Alfalfa (Medicago sativa) is a globally distributed economic legume crop used for forage and ecological restoration. We aimed to explore the mechanisms underlying the cold acclimation observed in this species. Our results for fall plant growth showed that non-dormant alfalfa (SD) maintained a vigorous growth rate compared to that of fall-dormant alfalfa (ZD); however, the winter survival rate of ZD was higher than that of SD. Among the ZD samples, the starch content first accumulated and then decreased; the sucrose content was consumed first along with simultaneous raffinose accumulation, which was followed by sucrose content accumulation, with consistent changes in the corresponding related synthase and hydrolase activity. SD exhibited the opposite trend. The transcriptome data showed that most of the differentially expressed genes were involved in carbon metabolism (ko01200), amino acid biosynthesis (ko01230), and starch and sucrose metabolism (ko00500). Our data clearly show that alfalfa’s cold acclimation mechanism is a complex process, with the establishment of stable carbon homeostasis; sucrose is first converted into starch and raffinose, and then, starch is converted into sucrose, which enables alfalfa’s cold resistance. The process is accompanied by CBF/DREB1A TF regulation. This study provides important insights into the cold acclimation mechanisms of alfalfa.

Chinese herbal medicine (CHM) residues represent a promising and sustainable category of silage additives, with the potential to modulate fermentation and enhance nutrient preservation. This study investigated the effects of two CHMs, Astragalus membranaceus L. (Astragali Radix, AR) and Inula helenium L. (Inulae Radix, IR), on the fermentation profile, nutritional composition, and bacterial community structure in barley silage. The forage was ensiled without additive (control, CK), or with 1% or 2% (w/w) of AR or IR for 75 days. The results showed that all additive treatments significantly improved fermentation quality, as evidenced by lower pH and reduced ammonia-nitrogen (NH₃-N) content compared to CK. The 2% IR treatment was most effective in promoting homolactic fermentation, yielding the highest lactic acid content and lactic acid-to-acetic acid ratio. Nutritionally, additives significantly increased dry matter, starch, and water-soluble carbohydrates, while decreasing neutral and acid detergent fiber contents. High-throughput sequencing of the 16S rRNA gene revealed that both herbal additives profoundly reshaped the microbial community. They suppressed undesirable bacteria and significantly enriched beneficial Lactobacillus species. Principal component analysis confirmed a distinct separation in microbial community structure between control and treated silages. These findings underscore the potential of these herbal residues as natural modulators of the silage microbiome for improved forage conservation.

Introduction: Freezing injury in winter is a major abiotic stress that significantly limits plant growth and survival. While nitrogen and phosphorus fertilizers have been demonstrated to alleviate the impact of freezing injury in various plant species, their role of fertilizers in the cold tolerance of Urtica spp. is still unknown.Methods: This study investigated the effects of fertilizers on the cold resistance of U. cannabina by comprehensively analyzing the physiological and biochemical indices, transcriptome, and metabolome of the U. cannabina under applications of 150 kg nitrogen ha-1 (N) and 90 kg phosphorus ha-1 (P), using “no fertilizer” (CK) as the control.Results: The results showed that applying nitrogen and phosphorus fertilizers reduced the malondialdehyde concentration and had much higher superoxide dismutase activity and soluble sugar and proline concentrations. Transcriptomics and metabolomics analysis revealed that applying nitrogen and phosphorus fertilizers tended to involve several critical regulatory pathways in the biosynthesis of secondary metabolites, flavonoid biosynthesis, and phenylpropanoid biosynthesis pathways. Concretely speaking, these fertilizers can affect the biosynthesis of naringenin, pinobanksin 3-acetate, galangin, and p-Coumaroyl shikimic acid and the expression of related genes to regulate the cold tolerance of U. cannabina. Moreover, through using weighted correlation network analysis (WGCNA), 4210 genes in response to nitrogen fertilizer and 5975 genes in response to phosphorus fertilizer, positively correlating with key metabolites, were identified. Several genes encoding enzymes including glucan endo-1,3-beta-glucosidase, pectinesterase, trehalase, hydroquinone glucosyltransferase, monodehydroascorbate reductase, tyrosine aminotransferase, and peroxidase were verified to be hub genes involved in the cold-stress response of U. cannabina.Discussion: Overall, these findings have laid a theoretical foundation for the highly efficient utilization of nitrogen and phosphorus in U. cannabina and provide novel insights into the regulatory network of U. cannabina in response to cold-temperature stress.