Integrative characterization of the heat shock transcription factors in Acorus: evolution and heat-responsive regulatory patterns.

With global climate change driving rising temperatures, the persistence and physiological performance of Acorus are increasingly threatened, highlighting the importance of understanding its molecular responses to heat stress. Heat shock transcription factors (HSFs) constitute a major family of transcriptional regulators involved in plant heat-stress responses and thermotolerance. Here, we identified 39 and 20 HSF genes in Acorus calamus and A. gramineus. Conserved motif and domain analyses revealed subfamily-specific structural features, while comparative synteny suggested that polyploidy and differential gene retention contributed to HSF family expansion in A. calamus. Subgenome-level analysis revealed no obvious difference in HSF gene number between subgenomes, whereas retained homeolog pairs exhibited tissue-dependent expression divergence. Promoter, predicted protein-protein interaction, and GO enrichment analyses linked AgHSFs to stress regulation and heat-response networks. Integrated analysis of time-series expression profiles, differential expression, weighted gene co-expression networks, and qRT-PCR revealed diverse temporal responses among AgHSFs and supported AgHSF07, AgHSF12, and AgHSF20 as strongly heat-responsive candidates for further characterization. AgHSF07 and AgHSF12 showed mainly nuclear YFP signals and strong autoactivation in the yeast system, supporting their potential roles as transcriptional activators. Together, this integrated genomic-transcriptomic framework provides insights into HSF evolution and heat-response regulation in Acorus and highlights AgHSF07 and AgHSF12 as promising candidate regulators for further functional studies of thermotolerance.