The homeostasis of ions and reactive oxygen species in root and shoot play crucial roles in the tolerance of alfalfa to salt alkali stress.

High pH saline-alkali stress, mainly NaHCO3, limited the development of animal husbandry in Songnen Plain. Ion imbalance and reactive oxygen species (ROS) metabolism disorder caused by saline-alkali stress inhibited plant growth. In this study, we compared the differences in ion absorption, transport and ROS metabolism between saline-tolerant alfalfa (ZD) and saline-sensitive alfalfa (ZM) under NaHCO3 stress using physiology and transcripomics techniques. WGCNA analysis identified key genes associated with NaHCO3 stress-induced changes. NaHCO3 stress inhibited the absorption of K+ and Mg2+, but activated Ca2+ signal. Furthermore, ZD maintained higher K+, Mg2+ and Ca2+ contents and the K+/Na+ ratio than ZM, this is mainly related to the higher expression of proteins or channel-encoding genes involved in ion absorption and transport in ZD. Antioxidant enzyme systems can be activated in response to NaHCO3 stress. Peroxidase (EC 1.11.1.6), catalase (EC 1.11.1.7) and glutathione transferase (EC 2.5.1.18) activities were higher in ZD than ZM, and most genes encoding the relevant enzymes also demonstrated a stronger up-regulation trend in ZD. Although NaHCO3 stress inhibited Trx-Prx pathway, ZD related enzymes and their genes were also inhibited less than ZM. WGCNA results identified many genes involved in ion absorption, transport and antioxidant systems that play an important role in NaHCO3 stress adaptation. Collectively, ZD has the stronger ion homeostasis regulation and ROS scavenging ability, so it's more resistant to NaHCO3. The results provide theoretical guidance for further understanding of the molecular mechanism of NaHCO3 resistance and provide potential genes for research to improve saline-alkali tolerance in alfalfa.

Responses of diazotrophic network structure and community diversity to alfalfa-maize intercropping are soil property-dependent.

Introduction: Intercropping and soil properties both affect soil diazotrophic communities. However, the specific effects that alfalfa-maize intercropping has on diazotrophic networks and community diversity under different soil properties remain unclear. Methods: In this study, we investigated the soil diazotrophic communities of two crop systems, alfalfa monoculture (AA) and alfalfa-maize intercropping (A/M), in two sites with similar climates but different soil properties (poor vs. average). Results and discussion: The diazotrophic network complexity and community diversity were higher at the site with poor soil than at the site with average soil (p < 0.05). Community structure also varied significantly between the sites with poor and average soil (p < 0.05). This divergence was mainly due to the differences in soil nitrogen, phosphorus, and organic carbon contents between the two sites. At the site with poor soil, the A/M system had lower diazotrophic diversity, lower network complexity and greater competition between diazotrophs than the AA system (p < 0.05) because intercropping intensified the soil phosphorus limitation under poor soil conditions. However, in the average soil, it was the A/M system that had an altered diazotrophic structure, with an increased abundance of 11 bacterial genera and a decreased abundance of three bacterial genera (p < 0.05). Conclusion: Our results indicated that the effects of alfalfa-maize intercropping on diazotrophic communities were soil property-dependent.

Comparative analysis of the physiological and transcriptomic profiles reveals alfalfa drought resistance mechanisms.

