Seed aging is a critical biological process that leads to progressive loss of seed vigor, thereby constraining germplasm conservation and agricultural productivity. To elucidate the molecular mechanisms underlying this process in grass species, we performed transcriptomic analyses to characterize regulatory networks underlying seed aging in Elymus sibiricus, a dominant forage species on the Qinghai–Tibet Plateau. Seeds were subjected to artificial accelerated aging (45 °C, 80% relative humidity, 1–6 days), followed by physiological evaluation and RNA sequencing. Seed vigor and germination percentage declined markedly with aging, accompanied by extensive transcriptional reprogramming. Integrative analyses identified pyruvate metabolism, MAPK signaling, and peroxisome function as key processes associated with vigor loss during late-stage aging. WGCNA further revealed that genes encoding heat shock proteins and glutathione metabolism-related enzymes were co-localized within the same module, suggesting a possible synergistic role in preserving seed viability during aging. In addition, WRKY24, ARF9, and ARF19 were identified as candidate hub transcription factors. WRKY24 may contribute to aging by modulating antioxidant defense-related genes (e.g., TRX1 and NRPC1), while ARF9 and ARF19 may regulate ROS homeostasis through predicted downstream targets, including FQR1, PER2, MAO1B, ANN5, and MT2B. Together, these findings support a hypothetical regulatory model in which WRKY and ARF transcription factors coordinate redox homeostasis and hormone signaling to regulate seed longevity in E. sibiricus. This study provides a systems-level framework for understanding seed aging in perennial grasses and identifies potential genetic targets for improving seed storability, with implications for germplasm conservation and alpine grassland sustainability.

Background: Artificial inoculation of Epichloë endophytes into elite forage germplasm aims to establish beneficial symbioses for developing high-yield, high-quality, and stress-tolerant cultivars, but host specificity of the fungi often causes compatibility issues in non-natural hosts. Methods: The E. bromicola isolated from native wild barley was inoculated into cultivated wild barley (Hordeum brevisubulatum) and cultivated barley (Hordeum valgare), forming Hb+Eb and Hv+Eb. The NHb+Eb (native wild barley naturally infected with E. bromicola) served as a control. We analyzed fungal colonization patterns and symbiotic gene regulation to clarify the compatibility between E. bromicola and non-natural hosts. Results: Compared with NHb+Eb and Hb+Eb, E. bromicola in Hv+Eb showed obvious hyphal vacuolization. E. bromicola colonization altered host trichome morphology and induced stomatal closure. Correspondingly, expression of the siderophore biosynthesis gene sidN and the NADPH oxidase complex genes (NoxA, NoxB, NoxR, RacA) was significantly lower (p < 0.05) in Hv+Eb than in Hb+Eb and NHb+Eb. Conclusions: This study reveals that the incompatibility between cultivated barley and E. bromicola is characterized by altered hyphal morphology, which is linked to the downregulation of sidN and Nox. These findings provide a critical theoretical foundation for developing highly compatible cereal-Epichloë germplasms.

Winter fallow fields are widely distributed in southern China, where legume–grass mixtures are considered a useful approach for improving land-use efficiency and forage productivity while maintaining agroecosystem functions. Previous studies on the use of winter fallow fields have generally focused either on monoculture or on crops subjected to a single cut. These studies have primarily assessed aboveground productivity, whereas soil bacterial community responses have received comparatively little attention. Consequently, limited information is available on how different seeding combinations and cutting times affect aboveground biomass and soil bacterial community structure in mixed-cropping systems. A fixed-site field experiment involving annual ryegrass (Lolium multiflorum L.) and Chinese milk vetch (Astragalus sinicus L.) was conducted on yellow clay soil. Five annual ryegrass-Chinese milk vetch seeding combinations (100:0, 75:25, 50:50, 25:75, and 0:100) were established as proportions of their respective monoculture seeding rates. The same plots were maintained for five consecutive winter growing seasons, from autumn 2021 to spring 2026. Plant and soil samples were collected only during the final growing season (2025–2026), at four cutting times in January, March, April, and May. These samples were used to assess the effects of seeding combination and cutting time on forage productivity, root traits, soil fertility, and soil bacterial community structure. Among all groups, the 75% annual ryegrass + 25% Chinese milk vetch group (R75M25) exhibited the best overall performance, with cumulative yields after four cuttings of 17,274.21 kg·ha⁻¹ dry matter, 2717.02 kg·ha⁻¹ crude protein, and 12,220.46 kg·ha⁻¹ digestible dry matter. In addition, the R75M25 group maintained relatively high contents of alkali-hydrolyzable nitrogen, available phosphorus, and available potassium in soil. The top three phyla of soil samples were Proteobacteria, Actinobacteria, and Acidobacteria, with a total relative abundance of >60%. Among these, Proteobacteria showed the greatest relative abundance in monoculture, whereas the seeding combination had a reduced proportion of it. In addition, its relative abundance rose steadily with cutting time. At the genus level, RB41, Gemmatimonas, and Sphingomonas dominated the soil bacterial community. The seeding combinations did not alter soil bacterial alpha diversity. Cutting times significantly reduced the phylogenetic diversity index and drove the temporal differentiation of taxa such as Actinobacteria and Bacteroidetes. The inclusion of an appropriate proportion of Chinese milk vetch improved forage nutritional value while maintaining high biomass accumulation in southern China, with the R75M25 group showing the best overall performance in winter fallow fields.