The Small Heat Shock Protein HSP17.2B is an Essential Component for HDS-Mediated Thermotolerance in Physcomitrium patens

Physcomitrium patens ( P. patens ), a model moss species occupying a unique transitional evolutionary position between aquatic and terrestrial plants, has evolved highly effective mechanisms to cope with heat stress and diverse environmental challenges. Previously, we identified the HDS regulatory module that coordinates thermal adaptation and growth, where MEcPP induces 81.25% of heat priming-competent HSP20 protein isoforms. In this study, we identified 26 HSP20 genes in P. patens , which are classified into three subfamilies (CI, CII, and P/MT). Focusing on the functional characterization of PpHSP17.2B , we found that its overexpression under heat stress enhances chlorophyll fluorescence and chlorophyll content, thereby improving thermal tolerance. Conversely, Pphsp17.2b knockout lines exhibited thermosensitive phenotypes. While the hds2 hds3 double mutant displayed significantly enhanced thermotolerance compared to the wild type (WT), the hds2 hds3 Pphsp17.2b triple mutant lines exhibited heat sensitivity indistinguishable from the Pphsp17.2b single mutant, completely abolished the resistance phenotype of the hds2 hds3 double mutant. This genetic evidence establishes PpHSP17.2B as an essential component downstream of HDS -mediated thermotolerance pathway. Furthermore, these findings expand the HDS module, and provide new insights into plant thermal regulatory networks. • 26 small heat shock protein genes were identified in Physcomitrium patens. • HSP17.2B overexpression confers thermotolerance, its mutant shows hypersensitivity. • The hds2hds3Pphsp17.2b abolished the resistance phenotype of the hds2hds3. • PpHSP17.2B is an essential component of HDS -mediated thermotolerance pathway.