Functions of the Sinorhizobium meliloti LsrB Substrate-Binding Domain in Oxidized Glutathione Resistance, Alfalfa Nodulation Symbiosis, and Growth.

To successfully colonize legume root nodules, rhizobia must effectively evade host-generated reactive oxygen species (ROS). LsrB, a redox regulator from Sinorhizobium meliloti, is essential for symbiosis with alfalfa (Medicago sativa). The three cysteine residues in LsrB's substrate domain play distinct roles in activating downstream redox genes. The study found that LsrB's substrate-binding domain, dependent on the cysteine residue Cys146, is involved in oxidized glutathione (GSSG) resistance and alfalfa nodulation symbiosis. LsrB homologues from other rhizobia, with Cys172/Cys238 or Cys146, enhance GSSG resistance and complement lsrB mutant's symbiotic nodulation. Substituting amino acids in Azorhizobium caulinodans LsrB with Cys restores lsrB mutant phenotypes. The lsrB deletion mutant shows increased sensitivity to NCR247, suggesting an interaction with host plant-derived NCRs in alfalfa nodules. Our findings reveal that the key cysteine residue in the LsrB's substrate domain is vital for rhizobium-legume symbiosis.

Screening of mtr-miR156a from exosomes of dairy cow blood to milk and its regulatory effect on milk protein synthesis in BMECs.

MicroRNA (miRNA) is a type of endogenous non-coding small RNA, which is abundant in living organisms. miRNAs play an important role in regulating gene expression and myriad cellular processes by binding to target messenger RNAs through complementary base pairing, and cross-species regulation mammalian cells by plant-derived xeno-miRNAs has been described. Here, we examined the miRNA species in two alfalfa (Medicago sativa, lucerne) cultivars commonly grown in Ningxia, China: cv. Zhongmu 1 and cv. Xinyan 52. Both cultivars have good salt and drought resistance. We found that the miRNA profiles were similar between the cultivars, with a slightly higher number of miRNAs present in the newer cv. Xinyan 52, which may contribute to its improved salt and drought tolerance. miRNAs were stable during drying, and some miRNAs were increased in dry versus fresh alfalfa, suggesting some miRNAs may be upregulated during drying. Alfalfa-derived miRNAs could be detected in exosomes from serum and whey collected from dairy cows, confirming the ability of the exogenous miRNAs (xeno-miRNAs) to enter the circulation and reach the mammary epithelium. In vitro studies confirmed that overexpression of mtr-miR156a could downregulate expression of Phosphatase 2 Regulatory Subunit B'gamma ( PPP2R5D) and Phosphoinositide-3-kinase Regulatory Subunit 2 (PIK3R2). Overexpression of mtr-miR156a also modulated PI3K-AKT-mTOR signaling as well as the casein content of milk produced by bovine mammary epithelial cells. Based on the known roles of PPP2R5D and PIK3R2 in regulating the PI3K-AKT-mTOR pathway as well as the effect of PI3K-AKT-mTOR on milk protein content, our findings implicate alfalfa-derived miR156a as a new cross-species regulator of milk quality in dairy cows.

MsABCG1, ATP-Binding Cassette G transporter from Medicago Sativa, improves drought tolerance in transgenic Nicotiana Tabacum.

Drought has a devastating impact, presenting a formidable challenge to agricultural productivity and global food security. Among the numerous ABC transporter proteins found in plants, the ABCG transporters play a crucial role in plant responses to abiotic stress. In Medicago sativa, the function of ABCG transporters remains elusive. Here, we report that MsABCG1, a WBC-type transporter highly conserved in legumes, is critical for the response to drought in alfalfa. MsABCG1 is localized on the plasma membrane, with the highest expression observed in roots under normal conditions, and its expression is induced by drought, NaCl and ABA signalling. In transgenic tobacco, overexpression of MsABCG1 enhanced drought tolerance, evidenced by increased osmotic regulatory substances and reduced lipid peroxidation. Additionally, drought stress resulted in reduced ABA accumulation in tobacco overexpressing MsABCG1, demonstrating that overexpression of MsABCG1 enhanced drought tolerance was not via an ABA-dependent pathway. Furthermore, transgenic tobacco exhibited increased stomatal density and reduced stomatal aperture under drought stress, indicating that MsABCG1 has the potential to participate in stomatal regulation during drought stress. In summary, these findings suggest that MsABCG1 significantly enhances drought tolerance in plants and provides a foundation for developing efficient drought-resistance strategies in crops.

