauOBP4, the preference and EAG values of M. saussurei to (Z)-3-Hexen-1-ol were significantly decreased and selection rate of M. saussurei to alfalfa flowers dropped to 57.50 % from 83.33 %. These findings indicated that (Z)-3-Hexen-1-ol is a crucial component in the host location process mediated by MsauOBP4.