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IntroductionSalt stress significantly affects plant growth, and Na+ has gained attention for its potential to enhance plant adaptability to saline conditions. However, the interactions between Na+, plants, and rhizosphere bacterial communities remain unclear, hindering a deeper understanding of how Na+ contributes to plant resilience under salt stress.MethodsThis study aimed to investigate the mechanisms through which Na+ promotes alfalfa's adaptation to salt stress by modifying rhizosphere bacterial communities. We examined the metabolic activity and community composition of both plant and rhizosphere bacteria under Na+ treatment.Results and discussionOur results revealed significant changes in the metabolism and community composition of both plant and rhizosphere bacteria following Na+ addition. Na+ not only promoted the growth of rhizosphere bacteria but also induced shifts in the plant-associated bacterial community, increasing the abundance of bacterial species linked to alfalfa's resistance to salt stress. Furthermore, the chemical characteristics of alfalfa were strongly correlated with the composition and network complexity of both plant and rhizosphere bacterial communities. These interactions suggest that Na+ plays a crucial role in enhancing alfalfa’s adaptability to salt stress by fostering beneficial bacterial communities in the rhizosphere. This finding highlights the potential of leveraging Na+ interactions with plant-microbe systems to improve crop resilience and productivity in saline agricultural environments.

Alfalfa (Medicago sativa L.) establishment is an effective strategy for grassland reconstruction in degraded ecosystems. However, the mechanisms underlying vegetation succession in reconstructed grasslands following alfalfa establishment remain elusive. In this study, we investigated vegetation community, soil quality and rhizosphere microbiota dynamics across a reconstructed grassland chronosequence in the loess region of Northwest China. A space-for-time substitution method was used to evaluate grassland vegetation coverage and alfalfa production performance in nine stands of different ages (1–50 years old). High-throughput sequencing was conducted to characterise rhizosphere microbial communities associated with alfalfa. The plant heights, yields and stem-to-leaf ratios of alfalfa all peaked in the 7-year-old stand and then decreased in older stands, with Stipa bungeana replacing alfalfa as the dominant species in the 50-year-old stand. Soil bulk density and major nutrient contents were highest in the artificial grassland (1–10 years). Soil enzyme activities (e.g., urease and sucrase) were enhanced in the transitional grassland (10–30 years), accompanied by enrichment of potentially beneficial microbial taxa (e.g., Actinobacteria and Mortierella) and functional fungi (e.g., saprotrophs and symbiotrophs) in the rhizosphere. Soil water content, total porosity and rhizosphere microbial diversity reached their maximum levels in the natural grassland (>30 years). The results indicate that alfalfa establishment alters soil structure and nutrient status in the short term, creating an optimal rhizosphere micro-environment. The improved soil conditions and rhizosphere microbiota are favourable for subsequent establishment of native grass species, leading to the formation of a stable semi-natural grasslands.

The key to restoring arid and semi-arid ecosystems is maintaining soil water and organic carbon contents. Alfalfa (Medicago sativa L.) is a high-yield perennial forage crop and performs ecological functions as a drought-resistance leguminous herb. It has been widely planted for reconstruction of degraded soils in the Loess Plateau in northwestern China, but long-term planting may affect soil carbon–water coupling and lead to crop yield reduction. To maximize the benefits of reconstructed grassland, this study explored the couplings of soil water, organic carbon, and alfalfa productivity along a reconstruction chronosequence in a semi-arid area of the Loess Plateau. Space-for-time substitution approach was used to select different-aged stands of reconstructed grassland (1, 5, 7, 10, 15, 20, 30 years old). Alfalfa above-ground biomass (AGB), soil water storage (SWS), organic carbon storage (SOCS), and carbon–water coupling coordination degree (D) were measured in the 0–100 cm soil profile. Alfalfa AGB reached a peak in the 7th year, and the degradation began in the 10th year. Both SWS and SOCS varied nonlinearly with stand age. The greatest loss of SWS occurred in the 15th year (80–100 cm depth), whereas the largest increase of SOCS occurred in the 30th year (0–20 cm depth). There was a negative feedback relationship between AGB and SWS over the 30-year study period (Pearson r = −0.835, P = 0.098). AGB and SOCS initially showed a trade-off within the first 10 years (Pearson r = −0.7431, P = 0.2569), in contrast to their positive feedback in the 20–30th years (Pearson r = 0.9978, P = 0.0421). A decoupling between SWS and SOCS (D < 0.6) was observed after 12 years of alfalfa planting. For agricultural production, a greater supply of water and organic fertilizer is required from the 7th year of alfalfa planting, and reseeding may be needed around the 10th year to prolong the life of alfalfa community. Alfalfa should be planted for no more than 12 consecutive years in the study area for ecological protection.

