Background: Drought is currently a global environmental problem, which inhibits plant growth and development and seriously restricts crop yields. Many plants exposed to drought stress can generate stress memory, which provides some advantages for resisting recurrent drought. DNA methylation is a mechanism involved in stress memory formation, and many plants can alter methylation levels to form stress memories; however, it remains unclear whether Medicago ruthenica exhibits drought stress memory, as the epigenetic molecular mechanisms underlying this process have not been described in this species. Methods: We conducted methylome and transcriptome sequencing to identify gene methylation and expression changes in plants with a history of two drought stress exposures. Results: Methylation analysis showed that drought stress resulted in an approximately 4.41% decrease in M. ruthenica genome methylation levels. The highest methylation levels were in CG dinucleotide contexts, followed by CHG contexts, with CHH contexts having the lowest levels. Analysis of associations between methylation and transcript levels showed that most DNA methylation was negatively correlated with gene expression except methylation within CHH motifs in gene promoter regions. Genes were divided into four categories according to the relationship between methylation and gene expression; the up-regulation of hypo-methylated gene expression accounted for the vast majority (692 genes) and included genes encoding factors key for abscisic acid (ABA) and proline synthesis. The hypo-methylation of the promoter and body regions of these two gene groups induced increased gene transcription levels. Conclusions: In conclusion, DNA methylation may contribute to drought stress memory formation and maintenance in M. ruthenica by increasing the transcription levels of genes key for ABA and proline biosynthesis.

Grazing disturbance can change the structure of plant rhizosphere microbial communities and thereby alter the feedback to promote plant growth or induce plant defenses. However, little is known about how such changes occur and vary under different grazing pressures or the roles of root metabolites in altering the composition of rhizosphere microbial communities. In this study, the effects of different grazing pressures on the composition of microbial communities were investigated, and the mechanisms by which different grazing pressures changed rhizosphere microbiomes were explored with metabolomics. Grazing changed composition, functions, and co-expression networks of microbial communities. Under light grazing (LG), some saprophytic fungi, such as Lentinus sp., Ramichloridium sp., Ascobolus sp. and Hyphoderma sp., were significantly enriched, whereas under heavy grazing (HG), potentially beneficial rhizobacteria, such as Stenotrophomonas sp., Microbacterium sp., and Lysobacter sp., were significantly enriched. The beneficial mycorrhizal fungus Schizothecium sp. was significantly enriched in both LG and HG. Moreover, all enriched beneficial microorganisms were positively correlated with root metabolites, including amino acids (AAs), short-chain organic acids (SCOAs), and alkaloids. This suggests that these significantly enriched rhizosphere microbial changes may be caused by these differential root metabolites. Under LG, it is inferred that root metabolites, especially AAs such as L-Histidine, may regulate specific saprophytic fungi to participate in material transformations and the energy cycle and promote plant growth. Furthermore, to help alleviate the stress of HG and improve plant defenses, it is inferred that the root system actively regulates the synthesis of these root metabolites such as AAs, SCOAs, and alkaloids under grazing interference, and then secretes them to promote the growth of some specific plant growth-promoting rhizobacteria and fungi. To summarize, grasses can regulate beneficial microorganisms by changing root metabolites composition, and the response strategies vary under different grazing pressure in typical grassland ecosystems.

Mowing significantly influences nutrient cycling and stimulates metabolic adjustments in plants to promote regrowth. Plant growth-promoting rhizobacteria (PGPR) are crucial for enhancing plant growth, nutrient absorption, and stress resilience; however, whether inoculation with PGPR after mowing can enhance plant regrowth capacity further, as well as its specific regulatory mechanisms, remains unexplored. In this study, PGPR Pantoea eucalyptus (B13) was inoculated into mowed Leymus chinensis to evaluate its effects on phenotypic traits, root nutrient contents, and hormone levels during the regrowth process and to further explore its role in the regrowth of L. chinensis after mowing. The results showed that after mowing, root nutrient and sugar contents decreased significantly, while the signal pathways related to stress hormones were activated. This indicates that after mowing, root resources tend to sacrifice a part of growth and prioritize defense. After mowing, B13 inoculation regulated the plant’s internal hormone balance by reducing the levels and signal of JA, SA, and ABA and upregulated the signal transduction of growth hormones in the root, thus optimizing growth and defense in a mowing environment. Transcriptomic and metabolomic analyses indicated that B13 promoted nutrient uptake and transport in L. chinensis root, maintained hormone homeostasis, enhanced metabolic pathways related to carbohydrates, energy, and amino acid metabolism to cope with mowing stress, and promoted root growth and regeneration of shoot. This study reveals the regenerative strategy regulated by B13 in perennial forage grasses, helping optimize resource utilization, increase yield, and enhance grassland stability and resilience.

Grazing, one of the primary utilization modes of grassland, is the main cause of grassland degradation. Historical overgrazing results in dwarf phenotype and decreased photosynthesis of perennial plants. However, it remains unknown what the mechanism underlying of this legacy effect is, and the role of stomata in the resulting decreased photosynthesis also remains unclear. To address these questions, differences in stomatal density, length and width on both adaxial and abaxial epidermis were compared between overgrazing and ungrazed Leymus chinensis offspring by using rhizome buds cultivated in a greenhouse, and the correlation between photosynthetic capacity and stomatal behavior was also investigated. Our results showed that historical grazing significantly impacted phenotype, photosynthesis and stomatal traits of L. chinensis. The offspring plants taken from overgrazed parents were dwarfed compared to those taken from ungrazed parents, and the photosynthesis and stomatal conductance of plants with a grazing history decreased by 28.6% and 21.3%, respectively. In addition, stomatal density and length on adaxial and abaxial leaf surfaces were significantly increased; however, stomatal width on abaxial leaf surfaces of overgrazed L. chinensis was significantly decreased compared with ungrazed individuals. Moreover, the expression patterns of eight genes related to stomatal regulation were tested: seven were down-regulated (2–18 times) and one was up-regulated (three times). Genes, involved in ABC transporter and receptor-like serine/threonine protein kinase were down-regulated. These results suggest that legacy effects of historical grazing affect the stomatal conductance by decreasing the stomatal width in progeny plants, which thus results in lower photosynthesis. Furthermore, changes of stomatal traits and function were regulated by the inhibition of ABC transporter and serine/threonine protein kinase. These findings are helpful for future exploration of the possible mechanisms underlying the response of grassland plants to long-term overgrazing.