In vivo, microspores in the anthers follow the gametophytic development pathway, culminating in the formation of pollen grains. Conversely, in vitro, under stress treatments, microspores can be reprogrammed into totipotent cells, initiating an embryogenic pathway that produces haploid and double-haploid embryos, which are important biotechnological tools in plant breeding. There is growing evidence that epigenetic reprogramming occurs during microspore embryogenesis through DNA methylation, but less is known about the role of histone modifications. This study investigates the dynamics of histone acetylation during the two microspore developmental pathways, microspore embryogenesis and pollen development, in Brassica napus. We analyzed histone H3 and H4 acetylation levels, histone acetyltransferase (HAT) activity and expression of HAC5 acetyltransferase using immunofluorescence, enzymatic activity assays, ELISA-like tests, and qPCR. Results showed a decrease in global histone acetylation levels during pollen maturation, correlated with reduced HAT activity and down- regulation of the BnHAC5-like gene. In contrast, stress-induced microspore reprogramming led to increased histone acetylation levels, enhanced HAT activity, and upregulation of BnHAC5-like. Treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor widely used in animal research but barely applied to plants, enhanced microspore embryogenesis initiation and proembryo formation, while increased histone acetylation levels. SAHA-treated proembryos showed higher expression than control of key embryo- genesis transcription factors BnFUS3, BnAGL15, and BnLEC2, suggesting that histone hyperacetylation facilitates transcriptional activation of essential genes for somatic embryogenesis initiation. These findings provided new insights into the epigenetic regulation of this process and revealed new opportunities with histone epigenetic regulator inhibitors, to improve microspore embryogenesis induction for crop improvement.