Abstract Background Auxin/induced-3-acetic acid (Aux/IAA) is an important plant hormone that affects plant growth and resistance to abiotic stresses. Drought stress is a vital factor in reducing plant biomass yield and production quality. Alfalfa (Medicago sativa L.) is the most widely planted leguminous forage and one of the most economically valuable crops in the world. Aux/IAA is one of the early responsive gene families of auxin, playing a crucial role in response to drought stress. However, the characteristics of the Aux/IAA gene family in alfalfa and its potential function in response to drought stress are still unknown. Result A total of 41 Aux/IAA gene members were identified in alfalfa genome. The physicochemical, peptide structure, secondary and tertiary structure analysis of proteins encoded by these genes revealed functional diversity of the MsIAA gene. A phylogenetic analysis classified the MsIAA genes into I-X classes in two subgroups. And according to the gene domain structure, these genes were classified into typical MsIAA and atypical MsIAA. Gene structure analysis showed that the MsIAA genes contained 1–4 related motifs, and except for the third chromosome without MsIAAs, they were all located on 7 chromosomes. The gene duplication analysis revealed that segmental duplication and tandem duplication greatly affected the amplification of the MsIAA genes. Analysis of the Ka/Ks ratio of duplicated MsAux/IAA genes suggested purification selection pressure was high and functional differences were limited. In addition, identification and classification of promoter cis-elements elucidated that MsIAA genes contained numerous elements associated to phytohormone response and abiotic stress response. The prediction protein–protein interaction network showed that there was a complex interaction between the MsAux/IAA genes. Gene expression profiles were tissue-specific, and MsAux/IAA had a broad response to both common abiotic stress (ABA, salt, drought and cold) and heavy metal stress (Al and Pb). Furthermore, the expression patterns analysis of 41 Aux/IAA genes by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Aux/IAA genes can act as positive or negative factors to regulate the drought resistance in alfalfa. Conclusion This study provides useful information for the alfalfa auxin signaling gene families and candidate evidence for further investigation on the role of Aux/IAA under drought stress. Future studies could further elucidate the functional mechanism of the MsIAA genes response to drought stress.

Abstract Background B-box (BBX) family is a class of zinc finger transcription factors (TFs) that play essential roles in regulating plant growth, development, as well as abiotic stress. However, no systematic analysis of BBX genes has yet been conducted in alfalfa (Medica go sativa L.), and their functions have not been elucidated up to now. Results In this study, 28 MsBBX genes were identified from the alfalfa genome, which were clustered into 4 subfamilies according to an evolutionary tree of BBX proteins. Exon-intron structure and conserved motif analysis reflected the evolutionary conservation of MsBBXs in alfalfa. Collinearity analysis showed that segmental duplication promoted the expansion of the MsBBX family. Analysis of cis-regulatory elements suggested that the MsBBX genes possessed many growth/development-, light-, phytohormone-, and abiotic stress-related elements. MsBBX genes were differentially expressed in leaves, flowers, pre-elongated stems, elongated stems, roots and nodules, and most MsBBXs were remarkably induced by drought, salt and various plant growth regulators (ABA, JA, and SA). Further functional verification demonstrated that overexpressing of the MsBBX11 gene clearly promoted salt tolerance in transgenic Arabidopsis by regulating growth and physiological processes of seedlings. Conclusions This research provides insights into further functional research and regulatory mechanisms of MsBBX family genes under abiotic stress of alfalfa.

