Integrated Multi-Omics Analysis to Reveal the Molecular Mechanisms of Inflorescence Elongation in Medicago sativa.

The morphological architecture of inflorescence influences seed production. The regulatory mechanisms underlying alfalfa (Medicago sativa) inflorescence elongation remain unclear. Therefore, in this study, we conducted a comparative analysis of the transcriptome, proteome, and metabolome of two extreme materials at three developmental stages to explore the mechanisms underlying inflorescence elongation in alfalfa. We observed the developmental processes of long and short inflorescences and found that the elongation capacity of alfalfa with long inflorescence was stronger than that of alfalfa with short inflorescences. Furthermore, integrative analysis of the transcriptome and proteome indicated that the phenylpropanoid biosynthesis pathway was closely correlated with the structural formation of the inflorescence. Additionally, we identified key genes and proteins associated with lignin biosynthesis based on the differential expressed genes and proteins (DEGs and DEPs) involved in phenylpropanoid biosynthesis. Moreover, targeted hormone metabolome analysis revealed that IAA, GA, and CK play an important role in the peduncle elongation of alfalfa inflorescences. Based on omics analysis, we detected key genes and proteins related to plant hormone biosynthesis and signal transduction. From the WGCNA and WPCNA results, we furthermore screened 28 candidate genes and six key proteins that were correlated with lignin biosynthesis, plant hormone biosynthesis, and signaling pathways. In addition, 19 crucial transcription factors were discovered using correlation analysis that might play a role in regulating candidate genes. This study provides insight into the molecular mechanism of inflorescence elongation in alfalfa and establishes a theoretical foundation for improving alfalfa seed production.

Effects of selenium enrichment on fermentation characteristics, selenium content and microbial community of alfalfa silage.

BACKGROUND: Selenium is essential for livestock and human health. The traditional way of adding selenium to livestock diets has limitations, and there is a growing trend to provide livestock with a safe and efficient source of selenium through selenium-enriched pasture. Therefore, this study was conducted to investigate the effects of selenium enrichment on fermentation characteristics, selenium content, selenium morphology, microbial community and in vitro digestion of silage alfalfa by using unenriched (CK) and selenium-enriched (Se) alfalfa as raw material for silage. RESULTS: In this study, selenium enrichment significantly increased crude protein, soluble carbohydrate, total selenium, and organic selenium contents of alfalfa silage fresh and post-silage samples, and it significantly decreased neutral detergent fiber and acid detergent fiber contents (p < 0.05). Selenium enrichment altered the form of selenium in plants, mainly in the form of SeMet and SeMeCys, which were significantly higher than that of CK (p < 0.05). Selenium enrichment could significantly increase the lactic acid content, reduce the pH value, change the diversity of bacterial community, promote the growth of beneficial bacteria such as Lactiplantibacillus and inhibit the growth of harmful bacteria such as Pantoea, so as to improve the fermentation quality of silage. The in vitro digestibility of dry matter (IVDMD), in vitro digestibility of acid detergent fibers (IVADFD) and in vitro digestibility of acid detergent fibers (IVNDFD) of silage after selenium enrichment were significantly higher than those of CK (p < 0.05). CONCLUSION: This study showed that the presence of selenium could regulate the structure of the alfalfa silage bacterial community and improve alfalfa silage fermentation quality. Selenium enrichment measures can change the morphology of selenium in alfalfa silage products, thus promoting the conversion of organic selenium.

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.

Exploring the Structure and Substance Metabolism of a Medicago sativa L. Stem Base.

The stem base of alfalfa is a critical part for its overwintering, regeneration, and yield. To better understand the specificity and importance of the stem base, we analyzed the structure, metabolic substances, and transcriptome of the stem base using anatomical techniques, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and RNA sequencing (RNA-seq), and compared it with stems and roots. The anatomical structure shows that the ratio of xylem to phloem changes at the base of the stem. A total of 801 compounds involved in 91 metabolic pathways were identified from the broadly targeted metabolome. Transcriptome analysis revealed 4974 differentially expressed genes (DEGs) at the stem base compared to the stem, and 5503 DEGs compared to the root. Comprehensive analyses of differentially accumulated compounds (DACs) and DEGs, in the stem base vs. stem, identified 10 valuable pathways, including plant hormone signal transduction, zeatin biosynthesis, α-Linolenic acid metabolism, histidine metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, pentose phosphate pathway, galactose metabolism, and fructose and mannose metabolism. The pathways of plant hormone signal transduction and carbon metabolism were also identified by comparing the stem base with the roots. Taken together, the stem base of alfalfa is the transition region between the stem and root in morphology; in terms of material metabolism, its growth, development, and function are regulated through hormones and sugars.

Multiomics Analyses Reveal MsC3H29 Positively Regulates Flavonoid Biosynthesis to Improve Drought Resistance of Autotetraploid Cultivated Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa subsp. sativa), the "queen of forage," is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29, a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.