BACKGROUND: Drought stress is a major limiting factor that affects forage yields, and understanding the drought resistance mechanism of plants is crucial for improving crop yields in arid areas. Alfalfa (Medicago sativa L.) is the most important legume plant, mainly planted in arid and semi-arid areas. However, the adaptability of alfalfa to drought stress and its physiological and molecular mechanisms of drought resistance remains unclear. RESULTS: In this study, we analyzed the physiological and transcriptome responses of alfalfa cultivars with different drought resistances (drought-sensitive Gannong No. 3 (G3), drought-resistant Gannong No. 8 (G8), and strong drought-resistant Longdong (LD)) under drought stress at 0, 6, 12, and 24 h. LD had higher catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities and a higher soluble protein content, lower malondialdehyde (MDA) content, a lower O2·- production rate, and a lower H2O2 content than G8 and G3 (P < 0.05). The functional enrichment analysis, temporal expression pattern analysis, and weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes (DEGs) showed phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, glutathione metabolism, and biosynthesis of amino acid responses to drought stress in alfalfa. The differential expression of genes during phenylpropanoid biosynthesis, starch and sucrose metabolism, and the glutathione metabolism pathway was further studied, and it was speculated that PAL, COMT, 4CL, CCR, CAD, HXK, INV, SUS, WAXY, AGP, GST, and APX1 played important roles in the alfalfa drought stress response. CONCLUSIONS: The aim of this study was to enhance alfalfa drought resistance by overexpressing positively regulated genes and knocking out negatively regulated genes, providing genetic resources for the subsequent molecular-assisted breeding of drought-resistant alfalfa crops.

Physiological and biochemical characteristics and microbial responses of Medicago sativa (Fabales: Fabaceae) varieties with different resistance to atrazine stress.

Atrazine, a commonly employed herbicide for corn production, can leave residues in soil, resulting in photosynthetic toxicity and impeding growth in subsequent alfalfa (Medicago sativa L.) crops within alfalfa-corn rotation systems. The molecular regulatory mechanisms by which atrazine affects alfalfa growth and development, particularly its impact on the microbial communities of the alfalfa rhizosphere, are not well understood. This study carried out field experiments to explore the influence of atrazine stress on the biomass, chlorophyll content, antioxidant system, and rhizosphere microbial communities of the atrazine-sensitive alfalfa variety WL-363 and the atrazine-resistant variety JN5010. The results revealed that atrazine significantly reduced WL-363 growth, decreasing plant height by 8.58 cm and root length by 5.42 cm (p < 0.05). Conversely, JN5010 showed minimal reductions, with decreases of 1.96 cm in height and 1.26 cm in root length. Chlorophyll content in WL-363 decreased by 35% under atrazine stress, while in JN5010, it was reduced by only 10%. Reactive oxygen species (ROS) accumulation increased by 60% in WL-363, compared to a 20% increase in JN5010 (p < 0.05 for both). Antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase (CAT), were significantly elevated in JN5010 (p < 0.05), suggesting a more robust defense mechanism. Although the predominant bacterial and fungal abundances in rhizosphere soils remained generally unchanged under atrazine stress, specific microbial groups exhibited variable responses. Notably, Promicromonospora abundance declined in WL-363 but increased in JN5010. FAPROTAX functional predictions indicated shifts in the abundance of microorganisms associated with pesticide degradation, resistance, and microbial structure reconstruction under atrazine stress, displaying different patterns between the two varieties. This study provides insights into how atrazine residues affect alfalfa rhizosphere microorganisms and identifies differential microbial responses to atrazine stress, offering valuable reference data for screening and identifying atrazine-degrading bacteria.

Arbuscular mycorrhizal fungi communities and promoting the growth of alfalfa in saline ecosystems of northern China.

