Inhibition of Histone H3K9 Methylation by BIX-01294 Promotes Stress-Induced Microspore Totipotency and Enhances Embryogenesis Initiation

Abstract

Microspore embryogenesis is a process of cell reprogramming, totipotency acquisition and embryogenesis initiation, induced in vitro by stress treatments and widely used in plant breeding for rapid production of doubled-haploids, but its regulating mechanisms are still largely unknown. Increasing evidence has revealed epigenetic reprogramming during microspore embryogenesis, through DNA methylation, but less is known about the involvement of histone modifications. In this study, we have analyzed the dynamics and possible role of histone H3K9 methylation, a major repressive modification, as well as the effects on microspore embryogenesis initiation of BIX-01294, an inhibitor of histone methylation, tested for the first time in plants, in Brassica napus and Hordeum vulgare. Results revealed that microspore reprogramming and initiation of embryogenesis involved a low level of H3K9 methylation. With the progression of embryogenesis, methylation of H3K9 increased, correlating with gene expression profiles of BnHKMT SUVR4-like and BnLSD1-like (writer and eraser enzymes of H3K9me2). At early stages, BIX-01294 promoted cell reprogramming, totipotency and embryogenesis induction, while diminishing bulk H3K9 methylation. DNA methylation was also reduced by short-term BIX-01294 treatment. By contrast, long BIX-01294 treatments hindered embryogenesis progression, indicating that H3K9 methylation is required for embryo differentiation. These findings open up new possibilities to enhance microspore embryogenesis efficiency in recalcitrant species through pharmacological modulation of histone methylation by using BIX-01294.

Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement

“…A recent report has shown that microspore reprogramming and the initiation of embryogenesis are associated with low levels of H3K9 methylation, while embryo differentiation progresses with increasing levels of this histone mark (Berenguer et al, 2017;Rodriguez-Sanz et al, 2014b), as demonstrated by biochemical and H3K9me2 immunofluorescence assays (Fig. 4).…”

“…Interestingly, the temporal profile of bulk H3K9 methylation during microspore embryogenesis correlates with the gene expression patterns of BnHKMT SUVR4-like and BnLSD1-like genes, 'writer' and 'eraser' enzymes of H3K9me2 (Berenguer et al, 2017), providing additional evidence of the developmental regulation of this histone modification during the process of microspore embryogenesis. Moreover, treatment of microspore cultures with the small molecule BIX-01294, a known inhibitor of H3K9 methylation in mammalian cells (Kubicek et al, 2007;Tachibana et al, 2002), promotes microspore reprogramming and embryogenesis initiation, in rapeseed and barley (Berenguer et al, 2017). Conversely, BIX-01294 treatment at advanced stages impairs embryo formation, showing that embryogenesis initiation involves low H3K9 methylation but embryo differentiation requires high levels of this repressive mark.…”

“…Here, we will review recent findings regarding the determinant factors that underlie stress-induced microspore embryogenesis induction and progression, with special emphasis on the role of autophagy, cell death, endogenous auxin, chromatin modifications, and cell wall remodelling. Information gained over the last few years has also shown that the modulation of these determinant factors by small bioactive molecules affects the process yield, emerging as a promising strategy to improve microspore embryogenesis efficiency (Barany et al, 2018;Berenguer et al, 2017;Solis et al, 2015); Pérez-Pérez et al, 2018a). This has opened up a completely new intervention pathway to increase the efficiency of the in vitro production of embryos and doubled-haploid plants in crop and forest species, especially recalcitrant ones, with the aim being their application in breeding programs.…”

Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley

“…3-Methyladenine (3-MA) and MnCl 2 were directly dissolved in the culture medium. Short treatments were carried out from culture initiation during 4 d. Mean percentages of ‘proembryos’ were quantified from random samples of three independent experiments, as previously described ( Berenguer et al , 2017 ).…”

“…RT-qPCR analyses were performed using the SsoAdvanced™ Universal SYBR ® Green Supermix on the iQ™5 Real-Time PCR Detection Sytem (Biorad). The oligonucleotides used are described in Supplementary Table S2 , and qPCR conditions were as previously described ( Berenguer et al , 2017 ). All qPCRs were run in duplicate, and the Cyclophilin gene was used as the internal reference gene.…”