Changes in histone methylation and acetylation during microspore reprogramming to embryogenesis occur concomitantly with BnHKMT and BnHAT expression and are associated to cell totipotency, proliferation and differentiation in Brassica napus

Abstract

In response to stress treatments, microspores can be reprogrammed to become totipotent cells that follow an embryogenic pathway producing haploid and double-haploid embryos, which are important biotechnological tools in plant breeding. Recent studies have revealed the involvement of DNA methylation in regulating this process, but no information is available on the role of histone modifications in microspore embryogenesis. Histone modifications are major epigenetic marks controlling gene expression during plant development and in response to environment. Lysine methylation of histones, accomplished by histone lysine methyltransferases (HKMTs), can occur on different lysine residues, with histone H3K9 methylation being mainly associated with transcriptionally silenced regions. In contrast, histone H3 and H4 acetylation is carried out by histone acetyltransferases (HATs) and is associated with actively transcribed genes. In this work we analyze three different histone epigenetic marks: dimethylation of H3K9 (H3K9me2) and acetylation of H3 and H4 (H3Ac and H4Ac) during microspore embryogenesis in Brassica napus, by Western blot and immunofluorescence assays. The expression patterns of histone methyltransferase BnHKMT and histone acetyltransferase BnHAT genes have been also analyzed by qPCR. Our results revealed different spatial and temporal distribution patterns for methylated and acetylated histone variants during microspore embryogenesis and their similarity with the expression profiles of BnHKMT and BnHAT, respectively. The data presented suggest the participation of H3K9me2 and HKMT in embryo cell differentiation and heterochromatinization events, whereas H3Ac, H4Ac and HAT would be involved in transcriptional activation, totipotency and proliferation events during cell reprogramming and embryo development.