Hao Sun

Soil salinization severely limits alfalfa productivity; however, the molecular mechanisms governing cultivar-specific differences in salt tolerance remain largely unclear. In this study, two alfalfa cultivars (Zhongmu No.3 and WL440-HQ) were exposed to 200 mM NaCl stress, followed by integrated transcriptome sequencing, weighted gene co-expression network analysis (WGCNA), and functional validation. In total, 3,517 salt-responsive differentially expressed genes (DEGs) were identified, including 795 shared DEGs and cultivar-specific DEGs (1,336 in Zhongmu No.3 and 1,386 in WL440-HQ). GO and KEGG enrichment revealed conserved stress-response pathways, including flavonoid biosynthesis and starch and sucrose metabolism, as well as cultivar-specific patterns, with Zhongmu No.3 strongly enriched in stimulus-responsive genes. WGCNA further identified phenotype-related modules and core hub genes, notably MsWRKY22 and MsPSK3. Overexpression of MsPSK3 enhanced salt-alkali tolerance in alfalfa by activating antioxidant systems. Dual-luciferase and yeast one-hybrid (Y1H) assays verified that MsMYC2 directly binds to and activates the MsPSK3 promoter. This study reveals the molecular regulatory network underlying alfalfa responses to salt–alkali stress and provides key candidate genes for breeding salt-tolerant alfalfa varieties.

Meat rabbits are ideal meat-producing animals. However, weaning-induced intestinal inflammation often leads to growth delays, and severe cases impair breeding efficiency. Alfalfa polysaccharides (APSs) have antioxidant and anti-inflammatory properties, making them potential natural alternatives to antibiotics. To date, relatively limited research has been conducted on APS in meat rabbits. This research investigated the effects of APS on growth performance, intestinal inflammation, and meat quality in rabbits. Eighty healthy rabbits were randomized into four treatment groups, each group consisting of five replicates, with four rabbits per replicate. The four experimental groups were the control group (CON, basal diet), 400 mg/kg APS group (basal diet + 400 mg/kg APS), 800 mg/kg APS group (basal diet + 800 mg/kg APS), and 1200 mg/kg APS group (basal diet + 1200 mg/kg APS). The results indicated that adding 800 mg/kg APS to the diet significantly increased ADG (p < 0.001) and reduced F/G (p = 0.008). With increasing APS supplementation levels, slaughter weight (p = 0.035), eviscerated weight (p = 0.020), semi-eviscerated weight (p = 0.015), and semi-eviscerated yield percentage (p = 0.035) were all significantly increased. Additionally, dripping loss in muscle was significantly reduced in the 800 mg/kg APS group (p = 0.006). In addition, the villus height of the small intestine and the expression of tight junctions were significantly increased by 800 mg/kg APS supplementation, which reduced intestinal permeability and lowered levels of intestinal inflammatory mediators by inhibiting the PPARγ/NF-κB pathway. Additionally, a diet with APS significantly increased the abundance of Flavonifractor, a butyrate-producing bacterium in the cecum. Cell assays further demonstrated that butyrate could inhibit the release of inflammatory cytokines from RAW264.7 via the PPARγ/NF-κB pathway. In conclusion, APS improved growth performance by reshaping the gut microbiota and increasing the level of butyrate in the cecum, further inhibiting intestinal inflammation through the PPARγ/NF-κB signaling pathway.

Composite saline-alkali stress severely limits alfalfa (Medicago sativa L.) productivity, but cultivar-specific responses and regulatory mechanisms remain unclear. Three cultivars, Zhongmu No.3 (ZM3), Yumu No.1 (YM1), and WL440-HQ, were evaluated under moderate composite saline-alkali stress (100 mmol/L NaCl + 100 mmol/L NaHCO₃). ZM3 and YM1 exhibited greater tolerance to saline-alkali stress than WL440-HQ. Transcriptome analysis of ZM3 seedlings treated with 75 mmol/L NaCl and 10 mmol/L NaHCO₃ identified 3,317 differentially expressed genes, including 1,622 upregulated and 1,695 downregulated genes. These genes were mainly enriched in oxidoreductase activity, phenylpropanoid and flavonoid biosynthesis, glutathione metabolism, MAPK signaling, and plant hormone signal transduction. Weighted Gene Co-expression Network Analysis identified nine co-expression modules, of which MEblue and MEturquoise were closely associated with growth and oxidative damage traits. Several hub transcription factors, including MsWRKY33, MsMYC2, MsERF1, bHLH family members, and the MsPSK3 gene, were identified. Heterologous overexpression assays showed that MsPSK3 enhanced saline-alkali tolerance by increasing superoxide dismutase and peroxidase activities and reducing reactive oxygen species and malondialdehyde accumulation. Yeast one-hybrid and dual-luciferase assays confirmed that MsMYC2 directly binds to the MsPSK3 promoter and activates its expression. In addition, an MsWRKY33-centered regulatory pathway involving ethylene signaling and melatonin biosynthesis was proposed to coordinate ROS scavenging, ion homeostasis, and osmotic adjustment. These results demonstrate that the MsMYC2–MsPSK3 and MsWRKY33-centered regulatory network could be potential key regulatory modules in ZM3, providing candidate genes for the genetic improvement of alfalfa.