The growth and biological decline of alfalfa may be linked to the rhizosphere microbiome. However, plant–microbe interactions in the rhizosphere of alfalfa and associated microbial community variations with stand age remain elusive. This study explored the successional pattern of rhizosphere microbial communities across different aged alfalfa stands and its relationship with alfalfa decline. Rhizosphere soils were collected from 2- and 6-year-old alfalfa stands. Control soils were collected from interspaces between alfalfa plants in the same stands. Soil bacterial and fungal communities were characterized by 16S and ITS rRNA gene sequencing, respectively. Specific microbial taxa colonized the rhizosphere soils, but not the control soils. The rhizosphere-specific taxa mainly included potentially beneficial genera (e.g., Dechloromonas, Verrucomicrobium) in the young stand and harmful genera (e.g., Peziza, Campylocarpon) in the old stand. Alfalfa roots regulated soil microbial communities by selective promotion or inhibition of distinct taxa. The majority of time-enriched taxa were reported as harmful fungi, whose relative abundances were negatively correlated with plant traits. Time-depleted taxa were mostly known as beneficial bacteria, which had relative abundances positively correlated with plant traits. The relative abundances of functional bacterial genes associated with vancomycin biosynthesis, zeatin biosynthesis, and amino acid metabolism trended lower in rhizosphere soils from the old stand. An upward trend was observed for fungal pathogens and wood saprotrophs with increasing stand age. The results suggest that root activity drives the negative succession of rhizosphere microbial communities during alfalfa decline in old stands.

Soil salinity is a major factor threatening the production of crops around the world. Smooth bromegrass (Bromus inermis L.) is a high-quality grass in northern and northwestern China. Currently, selecting and utilizing salt-tolerant genotypes is an important way to mitigate the detrimental effects of salinity on crop productivity. In our research, salt-tolerant and salt-sensitive varieties were selected from 57 accessions based on a comprehensive evaluation of 22 relevant indexes, and their salt-tolerance physiological and molecular mechanisms were further analyzed. Results showed significant differences in salt tolerance between 57 genotypes, with Q25 and Q46 considered to be the most salt-tolerant and salt-sensitive accessions, respectively, compared to other varieties. Under saline conditions, the salt-tolerant genotype Q25 not only maintained significantly higher photosynthetic performance, leaf relative water content (RWC), and proline content but also exhibited obviously lower relative conductivity and malondialdehyde (MDA) content than the salt-sensitive Q46 (p < 0.05). The transcriptome sequencing indicated 15,128 differentially expressed genes (DEGs) in Q46, of which 7,885 were upregulated and 7,243 downregulated, and 12,658 DEGs in Q25, of which 6,059 were upregulated and 6,599 downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the salt response differences between Q25 and Q46 were attributed to the variable expression of genes associated with plant hormone signal transduction and MAPK signaling pathways. Furthermore, a large number of candidate genes, related to salt tolerance, were detected, which involved transcription factors (zinc finger proteins) and accumulation of compatible osmolytes (glutathione S-transferases and pyrroline-5-carboxylate reductases), etc. This study offers an important view of the physiological and molecular regulatory mechanisms of salt tolerance in two smooth bromegrass genotypes and lays the foundation for further identification of key genes linked to salt tolerance.

Alfalfa is an excellent leguminous forage crop that is widely cultivated worldwide, but its yield and quality are often affected by drought and soil salinization. Hyperosmolality-gated calcium-permeable channel (OSCA) proteins are hyperosmotic calcium ion (Ca2+) receptors that play an essential role in regulating plant growth, development, and abiotic stress responses. However, no systematic analysis of the OSCA gene family has been conducted in alfalfa. In this study, a total of 14 OSCA genes were identified from the alfalfa genome and classified into three groups based on their sequence composition and phylogenetic relationships. Gene structure, conserved motifs and functional domain prediction showed that all MsOSCA genes had the same functional domain DUF221. Cis-acting element analysis showed that MsOSCA genes had many cis-regulatory elements in response to abiotic or biotic stresses and hormones. Tissue expression pattern analysis demonstrated that the MsOSCA genes had tissue-specific expression; for example, MsOSCA12 was only expressed in roots and leaves but not in stem and petiole tissues. Furthermore, RT–qPCR results indicated that the expression of MsOSCA genes was induced by abiotic stress (drought and salt) and hormones (JA, SA, and ABA). In particular, the expression levels of MsOSCA3, MsOSCA5, MsOSCA12 and MsOSCA13 were significantly increased under drought and salt stress, and MsOSCA7, MsOSCA10, MsOSCA12 and MsOSCA13 genes exhibited significant upregulation under plant hormone treatments, indicating that these genes play a positive role in drought, salt and hormone responses. Subcellular localization results showed that the MsOSCA3 protein was localized on the plasma membrane. This study provides a basis for understanding the biological information and further functional analysis of the MsOSCA gene family and provides candidate genes for stress resistance breeding in alfalfa.

The safety and reliability of battery storage systems are critical to the mass roll-out of electrified transportation and new energy generation. To achieve safe management and optimal control of batteries, the state of charge (SOC) is one of the important parameters. The machine-learning based SOC estimation methods of lithium-ion batteries have attracted substantial interests in recent years. However, a common problem with these models is that their estimation performances are not always stable, which makes them difficult to use in practical applications. To address this problem, an optimized radial basis function neural network (RBF-NN) that combines the concepts of Golden Section Method (GSM) and Sparrow Search Algorithm (SSA) is proposed in this paper. Specifically, GSM is used to determine the optimum number of neurons in hidden layer of the RBF-NN model, and its parameters such as radial base center, connection weights and so on are optimized by SSA, which greatly improve the performance of RBF-NN in SOC estimation. In the experiments, data collected from different working conditions are used to demonstrate the accuracy and generalization ability of the proposed model, and the results of the experiment indicate that the maximum error of the proposed model is less than 2%.

Drought is one of the most common abiotic stressors in plants. Melatonin (MT) is a high-efficiency and low-toxicity growth regulator that plays an important role in plant responses to drought stress. As a wild relative of wheat, Agropyron mongolicum has become an important species for the improvement of degraded grasslands and the replanting of sandy grasslands. However, the physiological and molecular mechanisms by which exogenous MT regulates drought stress in A. mongolicum remain unclear. To assess the effectiveness of MT intervention (100 mg·L−1), polyethylene glycol 6000 was used to simulate drought stress, and its ameliorating effects on drought stress in A. mongolicum seedlings were investigated through physiology, transcriptomics, and metabolomics. Physiological analysis indicated that MT treatment increased the relative water content and chlorophyll content and decreased the relative conductivity of A. mongolicum seedlings. Additionally, MT decreased malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation by enhancing antioxidant enzyme activities. The transcriptome and metabolite profiling analysis of A. mongolicum seedlings treated with and without MT under drought stress identified the presence of 13,466 differentially expressed genes (DEGs) and 271 differentially expressed metabolites (DEMs). The integrated analysis of transcriptomics and metabolomics showed that DEGs and DEMs participated in diverse biological processes, such as flavonoid biosynthesis and carbohydrate metabolism. Moreover, MT may be involved in regulating the correlation of DEGs and DEMs in flavonoid biosynthesis and carbohydrate metabolism during drought stress. In summary, this study revealed the physiological and molecular regulatory mechanisms of exogenous MT in alleviating drought stress in A. mongolicum seedlings, and it provides a reference for the development and utilization of MT and the genetic improvement of drought tolerance in plants from arid habitats.

