ANTI-SILENCING FUNCTION 1 ( ASF1) is an evolutionarily conserved histone chaperone involved in diverse chromatin-based processes in eukaryotes. Yet, its role in transcription and the underlying molecular mechanisms remain largely elusive, particularly in plants. Here, we show that the A rabidopsis thaliana ASF1 homologous genes, AtASF1A and AtASF1B, are involved in gene transcription activation in response to heat stress. The A tasf1ab mutant displays defective basal as well as acquired thermotolerance phenotypes. Heat-induced expression of several key genes, including the HEAT SHOCK PROTEIN ( HSP) genes H sp101, H sp70, H sa32, H sp17.6A and H sp17. 6B - CI, and the HEAT SHOCK FACTOR ( HSF) gene HsfA2 but not HsfB1 is drastically impaired in A tasf1ab as compared with that in wild type. We found that AtASF1A/ B proteins are recruited onto chromatin, and their enrichment is correlated with nucleosome removal and RNA polymerase II accumulation at the promoter and coding regions of HsfA2 and H sa32 but not HsfB1. Moreover, AtASF1A/ B facilitate H3K56 acetylation ( H3K56ac), which is associated with HsfA2 and H sa32 activation. Taken together, our study unravels an important function of AtASF1A/ B in plant heat stress response and suggests that AtASF1A/ B participate in transcription activation of some but not all HSF and HSP genes via nucleosome removal and H3K56ac stimulation. [ABSTRACT FROM AUTHOR]