Background: Salt stress is a major limiting factor for alfalfa yield due to its relatively low salt tolerance. High soil salinity adversely impacts alfalfa growth and development, leading to reduced yield. Melatonin (MT) is known to play a significant role in enhancing plant resistance to abiotic stresses. However, the mechanisms underlying melatonin-mediated abiotic stress responses, particularly salt stress, are not well understood. To address this gap, we conducted a study focusing on germinating alfalfa seeds under salt stress. Methods: Physiological indexes and transcriptomics analyses were carried out on the germinated seeds to investigate the effects of melatonin on alfalfa seedlings under salt stress conditions. Result: Our results revealed that the application of melatonin led to an increase in shoot length and fresh weight of alfalfa seedlings under salt stress. Moreover, key physiological indexes such as peroxidase (POD) activity and glutathione (GSH) content were increased, while content of malondialdehyde and superoxide anions decreased. Transcriptomic analysis identified a total of 2,131 differentially expressed genes (DEGs) in the salt-treated group, with 726 up-regulated and 1,405 down-regulated genes, while the MT-treated group showed 2,896 DEGs, of which 1,097 were up-regulated and 1,799 were down-regulated. Further, KEGG enrichment analysis highlighted the enrichment of DEGs in pathways including flavonoid biosynthesis, ABC transporter, glutathione metabolism and the MAPK signaling pathway, with these pathways more significantly enriched in the MT-treated group. These findings collectively indicate that melatonin plays a crucial role in the response of alfalfa to salt stress and provide new insights into melatonin mediated gene expression of alfalfa salt tolerance.

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.

Salt stress poses a significant threat to crop yields worldwide. Melatonin (MT), an endogenous hormone synthesized in plants, has emerged as a crucial player in plant responses to various abiotic stresses, including drought, salinity, heat, and cold. However, the precise molecular mechanisms underlying MT-mediated abiotic stress responses remain incompletely understood. To elucidate the key genes and pathways involved in MT-mediated alleviation of salt stress, we conducted physiological, biochemical, and transcriptomic analyses on alfalfa seedlings. Our results demonstrated that alfalfa seedlings treated with melatonin exhibited higher germination rates, longer bud lengths, and greater fresh weights compared to those subjected to salt stress alone. Furthermore, the levels of malondialdehyde (MDA) and superoxide anion (O²⁻) were reduced, while the activities and contents of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and glutathione (GSH) increased in response to melatonin treatment. Transcriptome analysis revealed 2181 differentially expressed genes (DEGs) in the salt-treated group, with 780 upregulated and 1401 downregulated genes. In contrast, the MT-treated group exhibited 4422 DEGs, including 1438 upregulated and 2984 downregulated genes. Functional annotation and pathway enrichment analysis indicated that DEGs were primarily involved in the biosynthesis of flavonoids, isoflavones, plant hormones, glutathione (GSH), soluble sugars, and other substances, as well as in ABC transporter and MAPK signaling pathways. Notably, the MT-treated group showed greater enrichment of DEGs in these pathways, suggesting that MT mitigates salt stress by modulating the expression of genes related to phytohormones and antioxidant capacity. Overall, our findings provide valuable insights into the molecular mechanisms underlying MT-mediated salt tolerance in alfalfa, with important implications for breeding salt-tolerant alfalfa and other crops.

