Integrating Transcriptome, Metabolome and Microbiome to Explore the Molecular Mechanism of Phenotypic Plasticity in...

The endangered Tibetan herb Phlomoides rotata is threatened by overharvesting and slow natural regeneration. To support its sustainable utilisation, we investigated the mechanisms underlying its phenotypic plasticity during low-altitude adaptation using an integrated multi-omics approach. Specifically, rhizosphere soils and leaf tissues were collected from P. rotata cultivated at high-, mid-, and low altitudes for multi-omics analysis, including bacterial and fungal profiling, and phenotypic, transcriptomic, and metabolomic assessments. Altitude-dependent shifts were observed in microbial community composition. Functional profiling suggests that rhizosphere microbial communities of P. rotata at low altitude possess enhanced metabolic activity and nutrient cycling capacity. Procrustes analysis revealed strong concordance between potential microbial indicators and phenotypic traits (R² = 0.84, p = 0.002 for bacteria, R² = 0.82, p = 0.005 for fungi). Transcriptomic analysis identified 3336 and 9208 unigenes associated with phenotypic variation. GO enrichment revealed that low-altitude samples were dominated by growth-related functions, while high-altitude samples favoured defence responses. KEGG enrichment of hub genes supported this pattern, highlighting enhanced developmental and biosynthetic pathways at low altitudes and stress-regulatory processes at high altitudes. Metabolomic analysis identified 658 altitude-associated differential metabolites. KEGG enrichment showed zeatin biosynthesis was prominent at high altitudes, while butanoate, starch, and sucrose metabolism were enriched at low altitudes. Furthermore, random forest analysis of phenotype-associated metabolites revealed that phenylpropanoids and organic acids were characteristic of high-altitude samples, while organoheterocyclic compounds were more typical of low-altitude environments. Mantel test and PLS-SEM modelling jointly revealed that altitude-driven shifts in rhizosphere microbiome function regulate host gene expression and secondary metabolism, ultimately shaping phenotypic variation. This comprehensive research provides novel insights into the environmentally induced phenotypic plasticity of alpine medicinal plants during low-altitude adaptation and offers a deeper understanding of the key drivers of this process.