An integration of physiology, transcriptomics, and proteomics reveals carbon and nitrogen metabolism responses in alfalfa (Medicago sativa L.) exposed to titanium dioxide nanoparticles.

Nanoparticle (NP) pollution has negative impacts and is a major global environmental problem. However, the molecular response of alfalfa (Medicago sativa L.) to titanium dioxide nanoparticles (TiO2 NPs) is limited. Herein, the dual effects of TiO2 NPs (0-1000 mg L-1) on carbon (C) and nitrogen (N) metabolisms in alfalfa were investigated. The results showed that 500 mg L-1 TiO2 NPs (Ti-500) had the highest phytotoxicity in the C/N metabolizing enzymes; and it significantly increased total soluble sugar, starch, sucrose, and sucrose-phosphate synthase. Furthermore, obvious photosynthesis responses were found in alfalfa exposed to Ti-500. By contrast, 100 mg L-1 TiO2 NPs (Ti-100) enhanced N metabolizing enzymes. RNA-seq analyses showed 4265 and 2121 differentially expressed genes (DEGs) in Ti-100 and Ti-500, respectively. A total of 904 and 844 differentially expressed proteins (DEPs) were identified in Ti-100 and Ti-500, respectively. Through the physiological, transcriptional, and proteomic analyses, the DEGs and DEPs related to C/N metabolism, photosynthesis, chlorophyll synthesis, starch and sucrose metabolism, and C fixation in photosynthetic organisms were observed. Overall, TiO2 NPs at low doses improve photosynthesis and C/N regulation, but high doses can cause toxicity. It is valuable for the safe application of NPs in agriculture.

Starch and sucrose metabolism plays an important role in the stem development in Medicago sativa.

The forage quality of alfalfa (Medicago sativa ) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.

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.

Genome-Wide Identification, Phylogenetic and Expression Analysis of Expansin Gene Family in Medicago sativa L.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa (Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members (MsEXPs). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis-acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

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.

Effects of Selenium on the Lignin Deposition Pattern and Stem Mechanical Properties of Alfalfa (Medicago sativa L.).

Lignin provides structural support to plants; however, it reduces their utilization rate. According to our previous studies, selenium (Se) reduces lignin accumulation in alfalfa, but the specific mechanism involved remains unclear. Therefore, at the seedling stage, four root irrigation treatments using 2.5, 50, and 5 µmol/L sodium selenite (S-RI), selenomethionine (SS-RI), Se nanoparticles (SSS-RI), and deionized water (CK-RI) were performed. At the branching stage, four treatments of foliar spraying with the three Se fertilizers described above at a concentration of 0.5 mmol/L (S-FS, SS-FS, and SSS-FS) and deionized water (CK-FS) were administered. The results revealed that all Se treatments chiefly reduced the level of deposition of syringyl (S) lignin in the first internode of alfalfa stems. SS-FS and SSS-FS treatments mainly reduced the deposition of S and guaiacyl (G) lignins in the sixth internode of alfalfa stems, respectively, while S-FS treatment only slightly reduced the deposition of G lignin. S, SS, and SSS-RI treatments reduced the level of deposition of S and G lignins in the sixth internode of alfalfa stems. Se application increased plant height, stem diameter, epidermis (cortex) thickness, primary xylem vessel number (diameter), and pith diameter of alfalfa but decreased primary xylem area and pith parenchyma cell wall thickness of the first internode, and SS(SSS)-FS treatment reduced the mechanical strength of alfalfa stems. Therefore, Se application could decrease lignin accumulation by regulating the organizational structure parameters of alfalfa stems and the deposition pattern of the lignin monomers.

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 P5CDH from Cleistogenes songorica improves alfalfa growth performance under field drought conditions.

Water stress affects the metabolic regulation and delays the growth and development of alfalfa, causing a reduction in biomass. New alfalfa germplasm was created with improved drought tolerance in greenhouse conditions by introducing the key gene P5CDH1 from C. songorica, a xerophytic grass. However, the field adaptability and response mechanism of new drought-tolerant alfalfa germplasms under water stress are still unclear. In the present study, the yield and quality traits of transgenic CsP5CDH1 alfalfa lines under water stress and normal irrigation conditions were measured and analyzed for two years. The genetic variance components of the tested traits were calculated from the data fitted by the mixed linear model. The plant height of all lines showed significant genotypic variation (σ2g) (P < 0.05), and the stem diameter, stem number, and dry weight of all lines had a significant genotype × environment interaction (σ2ge) (P < 0.05). The heritability (H) of plant height, stem diameter, stem number, dry weight and leaf-to-stem ratio of alfalfa lines were 0.87, 0.52, 0.59, 0.52 and 0.50, respectively. There were significant genotype × environment interactions (σ2ge) (P < 0.05) for the quality traits of all lines. The heritabilities (H) of acid detergent fiber and neutral detergent fiber were 0.65 and 0.64, respectively. The results of transcriptional expression analysis with RNA-seq showed that the genes MsProDH1, MsProDH4, MsProDH5, MsP5CDH1, MsP5CS5, MsP5CS9, and MsP5CR1, which are involved in the proline metabolism pathway, played an important role in the drought tolerance of innovative alfalfa germplasm. Under water stress, with the regulation of key genes in the proline metabolism pathway, the proline content of all alfalfa lines increased to varying degrees. Among them, the proline content in the shoots and roots of transgenic line L6 was 7.29 times and 12.22 times that under normal irrigation conditions, respectively. The present study helped to clarify that the new germplasm of alfalfa transformed with the CsP5CDH gene synthesized a large amount of proline under water stress, and effectively slowed leaf water loss, thus improving the drought resistance of alfalfa.

