Alfalfa (Medicago sativa) is a perennial forage crop with significant economic and ecological significance. If alfalfa can be planted in saline-alkali land, it will not only improve the utilization rate of marginal land and alleviate the competition between forage and cereal crops for arable land but will also increase the yield of high-quality domestic forage. In this study, we conducted transcriptomic analysis on the saline-alkali-tolerant alfalfa cultivar NQ-1 and compared its metabolite accumulation levels with saline-alkali-sensitive cultivars. The results showed that under saline-alkali stress, the photosynthesis and some secondary metabolic pathways in NQ-1 were activated, such as α-Linolenic acid metabolism, Phenylpropanoid and Flavonoid biosynthesis, and Photosynthesis-related pathways, providing substances and energy for enhancing NQ-1 stress tolerance. Furthermore, some specific flavonoids were detected that may contribute to the saline-alkali tolerance of NQ-1. In addition, transcription factors that may regulate flavonoid biosynthesis in NQ-1 under saline-alkali stress were also identified. This study deepens the understanding of the resistance mechanism of saline-alkali-tolerant cultivars of alfalfa and provides valuable information for molecular design breeding strategies for stress-resistant alfalfa.

Auxin response factors (ARFs) have been reported to play vital roles during plant growth and development. In order to reveal specific functions related to vegetative organs in grasses, an in-depth study of the ARF gene family was carried out in switchgrass (Panicum virgatum L.), a warm-season C4 perennial grass that is mostly used as bioenergy and animal feedstock. A total of 47 putative ARF genes (PvARFs) were identified in the switchgrass genome (2n = 4x = 36), 42 of which were anchored to the seven pairs of chromosomes and found to be unevenly distributed. Sixteen PvARFs were predicted to be potential targets of small RNAs (microRNA160 and 167). Phylogenetically speaking, PvARFs were divided into seven distinct subgroups based on the phylogeny, exon/intron arrangement, and conserved motif distribution. Moreover, 15 pairs of PvARFs have different temporal-spatial expression profiles in vegetative organs (2nd, 3rd, and 4th internode and leaves), which implies that different PvARFs have specific functions in switchgrass growth and development. In addition, at least 14 pairs of PvARFs respond to naphthylacetic acid (NAA) treatment, which might be helpful for us to study on auxin response in switchgrass. The comprehensive analysis, described here, will facilitate the future functional analysis of ARF genes in grasses.

Soil salinization–alkalization is a critical abiotic constraint on global agriculture, threatening agroecosystem sustainability. Leymus chinensis, a high–quality perennial forage with strong stress resilience, is an ideal model for studying saline–alkali tolerance in graminaceous crops. We integrated physiological, transcriptomic, and metabolomic profiling to dissect its responses under moderate vs. severe carbonate stress, mimicking natural saline–alkali soils rather than single salt stress treatments. Multi–omics analysis revealed drastic reprogramming of energy metabolism, carbohydrate homeostasis, water transport, and secondary metabolism. Our novel finding reveals that L. chinensis uses stress–severity–dependent mechanisms, with flavonoid biosynthesis as a central “regulatory hub”: moderate saline–alkali stress acts as a stimulus for “Adaptive Activation” (energy + antioxidants), promoting growth, while severe stress exceeds tolerance thresholds, causing “systemic imbalance” (oxidative damage + metabolic disruption) and growth retardation. Via WGCNA and metabolome–transcriptome modeling, 22 transcription factors linked to key flavonoid metabolites were identified, functioning as molecular switches for stress tolerance. Our integrated approach provides novel insights into L. chinensis’ tolerance networks, and the flavonoid biosynthesis pathways and regulatory genes offer targets for precision molecular breeding to enhance forage stress resistance and mitigate yield losses from salinization–alkalization.

