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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.

Comparison of the effects of alfalfa meal and sorghum distillery residue supplementation on the methane emissions in black-feathered Taiwan native chicken.

The issue of global warming, primarily fueled by anthropogenic greenhouse gas emissions, necessitates effective strategies to address methane (CH4) emissions from both ruminants and nonruminants. Drawing inspiration from successful approaches employed in ruminants, this study evaluates the impact of supplementing the diets of Taiwan's native black-feathered chickens with alfalfa meal and sorghum distillery residues (SDRs) on CH4 emissions. Using a respiration chamber the results reveal a significant reduction in CH4 emissions when incorporating either 30% alfalfa meal or 30% SDRs into the chicken diet, demonstrating a 59% and 49% decrease, respectively, compared to the control group (P < 0.05). Considering that alfalfa meal contains saponins and SDRs contain tannins, the study delves into the mechanism through which these components mitigate CH4 production in chickens. Incorporating saponins or tannins shows that groups supplemented with these components exhibit significantly lower CH4 emissions compared to the control group (P < 0.05), with a consistent linear decrease as the concentration of the feed additive increases. Further in vitro analysis of chicken cecal contents indicates a proportional reduction in CH4 production with increasing levels of added saponins or tannins (P < 0.05). These findings suggest that the CH4-reducing effects of alfalfa meal and SDRs can be attributed to their saponins and tannin content. However, caution is warranted as excessive alfalfa meal supplementation may adversely impact poultry growth. Consequently, sorghum distillery residue emerges as a more suitable feed ingredient for mitigating CH4 emissions in Taiwan's native black-feathered chickens compared to alfalfa. Additionally, substituting SDRs for conventional commercial chicken feed not only reduces CH4 emissions but also enhances the utilization of byproducts.

In this study, researchers focused on addressing the crucial issue of global warming by tackling methane (CH4) emissions from chickens, an important source of greenhouse gases. We investigated the impact of adding alfalfa meal and sorghum distillery residues (SDRs) to the diets of Taiwan's native black-feathered chickens. The results showed a reduction in CH4 emissions when incorporating either 30% alfalfa meal or 30% SDRs into the chicken diet. This led to a 59% and 49% decrease, respectively, compared to the control group. The study explored the mechanism behind this reduction, pointing to the presence of saponins in alfalfa meal and tannins in SDRs as key components influencing CH4 production. However, the study also highlighted a potential concern regarding excessive alfalfa meal supplementation, which could negatively impact poultry growth. On the other hand, SDRs were found to have no detrimental effects, making them a more suitable feed ingredient for mitigating CH4 emissions in Taiwan's native black-feathered chickens compared to alfalfa. Moreover, substituting SDRs for conventional commercial chicken feed not only reduces CH4 emissions but also contributes to the enhanced utilization of byproducts, promoting a more sustainable approach to poultry farming.

Comparative proteomic discovery of salt stress response in alfalfa roots and overexpression of MsANN2 confers salt tolerance.

Soil salinity constrains growth, development and yield of alfalfa (Medicago sativa L.). To illustrate the molecular mechanisms responsible for salt tolerance, a comparative proteome analysis was explored to characterize protein profiles of alfalfa seedling roots exposed to 100 and 200 mM NaCl for three weeks. There were 52 differentially expressed proteins identified, among which the mRNA expressions of 12 were verified by Real-Time-PCR analysis. The results showed increase in abundance of ascorbate peroxidase, POD, CBS protein and PR-10 in salt-stressed alfalfa, suggesting an effectively antioxidant and defense systems. Alfalfa enhanced protein quality control system to refold or degrade abnormal proteins induced by salt stress through upregulation of unfolded protein response (UPR) marker PDIs and molecular chaperones (eg. HSP70, TCP-1, and GroES) as well as the ubiquitin-proteasome system (UPS) including ubiquitin ligase enzyme (E3) and proteasome subunits. Upregulation of proteins responsible for calcium signal transduction including calmodulin and annexin helped alfalfa adapt to salt stress. Specifically, annexin (MsANN2), a key Ca2+-binding protein, was selected for further characterization. The heterologous of the MsANN2 in Arabidopsis conferred salt tolerance. These results provide detailed information for salt-responsive root proteins and highlight the importance of MsANN2 in adapting to salt stress in alfalfa.

