Epigenetic Regulation Of Maize Abiotic Stress Response

Plants, responding to unfavorable environmental conditions such as drought, high salinity, heat, cold and heavy metal pollution in the long process of natural selection, have evolved sophisticated sensing and responsive mechanisms to adapt to diverse environmental. A fast, flexible and efficient molecular mechanism is essential for plants'successful survive in the changed environmental conditions. In recent years, epigenetics was gradually significantly concerned. The changes of chromatin state and histone modifications regulate the activity of eukaryotic genes. The extensive and in-depth research of plant epigenetics promotes the improvement and development of molecular regulatory networks and explains the mechanisms of gene regulation better. In plant vernalization and flowering process, the level of histone modifications is closely related to gene activation or inhibition, but little information about changes of histone modifications and nucleosome structure under abiotic stress is known. In this paper, we use maize (Zea mays L.) as materials to systematically analyze the epigenetic regulation under abiotic stress, from two aspects, cell cycle and molecular signaling pathways. The results of this research are as follows:1. Epigenetic regulation of cell cycle progression in response to different types of abiotic stressWe used Flow Cytometry and Paraffin wax sections analysis to compare and detect the maize physiological state and cell cycle arrest under different types of abiotic stress. The results showed that the cell number, size and arrangement were affected by abiotic stress. Inhibition of root elongation is a main consequence of prolonged cell cycle duration, but different types of abiotic stress block cell cycle at different phases and cell cycle gene expression in response to abiotic stress is significantly different. Western blot and chromatin immunoprecipitation (ChIP) experiments further confirmed that abiotic stress-induced histone acetylation dynamic change is involved in the transcriptional regulation of cell cycle genes, resulting in cell cycle progression disturbance, thereby affecting the growth inhibition of maize seedling root. These results reveal that different histone N-terminal lysine sites acetylation is selective and targeted to specific gene and plays a key role in regulating the cell cycle genes.2. Epigenetic regulation of the ZmDREB2A gene under osmotic stress By Immunostaining and western blot, we detected the changes of global histone acetylation (H3K9ac and H4K5ac) level of maize seedlings under osmotic stress (mannitol treatment). The results showed that mannitol treatment significantly increased global H3K9and H4K5acetylation levels, exogenous ABA treatment significantly reduced the global H3K9and H4K5acetylation levels. Global H3K9and H4K5acetylation levels compared to the control group did not increase under exogenous ABA plus mannitol treatment. We used quantitative real-time PCR (qRT-PCR) to detect the expression of acetyltransferase and deacetylase in the different treatment groups. The results showed mannitol treatment increased the acetyltransferase and deacetylase expression, but the expression of two enzymes was decreased after the appliance of exogenous ABA. Meanwhile, exogenous ABA inhibited the expression of gene ZmDREB2A. We further use Chromatin Immunoprecipitation (ChIP) and CHART-PCR (Chromatin Accessibility by Real-Time PCR) to detect the histone acetylation and chromatin condensation state in the promoter region of ZmDREB2A. The results showed that the promoter-associated histone acetylation is involved in transcription regulation of the ZmDREB2A gene under osmotic stress.3. Epigenetic regulation of rDNA chromatin decondensation and HSF gene expression under heat stressUsing fluorescence in situ hybridization (FISH) and quantitative real-time PCR analysis, we detected45S rDNA chromatin structure and rRNA gene expression of maize seedlings leaves during the process of24h treatment and24h recovery from heat stress. The results showed that heat stress significantly induced45S rDNA chromatin decondensation, accompanied by the increased expression of rRNA genes. In the leaves of maize seedlings, the nucleus with45S rDNA decondensation is the highest proportion at5h of heat stress. During the recovery process,45S rDNA decondensation phenomenon is still very significant. Using western blot, we analyzed the dynamic alterations of global histone modifications (H3K9ac and H3K4me2) during the process of24h treatment and24h recovery from heat stress. The results showed that H3K9ac was involved in heat response and H3K4me2might function as an epigenetic mark of stress memory. ChIP assay indicated that histone modifications at the promoter region were relevant to HSF gene expression. These results suggest that histone acetylation (H3K9ac) and methylation (H3K4me2) have different response mechanism and memory mechanisms in leaves of maize seedling under heat stress, which control the45S rDNA decondensation state and the expression change of heat-response genes.