Background: The Songnen Plain in China contains soda saline–alkaline soil, wherein salinity and alkalinity severely constrain crop productivity. Alfalfa (Medicago sativa L.) is a forage legume that has adapted to moderate saline–alkaline conditions, but how its rhizosphere microbial community facilitated this adaptation remains unclear. Methods: Using 16S rRNA gene sequencing, we compared alfalfa rhizosphere bacteria in saline–alkaline soil (AS) and control soil. Bacteria isolated from AS were screened for plant growth-promoting traits, with the most effective strains validated in pot experiments involving 50 mM NaHCO₃. Results: Compared with the control soil bacterial community, the AS bacterial community was significantly enriched with Methylomirabilota and unclassified bacteria (phylum level), with the genus RB41 identified as the most discriminative biomarker. Gene functions predicted using PICRUSt2 reflected the responsiveness of this community to environmental stressors. Inoculations with Pseudomonas laurentiana strain M73 and Stenotrophomonas maltophilia strain M81, which were isolated from AS, significantly improved alfalfa growth and health under NaHCO₃ stress. Conclusions: Saline–alkaline conditions in the Songnen Plain reshape the alfalfa rhizosphere bacterial community, enriching for specific taxa and potentially enhancing microbial functions associated with stress resistance. Strains M73 and M81 can effectively promote stress tolerance, making them useful microbial resources for improving soil conditions.

Alfalfa (Medicago sativa L.), a perennial leguminous herb, exhibits robust cold and saline-alkali tolerance. In this study, two alfalfa cultivars, LJ and 218TR, were treated with saline-alkali stress, cold stress, and combined saline-alkali-cold stress , and p henotype, physiology , key metaboli te and stress-responsive genes were analyzed . The results showed m alondialdehyde, soluble sugar, proline content, and the activities of phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase initially increased under individual stresses but declined after combined stress . T he maximum photochemical efficiency of photosystem II and chlorophyll content declined under individual and combined stresses. The staining of leaves revealed that combined stress induced significantly higher cell mortality and accumulation of superoxide anio n compared to individual stresses. LJ exhibited superior resistant to saline-alkali, cold, and combined stress compared to 218TR . Metabolite analysis showed salicylic acid (SA) in two alfalfa wa s the most responsive metabolite to combined stress. The isochorismate synthase (ICS) and PAL as critical genes for SA biosynthesis were up-regulated in expression under single or combined stress, and promote d SA accumulation , thereby improving alfalfa resilience to combined saline-alkali-cold stress. This study elucidates the physiological and molecular mechanisms underlying alfalfa’s response to combined saline-alkali and cold stress, providing a theoretical basis for breeding stress-tolerant cultivars.

Alfalfa (Medicago sativa L.), a perennial leguminous herb, can tolerate cold and saline–alkaline conditions. In this study, alfalfa cultivars LJ and 218TR were exposed to saline–alkaline, cold, and saline–alkaline–cold conditions and compared in terms of phenotypes, physiological indices, key metabolite contents, and stress-responsive gene expression. Malondialdehyde, soluble sugar, proline contents and phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase activities initially increased under individual stress conditions, but decreased when stresses were combined. Photosystem II maximum photochemical efficiency and chlorophyll contents decreased under individual and combined stress conditions. Nitroblue tetrazolium-stained leaves revealed that the combined stress treatment significantly increased cell mortality rates and superoxide anion levels. LJ was more tolerant to saline–alkaline, cold, and combined stress treatments than 218TR. Metabolite analyses indicated that for LJ and 218TR, salicylic acid (SA) was the most responsive metabolite to combined stress conditions. Additionally, the expression of isochorismate synthase (ICS) and PAL genes critical for SA biosynthesis was upregulated under single or combined stress conditions, leading to SA accumulation and improved tolerance to saline–alkaline–cold conditions. This study revealed the physiological indices and molecular changes underlying alfalfa responses to saline–alkaline stress combined with cold stress, providing a theoretical basis for breeding stress-tolerant cultivars.