Overexpression of MsNIP2 improves salinity tolerance in Medicago sativa.

Alfalfa (Medicago sativa) is one of the most widely cultivated forage crops in the world. However, alfalfa yield and quality are adversely affected by salinity stress. Nodulin 26-like intrinsic proteins (NIPs) play essential roles in water and small molecules transport and response to salt stress. Here, we isolated a salt stress responsive MsNIP2 gene and demonstrated its functions by overexpression in alfalfa. The open reading frame of MsNIP2 is 816 bp in length, and it encodes 272 amino acids. It has six transmembrane domains and two NPA motifs. MsNIP2 showed high identity to other known NIP proteins, and its tertiary model was similar to the crystal structure of OsNIP2-1 (7cjs) tetramer. Subcellular localization analysis showed that MsNIP2 protein fused with green fluorescent protein (GFP) was localized to the plasma membrane. Transgenic alfalfa lines overexpressing MsNIP2 showed significantly higher height and branch number compared with the non-transgenic control. The POD and CAT activity of the transgenic alfalfa lines was significantly increased and their MDA content was notably reduced compared with the control group under the treatment of NaCl. The transgenic lines showed higher capability in scavenging oxygen radicals with lighter NBT staining than the control under salt stress. The transgenic lines showed relative lower water loss rate and electrolyte leakage, but relatively higher Na+ content than the control line under salt stress. The relative expression levels of abiotic-stress-related genes (MsHSP23, MsCOR47, MsATPase, and MsRD2) in three transgenic lines were compared with the control, among them, only the expression of MsCOR47 was up-regulated. Consequently, this study offers a novel perspective for exploring the function of MsNIP2 in improving salt tolerance of alfalfa.

Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO2.

Understanding crop responses to elevated CO2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild-type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO2, whereas the effect of BNF on biomass was only observed at elevated CO2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome-wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.

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.

The effects of including sprouted barley with alfalfa hay in the diet on ruminal health and performance of cow-calf pairs.

The world population is growing exponentially, increasing demand to produce high-quality protein for human consumption. Changes in weather patterns, drought, and decreased land resources due to urbanization have increased the strain on the agriculture sector to meet world demands. An alternative method to combat these issues and continue to produce high-quality livestock feed would be through a controlled environment vertical farming system. Commonly, cereal grains, such as barley, are used in these systems to produce livestock feed. However, there is little information on the viability of feeding sprouted grains to beef cattle. Two diets of either feeder-quality alfalfa hay (n = 10 pairs; ALF) or the same alfalfa hay and sprouted barley (SB; 12.6% dry matter [DM]; n = 10 pairs) were fed for 90 d to Angus pairs with a steer calf during mid to late lactation. On days 0 and 90, body weight (BW), milk, rumen fluid, and body condition score were collected from cows and hip height and BW were recorded for calves. On day 10, BW was recorded for cows and calves and rumen fluid was collected from cows. Rumen fluid was also collected from cows on day 45. On day 55, BW was collected for both cows and calves and milk from cows. Intake was recorded throughout the trial via bunks with Vytelle technology. The PROC MIXED procedure of SAS was used to analyze all data with the day as a repeated measure to determine the main effect of diet. Individual volatile fatty acids (VFA) were measured as a percent of total VFA. No differences (P ≥ 0.16) were observed in calf BW, hip height, milk protein, fat, lactose, calf DM intake (DMI), or cow DMI. Cows fed SB tended (P = 0.08) to have a decreased somatic cell count compared to ALF. Percent butyrate was impacted by diet × day (P = 0.02), but no difference (P > 0.09) at any time points were detected. Additionally, a diet × day effect (P = 0.001) on rumen pH demonstrated that both groups stayed consistent until day 45 and then SB pH decreased the last 45 d. There was a day effect for total VFA (P = 0.0009), acetate:propionate (Ac:Pr; P < 0.0001), acetate (P < 0.0001), and propionate (P < 0.0001) demonstrating that total VFA, acetate, and Ac:Pr all increased throughout the trial, while propionate decreased. These results indicate that SB can be a potential alternative feed at this stage of production as it does not negatively impact health or production, but does affect the rumen pH and proportion of some VFA.