Comparative role of calcium oxide nanoparticles and calcium bulk fertilizer to alleviate cadmium toxicity by modulating oxidative stress, photosynthetic performance and antioxidant-defense genes expression in alfalfa.

Cadmium (Cd) toxicity poses a significant threat to soil health and sustainable food production. Its bioaccumulation in plant tissues induces phytotoxicity by affecting physiological and biochemical attributes, leading to a reduction in plant biomass and production. Recently, nanotechnology has emerged as a promising approach for addressing heavy metal toxicity in an eco-friendly manner to enhance crop production. However, the comparative role of foliar applied calcium oxide nanoparticles (CaO-NPs) and bulk calcium fertilizer under Cd stress in alfalfa remains unexplored. Herein, we studied the ameliorative role of CaO-NPs and bulk calcium (50 and 100 mg L-1) to alleviate Cd stress (30 mg kg-1) in alfalfa seedlings. Plants exposed to Cd exhibited significant decreases in morpho-physiological traits, gas exchange attributes, and pigment contents as well as increase in Cd bioaccumulation in plant tissues. Notably, exogenous application of CaO-NPs ameliorates the toxic impact of Cd by enhancing plant biomass (45%), fluorescence efficiency and gaseous exchange attributes. The maximum dose of CaO-NPs induced Cd-tolerance response accompanied by a significant increase in antioxidative enzyme activities, such as superoxide dismutase (SOD; 29%), peroxidase (POD; 41%), catalase (CAT; 36%) and ascorbate peroxidase (APX; 49%), which play positive roles in ROS scavenging. TEM examination further revealed the protective role of these NPs in averting Cd-induced damage to leaf ultrastructure and mesophyll cells. Furthermore, CaO-NPs had a substantial influence on both Cd and Ca2+ accumulation in plant tissues, while qRT‒PCR analysis demonstrated higher expression of antioxidant defense genes viz. Cu/ZnSOD (0.38 fold change (FC)), MtPOD (0.51 FC), MtCAT (0.61 FC) and MtAPX (0.79 FC) under CaO-NPs application, over Cd control. Overall, our findings suggested that exogenous CaO-NPs could be effective in alleviating the adverse effects of Cd on alfalfa seedlings to ensure food safety and support sustainable agriculture.

Bioaugmentation protocols involving Methanobrevibacter thaueri and Pecoramyces ruminantium for investigating lignocellulose degradation and methane production from alfalfa stalks.

Two protocols involving batch cultures were used to investigate the bioaugmentation of methane production by Pecoramyces ruminantium, and Methanobrevibacter thaueri. Protocol I examined the effect of altering the proportion of the microbial constituents in inoculum on alfalfa stalk fermentations and showed a 25 % improvement in dry matter loss in cultures where the inoculum contained just 30 % of co-culture and 70 % of fungal monoculture. Protocol II involved consecutive cultures and alternating inoculations. This protocol resulted in 17-22 mL/g DM methane production with co-cultures a 30 % increase in methane relative to the fungal monoculture. Both protocols indicate that the co-culture rapidly dominated and was more resilient than the monoculture. Synergistic interaction between fungus and methanogen, promoted more efficient lignocellulose degradation and higher methane yield. This study highlighted the potential of microbial co-cultures for enhancing methane production from lignocellulosic biomass, offering a promising bioaugmentation strategy for improving biogas yields and waste valorization.

Performance and mechanistic study of biochar and magnesium-enhanced phytoremediation in cadmium-contaminated soil by alfalfa.

