The Functional Analysis Of Promoter DNA Methylation In Regulating Shoot Regeneration Of Arabidopsis Thaliana

In vitro organogenesis refers to the regeneration processes of shoots, roots or other organs from plant tissue or callus, its theoretical basis is the plant celltotipotent. The differentiated cells can be reversed to totipotency status and then differentiate into new tissue, organ and whole plants. Shoot regeneration is one important way of in vitro organogenesis.DNA methylation is an important style of epigenetic modification, it does not change the primary structure of DNA but to regulate gene's expression or maintain the stability of the genome in a tissue specific manner. Previous researches mainly focused on the relationship between the genomic DNA methylation and plant development. Little is known about DNA methylation in affecting specific gene expression and further influencing the plant growth and development and shoot regeneration in vitro.Here, we set out to explore the machanism of DNA methylation in affecting shoot regeneration and to make better understanding of the molecular machanism involved in the cell totipotent in Arabidopsis from in vitro culture system. The research mainly includes:1) Selection of the candidate DNA methylation-dependent shoot regeneration related genes (including:the gene expression profiles of RC and NRC; selection of the shoot regeneration related candidate genes; DNA methylation status analysis of the candidate genes; identification of the relationship among the target genes'DNA methylation status, expression and histone modifications);2) The function of DNA methylation in shoot regeneration and its molecular machanism.(including:the shoot regenerability analysis of the target genes' loss of function mutants; demethylation and up-regulated of SRF in affecting the shoot regeneration; identification of SRF target genes that involved in the shoot regeneration; the binding analysis of SRF with the promoters of its target genes). This work will lay foundation information in understanding the molecular manchanism of DNA methylation in affecting shoot regeneration in vitro.In addition, a set of differentially expressed transcription factors were identified by comparing the soybean Affymetrix microarray profiles generated from salinity-stressed and non-stressed plants. Their expression, DNA methylation level, histone modifications were analyzed in the non-stressed and slinity stressed seedlings. The dynamics and reversibility of both DNA and histone modification during the stress episode in regulating their expressions was also investigated.The main processes and results of this research were as follows:1. Role of the promoter DNA methylation in shoot regeneration in Arabidopsis1.1Identification of the DNA methylation-dependent shoot regeneretion related genes1.1.1The screening of candidate genes involved in shoot regenerationThe embryonic calli (RC), which were yellow-green in colour, contained embryonic cell mass, somatic embryos and regenerated shoots. The non-embryonic calli (NRC), which were pale green and fragile, was characterized by loosely packed, irregularly shaped cells. NRC completely lost the shoot regenerat ability.To identify candidate genes involved in shoot regeneration:1)1,432up-regulated and1,211down-regulated genes in RC were selected using gene expression profiles..2) The results of microarray-based selection were validated by qRT-PCR.3) By functional annotation,238genes were selected as candidate genes that involved in the shoot regeneration.1.1.2Identification of the candidate promoter methylated genesThe content and distribution of cytosine in the promoters of the238genes identified above were downloaded from www.urogene.org/methprimer/.28of them presented high cytosine content or CG islands in their promoter region, suggesting that they might be regulated by DNA methylation. Their expressions in DNA methyltransferase mutants as met1, cmt3, drm2, ddm1and wild type callus were analyzes by qRT-PCR.20of them were more highly expressed in one or two met1, cmt3, drm2and ddml calli than in the wild type, suggesting that their transcription might be regulated by DNA methylation.1.1.3Methylation status of the candidate promoters in RC and NRC1) Promoter DNA methylation status of the20candidate genes were examined by BSP (Bisulfite sequencing PCR). The promoters of6genes(SRF, MAX3, TFL1, ACC1, At2g05520and At5g55450) showed different methylation level between RC and NRC. Such as:SRF was hypermethylated in RC, while demethylated in NRC; other promoters showed lower mythylation level in RC than in NRC.2) The methylation patterns of above genes occurred in CG, CNG and CHH context in RC and/or NRC. In the SRF promoter, almost all the methylation was at CG dinucleotides other than at CNG and CHH sites. However, the methylation in the promoters of the other candidate genes occurred at not only CG site but also CNG and CHH sites.1.1.4The relationship between DNA methylation and expressionFor SRF, ACC1, MAX3, TFL1and At2g055520, the transcription level and methylation level of their promoters showed a negative correlation. In RC, SRF promoter was hypermethylated and its expression was greatly down-regulated, and in NRC, the ACC1, MAX3, TFL1and At2g05520promoters were all methylated, and the genes were down-regulated.1.1.5Expression and methylation status of the candidate genes in methyltransferase mutantsExpression and methylation status of the candidate genes were analysed by qRT-PCR and BSP. It was found that in comparison with Col-0, SRF and TFL1were both demethylated and up-regulated in met1and ddml mutants, At2g05520was demethylated and up-regulated in cmt3, ACC1, MAX3and At5g55450showed no change in methylation level, while ACC1, MAX3were up-regulated in met1and cmt3. At5g55450was up-regulated in drm2. This suggested that the transcription level of ACC1and TFL1were regulated by MET1and DDM1, At2g055520were regulated by CMT3, however, the transcription level of ACC1, MAX3. 