Salt stress is a major limiting factor for alfalfa yield due to its relatively low salt tolerance. High soil salinity adversely impacts alfalfa growth and development, leading to reduced yield. Melatonin (MT) is known to play a significant role in enhancing plant resistance to abiotic stresses. However, the mechanisms underlying melatonin-mediated abiotic stress responses, particularly salt stress, are not well understood. To address this gap, we conducted a study focusing on germinating alfalfa seeds under salt stress. Physiological indexes and transcriptomics analyses were carried out on the germinated seeds to investigate the effects of melatonin on alfalfa seedlings under salt stress conditions. Our results revealed that the application of melatonin led to an increase in shoot length and fresh weight of alfalfa seedlings under salt stress. Moreover, key physiological indexes such as peroxidase (POD) activity and glutathione (GSH) content were enhanced, while levels of malondialdehyde and superoxide anions decreased. Transcriptomic analysis identified a total of 2,131 differentially expressed genes in the salt-treated group, with 726 up-regulated and 1,405 down-regulated genes, while the MT-treated group showed 2896 differentially expressed genes, of which 1,097 were up-regulated and 1,799 were down-regulated. Further, KEGG enrichment analysis highlighted the enrichment of differentially expressed genes in pathways including flavonoid biosynthesis, ABC transporter, glutathione metabolism, and the MAPK signaling pathway, with these pathways more significantly enriched in the MT-treated group. These findings collectively suggest that melatonin plays a crucial role in alfalfa's response to salt stress and offer new insights into the genomic mechanisms underlying melatonin-mediated salt tolerance in alfalfa.

The type and composition of food strongly affect the variation and enrichment of the gut microbiota. The gut–microbiota–spleen axis has been developed, incorporating the spleen’s function and maturation. However, how short-chain fatty-acid-producing gut microbiota can be considered to recover spleen function, particularly in spleens damaged by changed gut microbiota, is unknown in geese. Therefore, the gut microbial composition of the caecal chyme of geese was assessed by 16S rRNA microbial genes, and a Tax4Fun analysis identified the enrichment of KEGG orthologues involved in lipopolysaccharide production. The concentrations of LPS, reactive oxygen species, antioxidant/oxidant enzymes, and immunoglobulins were measured from serum samples and spleen tissues using ELISA kits. Quantitative reverse transcription PCR was employed to detect the Kelch-like-ECH-associated protein 1–Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2), B cell and T cell targeting markers, and anti-inflammatory/inflammatory cytokines from the spleen tissues of geese. The SCFAs were determined from the caecal chyme of geese by using gas chromatography. In this study, ryegrass-enriched gut microbiota such as Eggerthellaceae, Oscillospiraceae, Rikenellaceae, and Lachnospiraceae attenuated commercial diet-induced gut microbial alterations and spleen dysfunctions in geese. Ryegrass significantly improved the SCFAs (acetic, butyric, propionic, isovaleric, and valeric acids), AMPK pathway-activated Nrf2 redox signaling cascades, B cells (B220, CD19, and IgD), and T cells (CD3, CD4, CD8, and IL-2, with an exception of IL-17 and TGF-β) to activate anti-inflammatory cytokines (IL-4 and IL-10) and immunoglobulins (IgA, IgG, and IgM) in geese. In conclusion, ryegrass-improved reprogramming of the gut microbiota restored the spleen functions by attenuating LPS-induced oxidative stress and systemic inflammation through the gut–microbiota–spleen axis in geese.

Introduction. Geese can naturally obtain dietary fiber from pasture, which has anti-inflammatory and antioxidant properties. This study aimed to investigate the inhibitory impacts of pasture on ameliorating LPS-ROS-induced gut barrier dysfunction and liver inflammation in geese. Materials and methods. The lipopolysaccharides (LPS), alkaline phosphatase (ALP), reactive oxygen species (ROS), tight junction proteins, antioxidant enzymes, immunoglobulins, and metabolic syndrome were determined using ELISA kits. The Kelch-like-ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) and inflammatory cytokines were determined using the quantitative reverse transcription PCR (RT-qPCR) method. The intestinal morphology was examined using the Hematoxylin and Eosin (H&E) staining method in ileal tissues. Results. Pasture significantly influences nutrient absorption (p < 0.001) by ameliorating LPS and ROS-facilitated ileal permeability (p < 0.05) and systemic inflammation (p < 0.01). Herein, the gut permeability was paralleled by liver inflammation, which was significantly mimicked by ALP-dependent Nrf2 (p < 0.0001) and antioxidant enzyme activation (p < 0.05). Indeed, the correlation analysis of host markers signifies the importance of pasture in augmenting geese’s health and production by averting gut and liver inflammation. Conclusions. Our results provide new insight into the mechanism of the pasture-induced ALP-dependent Nrf2 signaling pathway in limiting systemic inflammation in geese.