Recently, phytoremediation has been regarded as a green and environment friendly technique to treat metals contaminated soils. Thus, in this study, pot experiments were designed to investigate the combine effects of biochar and magnesium (MPs) to purify cadmium (Cd)-contaminated soils by Medicago sativa L. (alfalfa). The results showed that the combined use of biochar and Mg significantly increased the accumulation of Cd and promoted the transport of Cd from root to shoot in alfalfa, simultaneously. Importantly, the combined use of biochar and Mg could increase the accumulation of Cd in shoot and whole plant (shoot + root) of alfalfa up-to 59.1% and 23.1%, respectively. Moreover, the enhancement mechanism can be analyzed from several aspects. Firstly, the photosynthesis was enhanced, which was beneficial to plant growth. The product of photosynthesis provided energy for uptake and transport of Cd. Meanwhile, its transport in phloem could promote the transport of Cd. Secondly, the enhancement of antioxidant capacity of alfalfa effectively protected the membrane structure of alfalfa, which indicated that Cd could enter alfalfa from the channel on the cell membrane. Lastly, the chemical form of Cd and microbial community structure in soil were changed. Overall, these changes reduced the Cd toxicity in soil, enhanced the resistance capability of alfalfa, increased the Cd uptake by alfalfa and promoted the growth of alfalfa. Thus, the obtained results suggested that the combined use of biochar and Mg is an effective approach to enhance phytoremediation performance for purifying Cd-contaminated soils.

Harnessing plant-beneficial bacterial encapsulation: A sustainable strategy for facilitating cadmium bioaccumulation in Medicago sativa.

Plant-beneficial bacteria (PBB) have emerged as a promising approach for assisting phytoremediation of heavy metal (HM)-contaminated soils. However, their colonization efficiency is often challenged by complex soil environments. In this study, we screened one rhizobacterium (Klebsiella variicola Y38) and one endophytic bacterium (Serratia surfactantfaciens Y15) isolated from HM-contaminated soils and plants for their high resistance to Cd and strong growth-promoting abilities. These strains were encapsulated individually or in combination with alginate and applied with Medicago sativa in Cd-contaminated soil pot experiments. The effectiveness of different bacterial formulations in promoting plant growth and enhancing Cd bioconcentration in M. sativa was evaluated. Results showed that PBB application enhanced plant growth and antioxidant capacity while reducing oxidative damage. Encapsulated formulations outperformed unencapsulated ones, with combined formulations yielding superior results to individual applications. Quantitative PCR indicated enhanced PBB colonization in Cd-contaminated soils with alginate encapsulation, potentially explaining the higher efficacy of alginate-encapsulated PBB. Additionally, the bacterial agents modified Cd speciation in soils, resulting in increased Cd bioaccumulation in M. sativa by 217-337 %. The alginate-encapsulated mixed bacterial agent demonstrated optimal effectiveness, increasing the Cd transfer coefficient by 3.2-fold. Structural equation modeling and correlation analysis elucidated that K. variicola Y38 promoted Cd bioaccumulation in M. sativa roots by reducing oxidative damage and enhancing root growth, while S. surfactantfaciens Y15 facilitated Cd translocation to shoots, promoting shoot growth. The combined application of these bacteria leveraged the benefits of both strains. These findings contribute to diversifying strategies for effectively and sustainably remediating Cd-contaminated soils, while laying a foundation for future investigations into bacteria-assisted phytoremediation.

Genome-Wide Analysis of BBX Gene Family in Three Medicago Species Provides Insights into Expression Patterns under Hormonal and Salt Stresses.

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.

Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicagosativa.

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/µg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/µg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.

MsWRKY44 regulates Mg-K homeostasis of shoots and promotes alfalfa sensitivities to acid and Al stresses.

Mg-K homeostasis is essential for plant response to abiotic stress, but its regulation remains largely unknown. MsWRKY44 cloned from alfalfa was highly expressed in leaves and petioles. Overexpression of it inhibited alfalfa growth, and promoted leaf senescence and alfalfa sensitivities to acid and Al stresses. The leaf tips, margins and interveins of old leaves occurred yellow spots in MsWRKY44-OE plants under pH4.5 and pH4.5 +Al conditions. Meanwhile, Mg-K homeostasis was substantially changed with reduction of K accumulation and increases of Mg as well as Al accumulation in shoots of MsWRKY44-OE plants. Further, MsWRKY44 was found to directly bind to the promoters of MsMGT7 and MsCIPK23, and positively activated their expression. Transiently overexpressed MsMGT7 and MsCIPK23 in tobacco leaves increased the Mg and Al accumulations but decreased K accumulation. These results revealed a novel regulatory module MsWRKY44-MsMGT7/MsCIPK23, which affects the transport and accumulation of Mg and K in shoots, and promotes alfalfa sensitivities to acid and Al stresses.