Arbuscular mycorrhizal fungi (AMF) are universally distributed in soils, including saline soils, and can form mycorrhizal symbiosis with the vast majority of higher plants. This symbiosis can reduce soil salinity and influence plant growth and development by improving nutrient uptake, increasing plant antioxidant enzyme activity, and regulating hormone levels. In this study, rhizosphere soil from eight plants in the Songnen saline-alkaline grassland was used to isolate, characterize, and screen the indigenous advantageous AMF. The promoting effect of AMF on alfalfa (Medicago sativa L.) under salt treatment was also investigated. The findings showed that 40 species of AMF in six genera were identified by high-throughput sequencing. Glomus mosseae (G.m) and Glomus etunicatum (G.e) are the dominant species in saline ecosystems of northern China. Alfalfa inoculated with Glomus mosseae and Glomus etunicatum under different salt concentrations could be infested and form a symbiotic system. The mycorrhizal colonization rate and mycorrhizal dependence of G.m inoculation were significantly higher than those of G.e inoculation. With increasing salt concentration, inoculation increased alfalfa plant height, fresh weight, chlorophyll content, proline (Pro), soluble sugar (SS), soluble protein (SP), peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activity while decreasing the malondialdehyde (MDA) content and superoxide anion production rate. The results highlight that inoculation with G.m and G.e effectively alleviated salinity stress, with G.m inoculation having a significant influence on salt resistance in alfalfa. AMF might play a key role in alfalfa growth and survival under harsh salt conditions.

Enhancing Water and Soil Resources Utilization via Wolfberry-Alfalfa Intercropping.

The impact of the intercropping system on the soil-plant-atmosphere continuum (SPAC), encompassing soil evaporation, soil moisture dynamics, and crop transpiration, remains an area of uncertainty. Field experiments were conducted for two years in conjunction with the SIMDualKc (Simulation Dual Crop Coefficient) model to simulate two planting configurations: sole-cropped wolfberry (Lycium barbarum L.) (D) and wolfberry intercropped with alfalfa (Medicago sativa L.) (J). These configurations were subjected to different irrigation levels: full irrigation (W1, 75-85% θfc), mild deficit irrigation (W2, 65-75% θfc), moderate deficit irrigation (W3, 55-65% θfc), and severe deficit irrigation (W4, 45-55% θfc). The findings revealed that the JW1 treatment reduced the annual average soil evaporation by 32% compared with that of DW1. Additionally, mild, moderate, and severe deficit irrigation reduced soil evaporation by 17, 24, and 36%, respectively, compared with full irrigation. The intercropping system exhibited a more efficient canopy structure, resulting in reduced soil evaporation and alleviation of water stress to a certain extent. In terms of temporal dynamics, monocropping resulted in soil moisture levels from 1% to 15% higher than intercropping, with the most significant differences manifesting in the mid to late stages, whereas differences in the early stages were not statistically significant. Spatially, the intercropping system exhibited 7-19% lower soil water contents (SWCs) than sole cropping, primarily within the root water uptake zone within the 0-60 cm soil layer. The intercropping system showed an enhanced water absorption capacity for plant transpiration, resulting in a 29% increase in transpiration compared with sole cropping, thereby achieving water-saving benefits. These findings contribute to our understanding of the agronomic and environmental implications of intercropping wolfberry and alfalfa in arid regions and provide insights into optimizing water and soil resource management for sustainable agricultural practices.

Energy utilization in lactating Jersey cows consuming a mixture of DDGS and straw replacing alfalfa hay.

Some forages require significant amounts of water to grow, causing the dairy industry to be dependent on a limited resource. Feeding crop residues and feed coproducts in dairy rations may represent opportunities when alfalfa is not readily available, and to reduce the industry's use of water. A study using indirect calorimetry and 12 multiparous lactating Jersey cows (BW = 447.5 ± 43.7 kg; DIM = 71 ± 11 d, mean ± SD) was conducted to determine the effect of feeding dried distillers grains and solubles (DDGS) and straw in replacement of alfalfa hay on milk production and energy utilization. A triplicated 4 × 4 Latin square design was used to evaluate the replacement of alfalfa hay with a coproduct mixture (COP) of wheat straw and DDGS. Animals were blocked by milk yield and randomly assigned to 1 of 4 experimental treatments including (proportions on a DM basis): a control diet (CON) containing 18.2% of alfalfa hay, a low-coproduct diet (LCOP) that contained 8.1% of COP, a medium-coproduct diet (MCOP) that contained 16.3% of COP, and a high-coproduct diet (HCOP) that contained 24.3% of COP. No differences were observed for daily dry matter intake or milk yield (mean ± SEM) 19.5 kg ± 0.60, 29.6 kg ± 0.91, respectively. A quadratic tendency was observed where increasing inclusion of COP up to 16.3% maintained ECM and milk fat yield but decreased when animals were fed 24.3% COP. Total methane production decreased linearly from 429.4 to 345.0 ± 22.8 L/d from CON to HCOP diets, respectively. The digestibility of CP increased linearly from 64.0 to 70.4 ± 0.95% and N balance increased linearly from 43.3 to 90.7 ± 15.0 g/d in animals consuming CON to HCOP diets. Total time spent ruminating was lowest in animals consuming the HCOP diet. A linear increasing tendency in digestible and metabolizable energy of 2.92 to 3.02 ± 0.041 Mcal/kg and 2.58 to 2.70 ± 0.047 Mcal/kg was observed in animals consuming CON to HCOP. The proportion ME from DE (ME/DE) tended to linearly increase from 88.3 to 89.4 ± 0.454 when COP was added to the diet. Results of this study indicate that alfalfa hay with a mixture of straw and DDGS can maintain milk production and DMI, but the partial or full replacement of alfalfa with the COP mixture may result in differences in energy utilization in part driven by effects on CH4 reduction.

Serological detection of a novel ipomo-like virus infecting alfalfa in the U.S.

This study describes production of polyclonal antibodies against recently reported novel potyvirid infecting alfalfa (Medicago sativa L.). The virus was first found in alfalfa seed material and later identified in plant samples collected from commercial alfalfa fields in Arizona, USA. It was classified as a novel species related to the members of the genus Ipomovirus and potentially representing a new genus in the family Potyviridae (Nemchinov et al., 2023b). Polyclonal antibodies were produced against the predicted viral coat protein expressed in bacterial cells and used in different types of immunoassays for specific detection of this emerging virus. They could be helpful in plant virus certification programs, screening of alfalfa germplasm, research on pathogenicity, biology, and geographic distribution of this emerging virus.

Effects of protein grass hay as alternative feed resource on lamb's fattening performance and meat quality.

Protein grass hay (PGH) was used as a new feed source for lambs to study its effect on fattening performance and meat quality. Fifty-six male lambs were allotted to four experimental groups and fed for eight weeks either alfalfa hay (AH)-based diet (control) or diets in which AH was replaced with 33 %, 66 %, or 99 % PGH. The inclusion of PGH did not affect final body weight, dry matter intake, average daily gain, feed conversion ratio, or carcass weight. Moreover, substituting AH with PGH at any level did not influence the ruminal fermentation or serum biochemical parameters, meat color, water holding capacity, shear force, or amino acid profile. However, relative liver weight was increased with 66 % substitutions. Furthermore, replacing 99 % AH with PGH decreased the meat's pH at 24 h. Higher levels of C18:3n-3, C20:5n-3, and total n-3 PUFA and a lower ratio of n-6: n-3 PUFA were also observed in meat from lambs fed PGH at 99 %. These findings suggest that PGH could be incorporated into the lamb's diet up to 99 % without compromising fattening performance and body health while improving their meat n-3 PUFA deposition.

Genome-wide investigation of the TIFY transcription factors in alfalfa (Medicago sativa L.): identification, analysis, and expression.

