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Key message All ten dehydrin genes from three Medicago species are responsive to different kinds of abiotic stress, and CAS31 confers transgenic plants salt tolerance by down-regulating HKT1 expression. Dehydrins are protective proteins playing crucial roles in the tolerance of plants to abiotic stresses. However, a full-scale and systemic analysis of total dehydrin genes in Medicago at the genome level is still lacking. In this study, we identified ten dehydrin genes from three Medicago species ( M . truncatula , M . ruthenica , and M . sativa ), categorizing the coding proteins into four types. Genome collinearity analysis among the three Medicago species revealed six orthologous gene pairs. Promoter regions of dehydrin genes contained various phytohormone- and stress-related cis -elements, and transcriptome analysis showed up-regulation of all ten dehydrin genes under different stress conditions. Transformation of dehydrin gene CAS31 increased the tolerance of transgenic seedlings compared with wild-type seedlings under salt stress. Our study demonstrated that transgenic seedlings maintained the more chlorophyll, accumulated more proline and less hydrogen peroxide and malondialdehyde than wild-type seedlings under salt stress. Further study revealed that CAS31 reduced Na + accumulation by down-regulating HKT1 expression under salt stress. These findings enhance our understanding of the dehydrin gene family in three Medicago species and provide insights into their mechanisms of tolerance.

Twelve alfalfa genotypes that were selected for biomass under salinity, differences in Na and Cl concentrations in shoots and K/Na ratio were evaluated in this long-term salinity experiment. The selected plants were cloned to reduce genetic variability within each genotype. Salt tolerance (ST) index of the genotypes ranged from 0.39 to 1. The most salt-tolerant genotypes SISA14-1 (G03) and AZ-90ST (G10), the top performers for biomass, exhibited the least effect on shoot number and height. SISA14-1 (G03) accumulated low Na and Cl under salinity. Most genotypes exhibited a net reduction in shoot Ca, Mg, P, Fe, and Cu, while Mn and Zn increased under salinity. Salinity reduced foliar area and stomatal conductance; while net photosynthetic rate and transpiration were not affected. Interestingly, salinity increased chlorophyll and antioxidant capacity in most genotypes; however neither parameter correlated well to ST index. Salt-tolerant genotypes showed upregulation of the SOS1, SOS2, SOS3, HKT1, AKT1, NHX1, P5CS1, HSP90.7, HSP81.2, HSP71.1, HSPC025, OTS1, SGF29 and SAL1 genes. Gene expression analyses allowed us to classify genotypes based on their ability to regulate different components of the salt tolerance mechanism. Pyramiding different components of the salt tolerance mechanism may lead to superior salt-tolerant alfalfa genotypes.

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.

Background Soil salinity is an important factor affecting growth, development, and productivity of almost all land plants, including the forage crop alfalfa ( Medicago sativa ). However, little is known about how alfalfa responds and adapts to salt stress, particularly among different salt-tolerant cultivars. Results Among seven alfalfa cultivars, we found that Zhongmu-1 (ZM) is relatively salt-tolerant and Xingjiang Daye (XJ) is salt-sensitive. Compared to XJ, ZM showed slower growth under low-salt conditions, but exhibited stronger tolerance to salt stress. RNA-seq analysis revealed 2237 and 1125 differentially expressed genes (DEGs) between ZM and XJ in the presence and absence of salt stress, among which many genes are involved in stress-related pathways. After salt treatment, compared with the controls, the number of DEGs in XJ (19373) was about four times of that in ZM (4833). We also detected specific differential gene expression patterns: In response to salt stress, compared with XJ, ZM maintained relatively more stable expression levels of genes related to the ROS and Ca 2+ pathways, phytohormone biosynthesis, and Na + /K + transport. Notably, several salt resistance-associated genes always showed greater levels of expression in ZM than in XJ, including a transcription factor. Consistent with the suppression of plant growth resulting from salt stress, the expression of numerous photosynthesis- and growth hormone-related genes decreased more dramatically in XJ than in ZM. By contrast, the expression levels of photosynthetic genes were lower in ZM under low-salt conditions. Conclusions Compared with XJ, ZM is a salt-tolerant alfalfa cultivar possessing specific regulatory mechanisms conferring exceptional salt tolerance, likely by maintaining high transcript levels of abiotic and biotic stress resistance-related genes. Our results suggest that maintaining this specific physiological status and/or plant adaptation to salt stress most likely arises by inhibition of plant growth in ZM through plant hormone interactions. This study identifies new candidate genes that may regulate alfalfa tolerance to salt stress and increases the understanding of the genetic basis for salt tolerance.