Heavy fuel oil-contaminated soil remediation by individual and bioaugmentation-assisted phytoremediation with Medicago sativa and with cold plasma-treated M. sativa.

Developing an optimal environmentally friendly bioremediation strategy for petroleum products is of high interest. This study investigated heavy fuel oil (HFO)-contaminated soil (4 and 6 g kg-1) remediation by individual and combined bioaugmentation-assisted phytoremediation with alfalfa (Medicago sativa L.) and with cold plasma (CP)-treated M. sativa. After 14 weeks of remediation, HFO removal efficiency was in the range between 61 and 80% depending on HFO concentration and remediation technique. Natural attenuation had the lowest HFO removal rate. As demonstrated by growth rate and biomass acquisition, M. sativa showed good tolerance to HFO contamination. Cultivation of M. sativa enhanced HFO degradation and soil quality improvement. Bioaugmentation-assisted phytoremediation was up to 18% more efficient in HFO removal through alleviated HFO stress to plants, stimulated plant growth, and biomass acquisition. Cold plasma seed treatment enhanced HFO removal by M. sativa at low HFO contamination and in combination with bioaugmentation it resulted in up to 14% better HFO removal compared to remediation with CP non-treated and non-bioaugmented M. sativa. Our results show that the combination of different remediation techniques is an effective soil rehabilitation strategy to remove HFO and improve soil quality. CP plant seed treatment could be a promising option in soil clean-up and valorization.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased ß-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Overexpression of the alfalfa (Medicago sativa) gene, MsKMS1, negatively regulates seed germination in transgenic tobacco (Nicotiana tabacum).

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-associated proteins are a class of transmembrane proteins involved in intracellular trafficking pathways. However, the functions of many SNARE domain-containing proteins remain unclear. We have previously identified a SNARE-associated gene in alfalfa (Medicago sativa ) KILLING ME SLOWLY1 (MsKMS1 ), which is involved in various abiotic stresses. In this study, we investigated the function of MsKMS1 in the seed germination of transgenic tobacco (Nicotiana tabacum ). Phylogenetic analysis showed that MsKMS1 was homologous to the SNARE-associated or MAPR component-related proteins of other plants. Germination assays revealed that MsKMS1 negatively regulated seed germination under normal, D-mannitol and abscisic acid-induced stress conditions, yet MsKMS1 -overexpression could confer enhanced heat tolerance in transgenic tobacco. The suppressive effect on germination in MsKMS1 -overexpression lines was associated with higher abscisic acid and salicylic acid contents in seeds. This was accompanied by the upregulation of abscisic acid biosynthetic genes (ZEP and NCED ) and the downregulation of gibberellin biosynthetic genes (GA20ox2 and GA20ox3 ). Taken together, these results suggested that MsKMS1 negatively regulated seed germination by increasing abscisic acid and salicylic acid contents through the expression of genes related to abscisic acid and gibberellin biosynthesis. In addition, MsKMS1 could improve heat tolerance during the germination of transgenic tobacco seeds.

Feeding alfalfa-grass or red clover-grass mixture baleage: Effect on milk yield and composition, ruminal fermentation and microbiota taxa relative abundance, and nutrient utilization in dairy cows.

Our goal was to investigate the effect of diets containing baleages harvested from alfalfa-grass or red clover-grass mixture on production performance, ruminal fermentation and microbiota taxa relative abundance, milk fatty acid profile, and nutrient utilization in dairy cows. Twenty Jersey cows (18 multiparous and 2 primiparous) averaging (mean ± SD) 148 ± 45.2 days in milk and 483 ± 65.4 kg of body weight in the beginning of the study were used in a randomized complete block design with repeated measures over time. The experiment lasted 9 wk, with a 2 wk covariate period followed by 7 wk of data and sample collection (wk 4 and 7 used in the statistical analyses). Cows were fed diets containing (dry matter basis) 35% of a concentrate mash and the following forage sources: (1) 65% second- and third-cut (32.5% each) alfalfa-grass mixture baleages (ALF) or (2) 65% second- and third-cut (32.5% each) red clover-grass mixture baleages (RC). Diets did not affect dry matter intake, milk yield, and concentrations of milk fat and true protein. In contrast, milk fat yield tended to decrease and energy-corrected milk yield decreased with feeding RC versus ALF. The apparent total-tract digestibilities of dry matter, organic matter, and ash-free neutral detergent fiber, milk proportions of trans-10 18:1, cis-9,cis-12,cis-15 18:3, and total n-3 fatty acids, ruminal molar proportion of acetate, and plasma concentrations of Leu, Phe, and Val all increased in RC versus ALF. Diet × week interactions were found for several parameters, most notably ruminal molar proportions of propionate and butyrate, ruminal NH3-N, milk urea N, plasma urea N, and plasma His concentrations, urinary N excretion, enteric CH4 production, and all energy efficiency variables. Specifically, ruminal NH3-N and plasma urea N concentrations, urinary excretion of N, and CH4 production decreased in cows fed RC in wk 4 but not in wk 7. Milk urea N concentration decreased and that of plasma His increased with feeding RC during wk 4 and 7, although the magnitude of treatments difference varied between the sampling periods. Efficiency of energy utilization calculated as milk energy/metabolizable energy decreased and that of tissue energy/ME increased in RC versus ALF cows in wk 4, suggesting that ME was portioned toward tissue and not milk in the RC diet. Interactions were also observed for the relative abundance of the rumen bacterial phyla Verrucomicrobiota and Fibrobacterota, with cows offered RC showing greater values than those receiving ALF in wk 4 but no differences in wk 7. Several diet × week interactions were detected in the present study implying short-term treatment responses and warranting further investigations.

Inflammatory mediators, oxidative stress and genetic disturbance in rheumatoid arthritis rats supported by alfalfa seeds metabolomic constituents via blocking interleukin-1receptor.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by aggressive cartilage and bone erosion. This work aimed to evaluate the metabolomic profile of Medicago sativa L. (MS) (alfalfa) seeds and explore its therapeutic impact against RA in rats. Arthritis was induced by complete Freund's adjuvant (CFA) and its severity was assessed by the arthritis index. Treatment with MS seeds butanol fraction and interlukin-1 receptor antagonist (IL-1RA) were evaluated through measuring interlukin-1 receptor (IL-1R) type 1 gene expression, interlukin-1 beta (IL-1ß), oxidative stress markers, C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), caspase-3 (Cas-3), intracellular adhesion molecule-1 (ICAM-1), DNA fragmentation, and chromosomal damage. Total phenolics/ flavonoids content in the ethyl acetate, butanol fraction and crude extract of MS seeds were estimated. The major identified compounds were Quercetin, Trans-taxifolin, Gallic acid, 7,4'-Dihydroxyflavone, Cinnamic acid, Kudzusaponin SA4, Isorhamnetin 3-O-beta-D-2'',3'',4''-triacetylglucopyranoside, Apigenin, 5,7,4'-Trihydroxy-3'-methoxyflavone, Desmethylxanthohumol, Pantothenic acid, Soyasapogenol E, Malvidin, Helilandin B, Stigmasterol, and Wairol. Treatment with MS seeds butanol fraction and IL-1RA enhanced all the biochemical parameters and the histopathological features of the ankle joint. In conclusion, Trans-taxifolin was isolated for the first time from the genus Medicago. MS butanol fraction seeds extract and IL-1 RA were considered as anti-rheumatic agents.

Physiological and Transcriptomic Analysis Reveals That Melatonin Alleviates Aluminum Toxicity in Alfalfa (Medicago sativa L.).