Comparative transcriptomic and metabolomic analyses provide insights into the responses to high temperature stress in Alfalfa (Medicago sativa L.).

High temperature stress is one of the most severe forms of abiotic stress in alfalfa. With the intensification of climate change, the frequency of high temperature stress will further increase in the future, which will bring challenges to the growth and development of alfalfa. Therefore, untargeted metabolomic and RNA-Seq profiling were implemented to unravel the possible alteration in alfalfa seedlings subjected to different temperature stress (25 ℃, 30 ℃, 35 ℃, 40 ℃) in this study. Results revealed that High temperature stress significantly altered some pivotal transcripts and metabolites. The number of differentially expressed genes (DEGs) markedly up and down-regulated was 1876 and 1524 in T30_vs_CK, 2, 815 and 2667 in T35_vs_CK, and 2115 and 2, 226 in T40_vs_CK, respectively. The number for significantly up-regulated and down-regulated differential metabolites was 173 and 73 in T30_vs_CK, 188 and 57 in T35_vs_CK, and 220 and 66 in T40_vs_CK, respectively. It is worth noting that metabolomics and transcriptomics co-analysis characterized enriched in plant hormone signal transduction (ko04705), glyoxylate and dicarboxylate metabolism (ko00630), from which some differentially expressed genes and differential metabolites participated. In particular, the content of hormone changed significantly under T40 stress, suggesting that maintaining normal hormone synthesis and metabolism may be an important way to improve the HTS tolerance of alfalfa. The qRT-PCR further showed that the expression pattern was similar to the expression abundance in the transcriptome. This study provides a practical and in-depth perspective from transcriptomics and metabolomics in investigating the effects conferred by temperature on plant growth and development, which provided the theoretical basis for breeding heat-resistant alfalfa.

Tandem mass tag (TMT)-based quantitative proteomics analysis reveals the different responses of contrasting alfalfa varieties to drought stress.

BACKGROUND: Drought stress restricts the growth, distribution and productivity of alfalfa (Medicago sativa L.). In order to study the response differences of alfalfa cultivars to drought stress, we previously carried out physiological and molecular comparative analysis on two alfalfa varieties with contrasting drought resistance (relatively drought-tolerant Longdong and drought-sensitive Algonquin). However, the differences in proteomic factors of the two varieties in response to drought stress still need to be further studied. Therefore, TMT-based quantitative proteomic analysis was performed using leaf tissues of the two alfalfa cultivars to identify and uncover differentially abundant proteins (DAPs). RESULTS: In total, 677 DAPs were identified in Algonquin and 277 in Longdong under drought stress. Subsequently, we conducted various bioinformatics analysis on these DAPs, including subcellular location, functional classification and biological pathway enrichment. The first two main COG functional categories of DAPs in both alfalfa varieties after drought stress were 'Translation, ribosomal structure and biogenesis' and 'Posttranslational modification, protein turnover, chaperones'. According to KEGG database, the DAPs of the two alfalfa cultivars after drought treatment were differentially enriched in different biological pathways. The DAPs from Algonquin were enriched in 'photosynthesis' and 'ribosome'. The pathways of 'linoleic acid metabolism', 'protein processing in endoplasmic reticulum' and 'RNA transport' in Longdong were significantly enriched. Finally, we found significant differences in DAP enrichment and expression patterns between Longdong and Algonquin in glycolysis/glycogenesis, TCA cycle, photosynthesis, protein biosynthesis, flavonoid and isoflavonoid biosynthesis, and plant-pathogen interaction pathway after drought treatment. CONCLUSIONS: The differences of DAPs involved in various metabolic pathways may explain the differences in the resistance of the two varieties to drought stress. These DAPs can be used as candidate proteins for molecular breeding of alfalfa to cultivate new germplasm with more drought tolerance to adapt to unfavorable environments.

Transcriptome-Wide Identification of Dark- and Salt-Induced Senescence-Related NAC Gene Family Members in Alfalfa.

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis-acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs. Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium-mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence.