Integration of HDS and HSFA1 Functions in MEcPP‐Mediated Thermotolerance and Growth Regulation in <i>Physcomitrium patens</i>

Land plants evolved mechanisms to cope with terrestrial challenges. The methylerythritol phosphate (MEP) pathway intermediate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) plays a central role in chloroplast retrograde signalling (CRS) and stress responses in Arabidopsis. However, its regulation in nonvascular plants remains underexplored. This study characterizes the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene family in Physcomitrium patens, uncovering unique evolutionary patterns with multiple HDS copies compared to the single-copy HDS in most angiosperms. We demonstrate that HDS catalyses a key step in MEP and interacts with heat shock transcription factor A1 (HSFA1) to modulate thermotolerance. CRISPR/LbCas12a-edited hds mutants exhibited enhanced thermotolerance and elevated MEcPP levels, with exogenous MEcPP mimicking this effect. Transcriptomic analysis identified upregulated stress-responsive genes, including small heat shock proteins, which prepare the plant for heat stress. This priming effect depends on HSFA1, which regulates MEP pathway genes, including HDS, thereby influencing MEcPP accumulation and thermotolerance. Furthermore, HDS and HSFA1 synergistically regulate growth in P. patens, with hds2 hds3 hsfa1 triple mutant displaying reduced size, lower IAA levels, and altered non-photochemical quenching. These findings highlight a novel HDS-HSFA1 regulatory module, expanding CRS paradigms and offering new insights into the evolution of stress adaptation in bryophytes, bridging gaps in understanding HDS function across species.