Heat-responsive lncRNA and miRNA modules associated with thermotolerance in Moso bamboo

Heat stress limits Moso bamboo (Phyllostachys edulis) growth and development, yet the roles of non-coding RNA (ncRNA) networks in the heat response remain unclear. Here, by integrating mRNA, lncRNA and small RNA transcriptomes from seedlings exposed to 42 °C, we identified 2,889 differentially expressed lncRNAs, 1,216 mRNAs, and 21 miRNAs, respectively. Target prediction suggested that differentially expressed lncRNAs potentially regulate 578 genes, whereas differentially expressed miRNAs target 300 lncRNAs and 186 mRNAs. These predicted targets were enriched in heat response, reactive oxygen species (ROS) metabolism, and protein-folding pathways. Based on target prediction, network features, and heat-response-related expression patterns, we prioritized two candidate modules: Lnc041788-HSF37 and miR166d-Lnc398037-HSP70-20. We tested these modules using transient reporter assays in tobacco and heterologous overexpression in rice. Tobacco GUS assays and qRT-PCR in Moso bamboo leaves supported a positive regulatory association between Lnc041788 and HSF37. Heterologous overexpression of either Lnc041788 or HSF37 in rice enhanced thermotolerance. For the miR166d-Lnc398037-HSP70-20 module, tobacco reporter assays showed that miR166d repressed HSP70-20 expression, whereas Lnc398037 partially relieved this repression, consistent with a ceRNA-like effect. Correspondingly, overexpression of HSP70-20 or Lnc398037 enhanced thermotolerance, whereas miR166d overexpression increased heat sensitivity in rice. These phenotypes were accompanied by changes in antioxidant enzyme activities, ROS accumulation, and lipid peroxidation. Together, these results identify ncRNA-associated candidate modules that may contribute to heat-stress responses in Moso bamboo.