Research On Rapeseed Floral Transition Regulated By Histone Methylation And Its Distribution In Male Gametophyte Development

Histone methylation plays an important role in plant growth and development.Arabidopsis AtSDG8 is a histone lysine methyltransferase,and affects floral transition in plants by specifically methylating H3K36 at the FLC chromatin.Rapeseed is an important oil crop,and its floral transition and male gametophyte development are closely related to yield and quality.However,the regulation of histone methylation on the rapeseed floral transition and its distribution during meiosis remain unknown.In this study,by dint of the epigenetics,cytology,molecular biology techniques and so on,the allotetraploid Brassica napus(XY15)has been studied to clarify the regulation mechanism of histone methylation on the rapeseed floral transition and its distribution characteristics in male gametophyte development.The results are as follows.First,the regulation mechanism of BnaSDG8-mediated histone methylation on the rapeseed floral transition.1)Two BnaSDG8 genes,BnaSDG8.A and BnaSDG8.C,were cloned from XY15,and bioinformatics analysis revealed that BnaSDG8.A and BnaSDG8.C contained the same four functional domains as AtSDG8: CW domain,AWS domain,SET domain,and post-SET(C)domain.Spatiotemporal expression analysis showed that BnaSDG8.A and BnaSDG8.C were expressed at different developmental stages and in different organs with differences.2)Phylogenetic analysis showed that the SDG8 ortholog was evolutionarily conserved and each of these representative species had only one copy.Though no BnaSDG8 gene duplication or triplication event has occurred,a 39-amino-acid sequence(termed NNH domain)in the N terminal of BnaSDG8.C duplicated once,namely,two NNH domains existed in BnaSDG8.C.3)The functional complementary experiment showed that the transgenic plants harboring 35s::BnaSDG8.A and 35s::BnaSDG8.C flowered later than sdg8-2 mutants and at the same time as Col-0,and the significantly down-regulated transcript levels of BnaSDG8.A and BnaSDG8.C in the transgenic rapeseed with the RNA interference vector could cause earlier flowering,indicating that both BnaSDG8.A and BnaSDG8.C were able to perform the role of AtSDG8 in control of flowering.4)The ChIP analysis on the flowering regulatory genes BnaFLCs further revealed that compared with XY15 control plants,the transgenic rapeseed with RNAi vector had increased levels of H3K36me1 at all BnaFLC loci,except for BnaFLC3-2,which exhibited no changes in H3K36me1.No significant changes in H3K36me2 were detected at any BnaFLC gene,apart from BnaFLC1-1 and BnaFLC5-2,which had dramatically increased levels.Reduced levels of H3K36me3 were observed at all BnaFLC genes,with the exception of a noticeable increase at BnaFLC3-1,BnaFLC4 and BnaFLC5-2.In addition to BnaFLC1-2 and BnaFLC3-1 with an increased level of H3K4me3,and BnaFLC1-3 and BnaFLC5-1 with a slightly decreased level of H3K4me3,the levels of H3K4me3 at all BnaFLC loci were unchanged.Taken together,these data indicated that the histone lysine methyltransferases,encoded by BnaSDG8.A and BnaSDG8.C,were not involved in H3K36me3 deposition at all BnaFLC loci,but only affect the level of H3K36me3 on the BnaFLC2 chromatin,associated with its down-regulated transcript level to regulate the rapeseed floral transition.Second,the distribution of histone methylation during the development of pollen mother cells into male gametes in rape.1)Histochemical and cytological microscopic observation revealed that pollen cells at different stages of male gametophyte development,such as pollen mother cells,dyads,tetrads,mononuclear pollens,and trinuclear pollens,could be collected in flower buds ranging from 1.0mm to 2.5mm in length,establishing the corresponding relationship between bud size and developmental stage of pollen mother cell meosis.2)By the ways of immunostaining and confocal,the distribution of histone methylation during male gametophyte development was observed and the results showed that the accumulation positions of these three histone methylations,H3K4me2,H3K27me2 and H3K36me2,were highly coincident with the locations of chromosomes during pollen mother cell meosis,and they also distributed in the nucleus of the mononuclear pollens but only existed in the nucleus of vegetative cells of mature trinuclear pollens.3)The enrichment intensity of H3K4me2 and H3K36me2 in all stages of the rape male gametophyte development was significantly stronger than that of H3K27me2.