BACKGROUND: Alfalfa (Medicago sativa L.) is an essential leguminous forage with high nutrition and strong adaptability. The TIFY family is a plant-specific transcription factor identified in many plants. However, few reports have been reported on the phylogenetic analysis and gene expression profiling of TIFY family genes in alfalfa. RESULT: A total of 84 TIFY genes belonging to 4 categories were identified in alfalfa, including 58 MsJAZs, 18 MsZMLs, 4 MsTIFYs and 4 MsPPDs, respectively. qRT-PCR data from 8 genes in different tissues revealed that most MsTIFY genes were highly expressed in roots. The expression of MsTIFY14 was up-regulated after different times in both thrips-resistant and susceptible alfalfa after thrips feeding, and the expression of the remaining MsTIFYs had a strong correlation with the time of thrips feeding. Different abiotic stresses, including drought, salt, and cold, could induce or inhibit the expression of MsTIFY genes to varying degrees. In addition, the eight genes were all significantly up-regulated by JA and/or SA. Interestingly, MsTIFY77 was induced considerably by all the biotic, abiotic, or plant hormones (JA or SA) except ABA. CONCLUSION: Our study identified members of the TIFY gene family in alfalfa and analyzed their structures and possible functions. It laid the foundation for further research on the molecular functions of TIFYs in alfalfa.

Three endogenous pararetrovirus genomes in Medicago sativa.

The genome sequences of three related endogenous pararetroviruses were obtained by high-throughput genomic sequencing of Medicago sativa. The genomes were found to be integrated within plant genes. The phylogeny revealed that Caulimovirus-MSa3 was closely related to caulimoviruses of petunia, whereas Caulimovirus-MSa1 and Caulimovirus-MSa2 were distinct from constructed clades.

Novel Tubeimoside I liposomal drug delivery system in combination with gemcitabine for the treatment of pancreatic cancer.

Aim: To evaluate the anti-pancreatic cancer effect of novel Tubeimoside I multifunctional liposomes combined with gemcitabine.Methods: Liposomes were prepared through the thin film hydration method, with evaluations conducted on parameters including encapsulation efficiency (EE%), particle size, polydispersity index (PDI), zeta potential (ZP), storage stability, and release over a 7-day period. The cellular uptake rate, therapeutic efficacy in vitro and in vivo and the role of immune microenvironment modulation were evaluated.Results: The novel Tubeimoside I multifunctional liposomal exhibited good stability, significant anti-cancer activity, and immune microenvironment remodeling effects. Furthermore, it showed a safety profile.Conclusion: This study underscores the potential of Novel Tubeimoside I multifunctional liposomal as a promising treatment option for pancreatic cancer.

[Box: see text].

Visible-NIR spectroscopic authentication assay for the classification of lamb meat according to pasture-finishing duration.

Spectral analysis of meat combined with chemometric analysis has been identified as a promising tool for authenticating livestock-animal diets. The objectives of this study were (i) to determine whether the visible-NIR spectrum of perirenal adipose tissue (PAT) and caudal adipose tissue (CAT) can reliably discriminate lambs pasture-finished for different durations before slaughter, and (ii) to analyze the kinetics of appearance and stabilization of the visible-NIR spectrum-based pasture signature in PAT and CAT. Four groups of 50-55 lambs were used over three years: lambs finished on lucerne pasture for 0 (L0, concentrate-fed in stall), 21 (L21), 42 (L42) and 63 (L63) days before slaughter. Partial least squares discriminant analysis was applied on PAT or CAT visible-NIR spectra to discriminate the groups. No one adipose tissue reliably discriminated the four groups, with less than 62 % lambs correctly classified. However, visible-NIR spectroscopy was able to discriminate stall-fed (L0) from pasture-finished (L21 + L42 + L63) lambs, with an accuracy of 93.8 % and 87.5 % lambs correctly classified based on PAT and CAT spectra, respectively. The lucerne pasture fingerprint (or signature) on visible-NIR spectrum appeared between 0 and 42 days in more than 95 % of lambs. It stabilized between 42 and 63 days in CAT, but had not stabilized within the range of grazing durations pre-slaughter explored in PAT. Further research into shorter and longer pasture-finishing durations could help determine more precisely the time required for the pasture signature to appear and stabilize in animal tissues.

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.

Multi-trait modeling and machine learning discover new markers associated with stem traits in alfalfa.