1.1.6Histone modifications of the target genes in RC and NRC.High level of H3K9methylation and low level of H3K9acetylation were confirmed to be present in the the SRF promoter in RC, on the contrary, high level of H3K9methylation and low level of H3K9acetylation happened in the MAX3, TFL1and ACC1promoters in NRC. This suggested that in addition to DNA methylation, some H3K9me2and H3K9ac also existed within the target genes'promoters.1.2Molecular mechanism of promoter DNA methylation in regulating the shoot regeneration1.2.1Shoot regenerability in the mutants of candidate genesThe regenerability of the knock-out mutants accl-1, max3-9, tfl1-1and At2g05520and the two gain-of-function demethylated SRF mutants srf-1and srf-2was analyzed. The result indicated that compared with the Col-0, shoot regenerability of srf-1and srf-2was much lower, while the other knock-out mutants showed no change. This suggested that demethylation of SRF promoter repressed the shoot regenerability.1.2.2The possible mechanism of SRF in regulating the shoot regeneration1.2.2.1Identification of SRF target genes1) sqRT-PCR analysis indicated that compared to Col-0, the expression of WUS, WOX2, WOX8, WOX9and PIN1were all markedly down-regulated in the two SRF mutants.2) GUS staining assay showed that the expression of WOX9and WUS was repressed in the srf-2×WOX9::GUS, srf-2×WUS::GUS hybrid lines, indicating that demethylation and ectopic expression of SRF might repress the shoot regenerability through down-regulated WOX9and WUS in srf-2.1.2.2.2Identification of the direct target genes of SRF1) ChIP-sqPCR analysis indicated that SRF could directly bind to the WOX9promoter other than the promoters of WOX8, WOX2, WUS and PIN1, suggesting that SRF might repress WOX9's transcription by directly binding to its promoter.2) ChIP-sequencing results indicated that SRF might bind to20genes throughout the genome of Arabidopsis. 1.2.2.3The shoot regenerability was repressed in WOX9loss-of-function mutantsThe shoot regenerability in wox9-1and wox9-2of the WOX9loss-of-function mutants, was found to be much lower than in Col-0when cultured in the (LSIM).1.2.2.4Shoot regenerability recovery of NRC1)After transfering RNAi-SRF construct into the NRC, the cultures were transferred onto the shoot inducion medium. Some shoot-like structures were observed in the loosely packed culture surface. This suggested that the shoot regenerability of NRC was partly recovered.2) RT-PCR result indicated that compared with the non-transgenic NRC, the expression of WOX9, WOX8and WUS were all up-regulated in transgenic NRC, suggesting that the up-regulating of them might play an important role in promotering the shoot regenerability in the trangenic NRC.2. The dynamic changes of DNA methylation and histone modifications in salinity stress induced transcription factors (TFs) under salinity stress in soybean2.1Identification of salinity stress responsive TFs in soybean1)49(15GmMYBs,9GmNACs,16GmAP2/DREBs and9Gmb-ZIPs) up-regulated TFs genes were identified by comparing the salinity-stressed and non-stressed soybean Affymetrix microarray profiles, suggesting that they were salinity induced genes.2) The expression of the above TFs was assayed by sqRT-PCR in both mock and salinity-stressed soybean seedlings. It was found that14of the GmMYBs,8of the GmNACs,15of the GmAP2/DREBs and8of the Gmb-ZIPs were confirmed to be markedly induced by salinity stress.2.2Identification of the DNA methylated TFsThe expression of the45salinity-induced TFs was then analyzed in the mock and5-ADC treated seedlings. As a result, ten of the them showed higher levels of expression in treated than in mock-treated seedlings, suggeating that their expression might be regulated by DNA methylation. 2.3Methylation status of the candidate genes under salinity stress soybean1) Bisulfite sequencing result indicated that the Glyma11g02400, Glyma08g41450, Glyma16g27950and Glyma20g30840promoters all appeared to be dynamic methylated by the imposition of salinity stress.2) The methylation status of the target genes were also analyzed by Southern-blot under salinity stress conditions and the outcomes were in general consistent with the bisulphite sequence data.3) Gene expression and DNA methylation status analysis indicated that when the seedlings were exposed to salinity stress, a markedly nagitive correlation was observed between Glma11g02400, Glma16g27950, Glma20g30840expression and DNA methylation, while this did not extend to Glma08g41450.2.4DNA Methylation status of the target genes under5-ADC1) The DNA methylation status of the target genes were also analyzed in5-ADC treated soybean seedlings. As a result, all four TFs were hypermethylated in non-treated seedlings; after a48h exposure to5-ADC, it was clear that a substantial level of demethylation had occurred, suggesting that they were demethylated under5-ADC treatment.2) The methylation status of the target genes were also analyzed by Southern-blot under5-ADC treatment and the outcomes were in general consistent with the bisulphite sequence data.2.5Histone modifications of candidate genes'promoters under salinity stressChIP results indicated that significant differences of H3K9me2, H3K9ac and H3K4me3modification in the promoter of candidate genes between mock treatment and1h,3h,6h,12h and24h's NaCl treatment. During the NaCl treatment, the H3K9me2modification level was dramatically decreased in the promoters of Glyma11g02400, Glyma20g30840and Glyma08g41450. The H3K9ac was increased in the promoters of Glyma20g30840and Glyma08g41450after6h's NaCl treatment. The H3K4me3was increased in the promoters of Glyma11g02400, Glyma20g30840 and Glyma08g41450after1h,3h and6h's NaCl treatment, respectively.