As a high-quality legume forage, alfalfa is restricted by various abiotic stresses during its growth and development. Melatonin is a multifunctional signaling molecule that involves in plant defense against multiple stresses. However, little is known about its downstream signaling pathway and regulatory mechanisms in salt stress of alfalfa. In this study, we investigated the protective effects and key regulatory pathways of melatonin on alfalfa under salt tolerance. The results showed that melatonin promoted the growth of alfalfa seedlings under salt stress, as demonstrated by higher plant height, leaf area, and fresh weight. Melatonin treatment resulted in an increase in the photosynthetic capacity and starch content of alfalfa. Moreover, melatonin decreased cell membrane damage and reactive oxygen species (ROS) accumulation by enhancing antioxidant defense activity under salt stress conditions. Transcriptome sequencing (RNA-seq) analysis revealed that melatonin mainly induced the transcription of genes involved in Ca2+ signaling (cyclic nucleotide gated channel, CNGCs; cam modulin/calmodulin-like protein, CAM/CMLs and calcium-dependent protein kinase, CDPKs), starch and sucrose metabolism (α-amylase, AMYs; β-amylase, BAMs; starch synthase, SSs and sucrose synthase, SUSs), plant hormone signal transduction (auxin/indole acetic acid protein, AUX/IAAs; ABA receptor, PYL4; protein phosphatase 2C, PP2Cs; scarecrow-like protein, SCLs and ethylene-responsive transcription factor 1B, ERF1B), and key transcription factors (C3Hs, MYBs, ERFs, and WRKYs). Specifically, we focused on starch and sucrose metabolism and plant hormone signal transduction pathways. The interactions between melatonin and other phytohormones occurred via regulation of the expression of genes involved in hormone signaling pathways. In addition, melatonin increased the contents of endogenous melatonin, auxin, gibberellic acid (GA3), salicylic acid, brassinosteroids, and ethylene, while decreasing the abscisic acid content under salt stress. In summary, this study established a regulatory network for melatonin-induced key signaling pathways and functional genes under salt stress and provided a theoretical basis for salt tolerance breeding in alfalfa.

Alfalfa (Medicago sativa L.) is an important forage crop, and its productivity is severely affected by salt stress. Although proline is a compatible osmolyte that plays an important role in regulating plant abiotic stress resistance, the basic mechanism of proline requires further clarification regarding the effect of proline in mitigating the harmful effects of salinity. Here, we investigate the protective effects and regulatory mechanisms of proline on salt tolerance of alfalfa. The results show that exogenous proline obviously promotes seed germination and seedling growth of salt-stressed alfalfa. Salt stress results in stunted plant growth, while proline application alleviates this phenomenon by increasing photosynthetic capacity and antioxidant enzyme activities and decreasing cell membrane damage and reactive oxygen species (ROS) accumulation. Plants with proline treatment maintain a better K+/Na+ ratio by reducing Na+ accumulation and increasing K+ content under salt stress. Additionally, proline induces the expression of genes related to antioxidant biosynthesis (Cu/Zn-SOD and APX) and ion homeostasis (SOS1, HKT1, and NHX1) under salt stress conditions. Proline metabolism is mainly regulated by ornithine-δ-aminotransferase (OAT) and proline dehydrogenase (ProDH) activities and their transcription levels, with the proline-treated plants displaying an increase in proline content under salt stress. In addition, OAT activity in the ornithine (Orn) pathway rather than Δ1-pyrroline-5-carboxylate synthetase (P5CS) activity in the glutamate (Glu) pathway is strongly increased under salt stress, made evident by the sharp increase in the expression level of the OAT gene compared to P5CS1 and P5CS2. Our study provides new insight into how exogenous proline improves salt tolerance in plants and that it might be used as a significant practical strategy for cultivating salt-tolerant alfalfa.