Alfalfa biomass can be fractionated into leaf and stem components. Leaves comprise a protein-rich and highly digestible portion of biomass for ruminant animals, while stems constitute a high fiber and less digestible fraction, representing 50 to 70% of the biomass. However, little attention has focused on stem-related traits, which are a key aspect in improving the nutritional value and intake potential of alfalfa. This study aimed to identify molecular markers associated with four morphological traits in a panel of five populations of alfalfa generated over two cycles of divergent selection based on 16-h and 96-h in vitro neutral detergent fiber digestibility in stems. Phenotypic traits of stem color, presence of stem pith cells, winter standability, and winter injury were modeled using univariate and multivariate spatial mixed linear models (MLM), and the predicted values were used as response variables in genome-wide association studies (GWAS). The alfalfa panel was genotyped using a 3K DArTag SNP markers for the evaluation of the genetic structure and GWAS. Principal component and population structure analyses revealed differentiations between populations selected for high- and low-digestibility. Thirteen molecular markers were significantly associated with stem traits using either univariate or multivariate MLM. Additionally, support vector machine (SVM) and random forest (RF) algorithms were implemented to determine marker importance scores for stem traits and validate the GWAS results. The top-ranked markers from SVM and RF aligned with GWAS findings for solid stem pith, winter standability, and winter injury. Additionally, SVM identified additional markers with high variable importance for solid stem pith and winter injury. Most molecular markers were located in coding regions. These markers can facilitate marker-assisted selection to expedite breeding programs to increase winter hardiness or stem palatability.

Replacing alfalfa-based total mixed ration with Moringa leaves for improving carcass and meat quality characteristics in lambs.

The expected increase in the population will put more pressure on resources, which means there will be a greater demand for food and the need for different ingredients to feed animals. This study aimed to explore the potential benefits of replacing alfalfa-based total mixed ration (TMR) with Moringa spp. On carcass characteristics and meat quality traits in lamb. The experiment involved 60 intact male lambs of the Harri breed, averaging 24 ± 1 kg in weight and approximately 5 months old. Lambs were randomly divided into five feeding groups: T1 (40 % alfalfa-based TMR), T2 (40 % M. oleifera), T3 (40 % M. peregrina), T4 (20 % alfalfa-based TMR + 20 % M. oleifera), and T5 (20 % alfalfa-based TMR + 20 % M. peregrina. The results confirmed that incorporating Moringa spp. Into lamb diets significantly reduced intramuscular fat content. Supplementation also improved tenderness, texture properties, and water-holding capacity. Additionally, the treatment groups exhibited a significant decrease in total non-carcass components compared to the control group. Overall, these findings suggest positive changes and enhancements in lamb carcass traits and meat quality attributes with Moringa spp. Supplementation, without observed adverse effects.

Halotolerant Enterobacter asburiae A103 isolated from the halophyte Salix linearistipularis: Genomic analysis and growth-promoting effects on Medicago sativa under alkali stress.

Soil salinization negatively affects plant growth and threatens food security. Halotolerant plant growth-promoting bacteria (PGPB) can alleviate salt stress in plants via diverse mechanisms. In the present study, we isolated salt-tolerant bacteria with phosphate-solubilizing abilities from the rhizosphere of Salix linearistipularis, a halophyte distributed in saline-alkali soils. Strain A103 showed high phosphate solubilization activity and was identified as Enterobacter asburiae based on genome analysis. In addition, it can produce indole-3-acetic acid (IAA), siderophores, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Genome mining has also revealed the presence of several functional genes involved in the promotion of plant growth. Inoculation with A103 markedly improved alfalfa growth in the presence of 100 mM NaHCO3. Under alkali stress, the shoot and root dry weights after bacterial inoculation improved by 42.9 % and 21.9 %, respectively. Meanwhile, there was a 35.9-37.1 % increase in the shoot and root lengths after treatment with A103 compared to the NaHCO3-treated group. Soluble sugar content, peroxidase and catalase activities increased in A103-inoculated alfalfa under alkaline stress. A significant decrease in the malondialdehyde content was observed after treatment with strain A103. Metabolomic analysis indicated that strain A103 positively regulated alkali tolerance in alfalfa through the accumulation of metabolites, such as homocarnosine, panthenol, and sorbitol, which could reduce oxidative damage and act as osmolytes. These results suggest that halophytes are valuable resources for bioprospecting halotolerant beneficial bacteria and that the application of halotolerant growth-promoting bacteria is a natural and efficient strategy for developing sustainable agriculture.