Background Alfalfa ( Medicago sativa L.) is a vital forage crop with substantial economic and ecological significance in agriculture and animal husbandry. However, atrazine, a widely used herbicide, negatively impacts the growth and yield of alfalfa due to its residual presence in the environment. Transcriptomic analysis was performed to investigate the differences in tolerance and uncover the potential molecular regulatory mechanisms between the tolerant variety JN5010 and the sensitive variety WL363 when subjected to atrazine stress, using RNA-seq on pooled samples. Results Based on the analysis of gene expression profiles, significant differences were observed between the tolerant variety JN5010 and the sensitive variety WL363 under atrazine stress: 2,297 upregulated and 3,167 downregulated in the shoot parts, and 3,232 upregulated and 4,907 downregulated in the roots of JN5010. In WL363, 2,937 genes were upregulated and 4,237 genes were downregulated in the shoot parts, while 5,316 genes were upregulated and 7,977 genes were downregulated in the roots. The DEGs in the shoot parts were mainly involved in biological regulation, metabolic processes, and cellular processes, including proline metabolic processes and S-adenosylmethionine cycle. The DEGs in the roots were predominantly associated with nitric oxide synthesis and metabolism, as well as processes related to cell wall biosynthesis and degradation. In the shoot parts of JN5010, six DEGs were mapped onto the proline metabolic pathway, including four upregulated genes involved in proline biosynthesis and two downregulated genes involved in proline catabolism. In the roots of WL363, eleven DEGs were mapped onto the phenylpropanoid biosynthesis pathway, including seven upregulated genes involved in flavonoid biosynthesis and four downregulated genes associated with lignin biosynthesis. These findings highlight the distinct genetic responses of the two alfalfa varieties to atrazine stress, with JN5010 exhibiting more consistent gene expression patterns compared to the sensitive variety WL363. Conclusions The tolerant variety JN5010 shows improved tolerance to atrazine stress by maintaining stable gene expression and precise regulation in various pathways, such as antioxidant processes, signaling, photosynthesis, and toxin removal. This differential gene expression helps JN5010 maintain stability in its functions under stress, demonstrating better adaptability. These findings enhance our understanding of how alfalfa tolerates atrazine stress and provide important insights for developing atrazine-tolerant varieties.

Applying non-thermal plasma (NTP) to seeds prior to sowing is recognized for its ability to enhance germination and promote plant growth. This study investigated the effects of NTP seed treatment on alfalfa seed surface characterization, germination, growth, and biochemical traits under varying water conditions. NTP modified seed surface properties by decreasing water contact angle, roughening the coat, and reducing O–H/N–H and C–H band intensities, while major functional groups remained intact. Short plasma exposures (<2 min) enhanced germination, whereas prolonged treatment (10 min) reduced viability, indicating embryo sensitivity. In pot experiments, both 1 and 5 min treatments improved fresh and dry weight, stem and root elongation, pigment accumulation, and protein content, particularly under normal and moderate water stress, while extended exposure (10 min) offered limited benefits and could be detrimental under severe drought. Root growth was most responsive, suggesting enhanced water and nutrient uptake. Plasma had modest effects on polyphenols and flavonoids but influenced early physiological responses and antioxidant activity. These findings highlight NTP as a promising seed priming tool to improve alfalfa performance, though further studies are needed to clarify the mechanisms and specific contributions of plasma components.

