Integrated transcriptomics and metabolomics reveal regulatory networks in Poa annua under combined drought and cold

In natural environments, plants are often exposed to multiple abiotic stresses simultaneously. Their combined effects usually cause more severe damage than a single stress. However, little is known about the coordinated response mechanisms of cool-season turfgrass to combined drought and cold stress. Two accessions of Annual bluegrass (Poa annua L.) with contrasting stress tolerance were used in this study: Huangzhong (HZ, tolerant) and Zhouqu (ZQ, sensitive). Physiological phenotyping, stomatal behavior observation, transcriptomics, and metabolomics were integrated to systematically compare their responses to drought, cold, and combined stress. The aim was to reveal the physiological and molecular regulatory differences between the two accessions and to identify the core pathways underlying combined stress responses. Results showed that combined stress significantly aggravated photosynthetic inhibition and oxidative damage. The sensitive accession ZQ exhibited much greater damage than the tolerant accession HZ. Transcriptomic and metabolomic analyses identified 11,440 and 13,631 differentially expressed genes, as well as 2585 and 2642 differentially accumulated metabolites in HZ and ZQ, respectively, under combined stress. Weighted gene co-expression network analysis (WGCNA) identified a core module (MEred) significantly correlated with photosynthetic efficiency and antioxidant capacity, with HCT and PAL as candidate hub genes. Meanwhile, multi-omics integration revealed that phenylpropanoid biosynthesis was strongly activated only in the tolerant accession HZ, and clustering analysis further demonstrated that the molecular profiles under combined stress closely resembled those under drought stress alone. This study provides new insights into the coordinated regulatory network of cool-season turfgrass in response to multiple abiotic stresses. It also offers potential targets for genetic improvement and functional utilization of stress-tolerance genes.