Cultivating new alfalfa (Medicago sativa L.) varieties with high yield and quality is of great significance for improving alfalfa yield and promoting the development of the grass and livestock industry. Plant height is an important indicator of alfalfa yield and is closely related to photosynthetic capacity, harvest index and yield. However, the underlying cause of the variation in height among alfalfa plants is not clear. In this paper, we measured the phenotypic traits, photosynthetic physiology and endogenous hormone content of tall- and short-stalked alfalfa materials and analyzed the important external and internal factors that caused the difference in plant height of alfalfa. We found that the phenotypic traits of tall- and short-stalked alfalfa materials showed significant differences, and dwarf alfalfa showed significant shortening of the main stem internode length. There were also some differences in light and physiological indicators and endogenous hormone contents between tall- and short-stalked alfalfa materials. Through correlation analysis, we found that the phenotypic traits and physiological indicators significantly correlated with alfalfa plant height were the number of internodes, stem diameter, average internode length, leaf–stem ratio, leaf area, Pn (net photosynthetic rate), Tr (transpiration rate), upper leaf SP (soluble protein), Suc (sucrose) content, middle stem Sta (starch) content, middle stem ZT (zeatin) and IAA (indole-3-acetic acid). Further analysis showed that Tr, IAA and LA played a direct role in plant height, with Tr contributing the most to plant height, followed by IAA. Finally, we found that the starch content of the middle stem had a significant impact on plant height through principal component analysis. These results provide new insights into the formation and genetic improvement of plant height traits in leguminous forages such as alfalfa.

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.

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.

Fatty acid and central carbon metabolism are crucial energy metabolism reactions. However, to date, few studies have examined their distribution characteristics within the alfalfa–rhizobia symbiotic system. To clarify the distributional differences and accumulation rates of fatty acids and central carbon with this system, we measured the plant phenotype, nodule formation, nitrogen fixation capacity, and key nitrogen metabolism enzyme activities of Medicago sativa ‘Gannong No. 9’ 35 days post-inoculation (dpi) with Sinorhizobia meliloti LL11. Additionally, we employed targeted metabolomics to analyze central carbon and fatty acid metabolites in various tissue samples of symbiotic and control (C.K.) plants, as well as in S. meliloti LL11. We found that plant height; root length; aboveground fresh and dry weights; underground fresh and dry weights; and nitrate reductase, nitrogen reductase, glutamine synthetase, and glutamate synthase activities were significantly higher in the leaves and roots of symbiotic plants than in those of C.K. plants. Compared to symbiotic plants, C.K. plants exhibited higher total central carbon and fatty acid metabolite content, accounting for 38.61% and 48.17% of C.K. plants, respectively. We detected 32 central carbon and 40 fatty acid metabolites in S. meliloti LL11, with succinate (343,180.8603 ng·mL⁻¹) and hexadecanoic acid (4889.7783 ng·mL⁻¹) being the most. In both symbiotic and C.K. plants, central carbon metabolite was considerably higher than the fatty acid metabolite central. Moreover, the carbon metabolites found in symbiotic plants were primarily distributed in pink nodule roots (PNRs), with malate exhibiting the highest content (4,800,612.3450 ng·g⁻¹), accounting for 53.09% of total central carbon metabolite content. Fatty acid metabolites were mainly found in pink root nodules (P.N.s), which are sites of nitrogen fixation. Trans-10-nonadecenoic acid and hexadecanoic acid exhibited the highest contents, comprising >15% of the total fatty acid metabolite content. We found that petroselaidic acid is only present in P.N., which seems to be closely related to the nitrogen fixation reaction in P.N. In general, symbiotic plants transfer central carbon metabolites to nodules via PNRs to drive nitrogen fixation. However, in P.N.s, these metabolites are limited, leading to accumulation in PNRs. Fatty acid metabolites, crucial for nitrogen fixation, are prevalent in P.N.s. Conversely, C.K. plants without nitrogen fixation distribute these metabolites primarily to the stems, emphasizing growth. This study provides new insights into the energy metabolism of symbiotic nitrogen fixation.

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.

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.