Aluminum (Al) toxicity is the most common factor limiting the growth of alfalfa in acidic soil conditions. Melatonin (MT), a significant pleiotropic molecule present in both plants and animals, has shown promise in mitigating Al toxicity in various plant species. This study aims to elucidate the underlying mechanism by which melatonin alleviates Al toxicity in alfalfa through a combined physiological and transcriptomic analysis. The results reveal that the addition of 5 µM melatonin significantly increased alfalfa root length by 48% and fresh weight by 45.4% compared to aluminum treatment alone. Moreover, the 5 µM melatonin application partially restored the enlarged and irregular cell shape induced by aluminum treatment, resulting in a relatively compact arrangement of alfalfa root cells. Moreover, MT application reduces Al accumulation in alfalfa roots and shoots by 28.6% and 27.6%, respectively. Additionally, MT plays a crucial role in scavenging Al-induced excess H2O2 by enhancing the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), consequently reducing malondialdehyde (MDA) levels. More interestingly, the RNA-seq results reveal that MT application significantly upregulates the expression of xyloglucan endotransglucosylase/hydrolase (XTH) and carbon metabolism-related genes, including those involved in the glycolysis process, as well as sucrose and starch metabolism, suggesting that MT application may mitigate Al toxicity by facilitating the binding of Al to the cell walls, thereby reducing intracellular Al accumulation, and improving respiration and the content of sucrose and trehalose. Taken together, our study demonstrates that MT alleviates Al toxicity in alfalfa by reducing Al accumulation and restoring redox homeostasis. These RNA-seq results suggest that the alleviation of Al toxicity by MT may occur through its influence on cell wall composition and carbon metabolism. This research advances our understanding of the mechanisms underlying MT's effectiveness in mitigating Al toxicity, providing a clear direction for our future investigations into the underlying mechanisms by which MT alleviates Al toxicity in alfalfa.

Enriched endogenous free Spd and Spm in alfalfa (Medicago sativa L.) under drought stress enhance drought tolerance by inhibiting H2O2 production to increase antioxidant enzyme activity.

Drought stress is a major factor limiting agricultural development, and exogenous polyamines (PAs) can increase plant drought resistance by enhancing antioxidant activity, but few studies have examined whether endogenous PAs enhance the plant antioxidant system. Here, to investigate the effects of endogenous PAs on the antioxidant system of alfalfa under drought stress and the underlying mechanisms, two alfalfa cultivars, Longzhong (drought resistant) and Gannong No. 3 (drought sensitive), were used as test materials, and their seedlings were treated with polyethylene glycol (PEG-6000) for 8 days at -1.2 MPa to simulate drought stress. The levels of free PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)], hydrogen peroxide (H2O2), malondialdehyde (MDA), key PA metabolism enzyme [arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), polyamine oxidase (PAO), and diamine oxidase (DAO)] activities, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)] activities were measured. These physiological indicators were used for correlation analysis to investigate the relationship between PA metabolism and the antioxidant enzyme system. The results showed that PA synthesis in alfalfa under drought stress was dominated by the ADC pathway. Spd and Spm played an important role in improving drought tolerance. The high levels of ADC and SAMDC activities were facilitated by the conversion of Put to Spd and Spm. H2O2 generation by oxidative decomposition of PAs was mainly dependent on the oxidative decomposition of DAO but not PAO. Low DAO activity favored low H2O2 production. Spd, Spm, ADC, ODC and SAMDC were positively correlated with the antioxidant enzymes SOD, CAT and POD in both cultivars under drought. Therefore, we concluded that high ADC and SAMDC activities in alfalfa promoted the conversion of Put to Spd and Spm, leading to high accumulation of Spd and Spm and low Put accumulation. Low Put levels led to low H2O2 production through low DAO activity, and low H2O2 levels induced the expression of antioxidant enzyme-encoding genes to improve antioxidant enzyme activity and reduce MDA accumulation and thereby enhanced drought resistance in alfalfa.

Elevated CO2 differentially attenuates beryllium-induced oxidative stress in oat and alfalfa.

Elevated CO2 (eCO2 ) is one of the climate changes that may benefit plant growth under emerging soil contaminants such as heavy metals. In this regard, the morpho-physiological mechanisms underlying the mitigating impact of eCO2 on beryllium (Be) phytotoxicity are poorly known. Hence, we investigated eCO2 and Be interactive effects on the growth and metabolism of two species from different groups: cereal (oat) and legume (alfalfa). Be stress significantly reduced the growth and photosynthetic attributes in both species, but alfalfa was more susceptible to Be toxicity. Be stress induced reactive oxygen species (ROS) accumulation by increasing photorespiration, subsequently resulting in increased lipid and protein oxidation. However, the growth inhibition and oxidative stress induced by Be stress were mitigated by eCO2 . This could be explained, at least partially, by the increase in organic acids (e.g., citric acid) released into the soil, which subsequently reduced Be uptake. Additionally, eCO2 reduced cellular oxidative damage by reducing photorespiration, which was more significant in alfalfa plants. Furthermore, eCO2 improved the redox status and detoxification processes, including phytochelatins, total glutathione and metallothioneins levels, and glutathione-S-transferase activity in both species, but to a greater extend in alfalfa. In this context, eCO2 also stimulated anthocyanin biosynthesis by accumulating its precursors (phenylalanine, coumaric acid, cinnamic acid, and naringenin) and key biosynthetic enzymes (phenylalanine ammonia-lyase, cinnamate hydroxylase, and coumarate:CoA ligase) mainly in alfalfa plants. Overall, this study explored the mechanistic approach by which eCO2 alleviates the harmful effects of Be. Alfalfa was more sensitive to Be stress than oats; however, the alleviating impact of eCO2 on Be stress was more pronounced in alfalfa.

Salicylic acid alleviates Zn-induced inhibition of growth via enhancing antioxidant system and glutathione metabolism in alfalfa.

Zinc (Zn) is considered as one of the heavy metal pollutants in soil affecting agriculture. Salicylic acid (SA) is an important phytohormone that can mitigate effects against various abiotic stresses in plants, however, its exploration to improve Zn stress tolerance in alfalfa plants is still elusive. Thus, in the present study, exogenous SA treatment was conducted on alfalfa plants under Zn stress. The effects of exogenous SA on the physiological effects of alfalfa plants and the expression levels related genes were studied. This study tested the biomass, relative water content, chlorophyll levels, photosynthetic capacity, proline and soluble sugar contents, detected the activity of antioxidant enzymes (such as peroxidase and superoxide dismutase), glutathione biosynthesis, and endogenous SA levels, and quantified the genes associated with the antioxidant system and glutathione metabolism-mediated Zn stress. The results showed that exogenous SA could elevate the physiological adaptability of alfalfa plants through enhancing photosynthesis, proline and soluble sugar levels, stimulating antioxidant system and glutathione metabolism, and inducing the transcription level of related genes, thereby diminishing oxidative stress, inhibiting excessive Zn accumulation of alfalfa plants, increasing tolerance to Zn stress, and reducing the toxicity of Zn. Collectively, the application of SA alleviates Zn toxicity in alfalfa plants. The findings gave first insights into the regulatory mechanism of the Zn stress tolerance of alfalfa by exogenous SA and this might have positive implications for managing other plants which are suffering Zn stress.

A 3-Ketoacyl-CoA Synthase 10 (KCS10) Homologue from Alfalfa Enhances Drought Tolerance by Regulating Cuticular Wax Biosynthesis.

Cuticular wax, forming the first line of defense against adverse environmental stresses, comprises very long-chain fatty acids (VLCFAs) and their derivatives. 3-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme for VLCFA biosynthesis. In this study, we isolated KCS10, a KCS gene from alfalfa, and analyzed the effect of gene expression on wax production and drought stress in transgenic plants. MsKCS10 overexpression increased compact platelet-like crystal deposition and promoted primary alcohol biosynthesis through acyl reduction pathways in alfalfa leaves. Overexpression of MsKCS10 induced the formation of coiled-rodlet-like crystals and increased n-alkane content through decarbonylation pathways in tobacco and tomato fruits. Overexpression of MsKCS10 enhanced drought tolerance by limiting nonstomatal water loss, improving photosynthesis, and maintaining osmotic potential under drought stress in transgenic tobacco. In summary, MsKCS10 plays an important role in wax biosynthesis, wax crystal morphology, and drought tolerance, although the mechanisms are different among the plant species. MsKCS10 can be targeted in future breeding programs to improve drought tolerance in plants.