Alfalfa ( Medicago sativa ) is known to release allelopathic substances to affect the germination and growth of other plants, which have the potential to be applied in controlling weeds. Green foxtail ( Setaria viridis ) and barnyardgrass ( Echinochloa crus-galli ), as malignant weeds worldwide, also pose a serious threat to alfalfa in northern China. In this study, the sensitivity of the two weeds to the extracts from the first, second, and third stubbles of six varieties were investigated to further reveal the allelopathic interference of different varieties of alfalfa on notorious weeds. The germination rate, the length and fresh weight of seedlings, the length and fresh weight of roots were measured to elucidate the allelopathy of alfalfa extracts on the two weeds. The results suggested that: (1) The allelopathy of six alfalfa varieties tested showed obvious intraspecific differences, the inhibition of Zhongmu No.3 on green foxtail and barnyardgrass was weaker than other varieties, with the values of synthetical allelopathic effect (SAE) were -0.55 and -0.29, respectively. (2) The inhibitory effect of alfalfa extracts on green foxtail was enhanced with the increase of stubbles, while the differences between three stubbles on barnyardgrass were not clear, especially between the first and second stubbles. (3) Compared with barnyardgrass (SAE = -0.39 ~ -0.29), green foxtail (SAE = -0.65 ~ -0.52) was generally more susceptible to the extracts. (4) The inhibitory effect of alfalfa extracts on root was stronger than seedling in the same weed. For example, the third stubble extracts of Baoding variety inhibited 88.00% of the roots at the concentration of 0.01 g mL -1 , but did not affect the seedlings of green foxtail. The study may help to comprehensively reveal the allelopathic effect of different alfalfa varieties in the first three stubbles on green foxtail and barnyardgrass, providing scientific evidence for weed control based on natural plant extracts in the future.

Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were grown in Cd-added soils. The fungus significantly colonized alfalfa roots by day 25 after planting. Colonization of alfalfa by G. intraradices in soils contaminated with Cd ranged from 17% to 69% after 25-60 days and then decreased to 43%. The biomass of plant shoots with AM fungi showed significant 1.7-fold increases compared to no AM fungi addition under the treatment of 20 mg kg(-1) Cd. Concentrations of Cd in the shoots of alfalfa under 0.5, 5, and 20 mgkg(-1) Cd without AM fungal inoculation are 1.87, 2.92, and 2.38 times higher, respectively, than those of fungi-inoculated plants. Fungal inoculation increased Cd (37.2-80.5%) in the cell walls of roots and shoots and decreased in membranes after 80 days of incubation compared to untreated plants. The proportion of the inactive forms of Cd in roots was higher in fungi-treated plants than in controls. Furthermore, although fungi-treated plants had less overall Cd in subcellular fragments in shoots, they had more inactive Cd in shoots than did control plants. These results provide a basis for further research on plant-microbe symbioses in soils contaminated with heavy metals, which may potentially help us develop management regimes for phytoremediation.

Aluminum toxicity severely inhibits root elongation and nutrient uptake, causing global agricultural yield losses. Dissolved Al3+ are accumulating in plants and subsequently entering food chains via crops and forage plants. Chronic dietary exposure to Al3+ poses a risk to human health. In this study, Pseudomonas sp. strain ADAl3–4, isolated from plant rhizosphere soil, significantly enhanced plant development and biomass. Phenotypic validation using Arabidopsis mutants showed that strain ADAl3–4 regulates plant growth and development under aluminum stress by reprogramming the cell cycle, regulating auxin and ion homeostasis, and enhancing the root absorption of Al3+ from the soil. Transcriptomic and biochemical analyses showed that strain ADAl3–4 promotes plant growth via regulating signal transduction, phytohormone biosynthesis, flavonoid biosynthesis, and antioxidant capacity, etc., under aluminum stress. Our findings indicate that Pseudomonas sp. strain ADAl3–4 enhances plant development and stress resilience under Al3+ toxicity through a coordinated multi-dimensional regulatory network. Furthermore, strain ADAl3–4 promoted the root absorption of aluminum rather than the transportation of Al to the aerial part, endowing it with application prospects.

This paper investigates how plants respond to stress caused by asbestos cement products in irrigation water. It presents a thorough evaluation of the exposure and risk factors for plants, water, and soil when exposed to these materials. The experimental results provide empirical evidence of plant stress responses based on physiological and germination parameters. The research is motivated by concerns about environmental contamination from asbestos cement in irrigation water, which can be toxic to plants and lead to soil pollution, negatively impacting vegetation and soil quality. When exposed to asbestos in water, plants experience toxic stress that can inhibit photosynthesis, nutrient uptake, and germination. Asbestos can also adversely affect cell division and metabolism, risking plant growth, reproduction, and overall health, as well as making them more susceptible to disease and pests under environmental stress. The paper examines the impact on germination and physiological parameters of Trifolium pratense , Medicago sativa , and Solanum lycopersicum , particularly how they were affected by pre-established concentrations of irrigation water mixed with asbestos cement during a controlled germination experiment. The research methodology was developed in the absence of established global practices, standards, and methods, creating an opportunity for further methodological advancement. The findings could serve as a situational analysis for professionals in environmental plant protection and analytical fields.