Thriving in a salty future: morpho-anatomical, physiological, and molecular adaptations to salt stress in alfalfa (Medicago sativa L.) and other crops.

BACKGROUND: With soil salinity levels rising at an alarming rate, accelerated by climate change and human interventions, there is a growing need for crop varieties that can grow on saline soils. Alfalfa (Medicago sativa) is a cool-season perennial leguminous crop, commonly grown as forage, biofuel feedstock, and soil conditioner. It demonstrates significant potential for agricultural circularity and sustainability, for example by fixing nitrogen, sequestering carbon, and improving soil structures. Although alfalfa is traditionally regarded as moderately salt-tolerant species, modern alfalfa varieties display specific salt-tolerance mechanisms, which could be used to pave alfalfa's role as a leading crop able to grow on saline soils. SCOPE: Alfalfa's salt tolerance underlies a large variety of cascading biochemical and physiological mechanisms. These are partly enabled by alfalfa's complex genome structure and out-crossing nature, which on the other hand entail impediments for molecular and genetic studies. This review first summarizes the general effects of salinity on plants and the broad-ranging mechanisms for dealing with salt-induced osmotic stress, ion toxicity, and secondary stress. Secondly, we address defensive and adaptive strategies that have been described for alfalfa, such as the plasticity of alfalfa's root system, hormonal crosstalk for maintaining ion homeostasis, spatiotemporal specialized metabolite profiles, and the protection of alfalfa-rhizobia associations. Finally, bottlenecks for research of the physiological and molecular salt-stress responses as well as biotechnology-driven improvements of salt tolerance are identified and discussed. CONCLUSION: Understanding morpho-anatomical, physiological, and molecular responses to salinity is essential for the improvement of alfalfa and other crops in saline land reclamation. This review identifies potential breeding targets for enhancing alfalfa performance stability and general crop robustness for rising salt levels as well as to promote alfalfa applications in saline land management.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana.

Analysis on morphological characteristics and identification of candidate genes during the flowering development of alfalfa.

Flower development is a crucial and complex process in the reproductive stage of plants, which involves the interaction of multiple endogenous signals and environmental factors. However, regulatory mechanism of flower development was unknown in alfalfa (Medicago sativa). In this study, the three stages of flower development of 'M. sativa cv. Gannong No. 5' (G5) and its early flowering and multi flowering mutant (MG5) were comparatively analyzed by transcriptomics. The results showed that compared with late bud stage (S1), 14287 and 8351 differentially expressed genes (DEGs) were identified at early flower stage (S2) in G5 and MG5, and 19941 and 19469 DEGs were identified at late flower stage (S3). Compared with S2, 9574 and 10870 DEGs were identified at S3 in G5 and MG5, respectively. Venn analysis revealed that 547 DEGs were identified among the three comparison groups. KEGG pathway enrichment analysis showed that these genes were involved in the development of alfalfa flowers through redox pathways and plant hormone signaling pathways. Key candidate genes including SnRK2, BSK, GID1, DELLA and CRE1, for regulating the development from buds to mature flowers in alfalfa were screened. In addition, differential expression of transcription factors such as MYB, AP2, bHLH, C2C2, MADS-box, NAC, bZIP, B3 and AUX/IAA also played an important role in this process. The results laid a theoretical foundation for studying the molecular mechanisms of the development process from buds to mature flowers in alfalfa.