Climate variability and uncertainty associated with weather patterns can greatly impact feed security for cattle producers. Flooding, drought, and temperature extremes can reduce a farmer's ability to produce a consistent crop, resulting in feed prices that can fluctuate greatly. Vertical farming systems that sprout cereal grains in a controlled environment, using precision irrigation, may alleviate the effects of external factors such as climate and resulting feed prices. The objective of this study was to determine if sprouted barley (SB) could be used as an effective alternative feed source for cow-calf pairs. Two diets were fed to 20 cow-calf pairs, a control diet consisting of 100% feeder-quality alfalfa hay, or an experimental diet comprised of feeder-quality alfalfa hay and a 12.6% dry matter inclusion of SB for 90 d. Body weight, feed intake, and feeding behavior were analyzed in the cows and calves. Ruminal health was also assessed in cows by analyzing the ruminal fluid for pH and volatile fatty acid composition. When health and performance metrics were analyzed, no differences were found between the two diets that were administered to the cattle.

Molybdenum-mediated nitrogen accumulation and assimilation in legumes stepwise boosted by the coexistence of arbuscular mycorrhizal fungi and earthworms.

Molybdenum (Mo) is a critical micronutrient for nitrogen (N) metabolism in legumes, yet the impact of Mo on legume N metabolism in the context of natural coexistence with soil microorganisms remains poorly understood. This study investigated the dose-dependent effect of Mo on soil N biogeochemical cycling, N accumulation, and assimilation in alfalfa under conditions simulating the coexistence of arbuscular mycorrhizal fungi (AMF) and earthworms. The findings indicated that Mo exerted a hormetic effect on alfalfa N accumulation, facilitating it at low concentrations (below 29.98 mg/kg) and inhibiting it at higher levels. This inhibition was attributed to Mo-induced constraints on C supply for nitrogen fixation. Concurrently, AMF colonization enhanced C assimilation in Mo-treated alfalfas by promoting nutrients uptake, particularly Mg, which is crucial for chlorophyll synthesis. This effect was further amplified by earthworms, which improved AMF colonization (p < 0.05). In the soil N cycle, these organisms exerted opposing effects: AMF enhanced soil nitrification and earthworms reduced soil nitrate (NO3--N) reduction to jointly increase soil phyto-available N content (p < 0.05). Their combined action improved alfalfa N assimilation by restoring the protein synthesis pathway that is compromised by high Mo concentrations, specifically the activity of glutamine synthetase. These findings underscored the potential for soil microorganisms to mitigate N metabolic stress in legumes exposed to elevated Mo levels.

MsSPL12 is a positive regulator in alfalfa (Medicago sativa L.) salt tolerance.

KEY MESSAGE: Over expression of MsSPL12 improved alfalfa salt tolerance by reducing Na+ accumulation and increasing antioxidant enzyme activity and regulating down-stream gene expression. Improvement of salt tolerance is one of the major goals in alfalfa breeding. Here, we demonstrated that MsSPL12, an alfalfa transcription factor gene highly expressed in the stem cells, plays a positive role in alfalfa salt tolerance. MsSPL12 is localized in the nucleus and shows transcriptional activity in the presence of its C-terminus. To investigate MsSPL12 function in plant response to salt stress, we generated transgenic plants overexpressing either MsSPL12 or a chimeric MsSPL12-SRDX gene that represses the function of MsSPL12 by using the Chimeric REpressor gene-Silencing Technology (CRES-T), and observed that overexpression of MsSPL12 increased the salt tolerance of alfalfa transgenic plants associated with an increase in K+/Na+ ratio and relative water content (RWC) under salt stress treatment, but a reduction in electrolyte leakage (EL), reactive oxygen species (ROS), malondialdehyde (MDA), and proline (Pro) compared to wild type (WT) plants. However, transgenic plants overexpressing MsSPL12-SRDX showed an inhibited plant growth and a reduced salt tolerance. RNA-sequencing and quantitative real-time PCR analyses revealed that MsSPL12 affected the expression of plant abiotic resistance-related genes in multiple physiological pathways. The potential MsSPL12-mediated regulatory pathways based on the differentially expressed genes between the MsSPL12 overexpression transgenics and WT controls were predicted. In summary, our study proves that MsSPL12 is a positive regulator in alfalfa salt tolerance and can be used as a new candidate for manipulation to develop forage crops with enhanced salt tolerance.

PECTIN ACETYLESTERASE12 regulates shoot branching via acetic acid and auxin accumulation in alfalfa shoots.

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa.