Time-series metatranscriptomics reveals differential salinity effects on the methanogenic food web in paddy soil

Saltwater intrusion and sea level rise (SWISLR) threaten coastal agroecosystems, yet their impact on the methanogenic food web in rice paddies remains virtually unknown. Using "double-RNA" metatranscriptomics (rRNA, mRNA), we investigated salinity effects on the methanogenic community in straw-amended slurries following different preincubation periods. Our results demonstrate that salt stress significantly inhibits methane (CH₄) production, with the degree of inhibition varying by the community's successional stage. Salinity fundamentally shifted the methanogenic food web toward Clostridiaceae dominance, altering key metabolic pathways including polymer breakdown, glycolysis, pyruvate metabolism, decarboxylation conversion of pyruvate to acetate, and ethanol fermentation. Notably, the accumulation of acetate under salt stress fueled acetoclastic methanogenesis by Methanosarcinaceae, mitigating CH₄ production inhibition after a 14-day preincubation. While the strong salinity inhibitory effect on the expression of the hydrogenotrophic methanogenesis pathway by Methanocellaceae diminished with preincubation time, syntrophic propionate oxidation activity was highly suppressed after 14-day preincubation. Additionally, the expression of the methanol-dependent methanogenesis pathway by Methanomassiliicoccales increased with prolonged preincubation time. These findings provide critical insights into the resilience of the anaerobic food web under salinity stress, with broader implications for understanding the impacts of sea level rise on global biogeochemical cycles.IMPORTANCESeawater intrusion and sea level rise (SWISLR), driven by climate change, pose significant threats to coastal agroecosystems, particularly salt-affected paddy soils. Despite the importance of these systems in global methane dynamics, the specific effects of salinity on the methanogenic food web in rice paddies remain poorly understood. Using a "double-RNA" metatranscriptomics approach, this study demonstrates that salinity markedly alters methane production and microbial community dynamics, with these effects varying across different successional stages of the microbial assemblage. The resilience of the methanogenic food web under salinity stress is governed by time-dependent metabolic shifts, offering critical insights into how SWISLR may influence methane emissions and broader biogeochemical processes in coastal agricultural landscapes. These findings highlight the urgent need to incorporate SWISLR-related impacts into assessments of coastal agroecosystems' contributions to global methane budgets and climate feedback mechanisms.