A Study Of The Function Of Epigenetic Modifications In The Heat-mediated Plant Programmed Cell Death

Among hotspots of foundmental research, mitosis and programmed cell death (PCD) are involved in many processes, including development, senescence, abiotic stress responses and pathogen infection. In animals, PCD is divided into apoptosis, autophage and necrosis. There are obvious cytological and physiological differences between these types of PCD. The molecular signalling pathways of apoptosis, autophage and necrosis are independent and closely related. In plants, the classification of PCD is not clear due to significant differences between molecular regulations in various PCD examples. Therefore, the researches on the molecular regulation of different PCD examples contribute a lot to the classification of plant PCD.Chromatin modification is an important part of epigenetics, regulating gene expressions via chromatin structure changes. Chromatin modification plays a key role in many biological processes, including iPS differentiation, germ cell formation, seed germination and plant response to stress. However, the research of chromatin modification in PCD is scanty so far. The function research of chromatin modification in the mitosis and heat-induced PCD processes contributes to the understanding of chromatin modification and molecular regulation in the mitosis and PCD processes. In this research, maize seedlings are used to investigate the molecular regulation, epigenetic regulation and rDNA regulation in the mitosis and heat-induced PCD processes. The following contents are included in the research:1. The chromatin regulation in heat-induced PCD in maize seedling leavesWe found that heat stress induced PCD in maize seedling leaves, which was characterized by chromatin DNA laddering and DNA strand breaks detected by a TdT-mediated dUTP nick end labeling (TUNEL) test. The activities of the reactive oxygen species (ROS)-related enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were progressively increased over time in the heat-treated seedlings. However, the concentration of H2O2 molecule kept relatively lower levels, while the concentration of superoxide anion (O2-) was accumulated, accompanied by the occurrence of higher ion leakage rates after heat treatment. The total acetylation levels of histones H3K9, H4K5 and H3 were significantly increased whereas the di-methylation level of histone H3K4 was unchanged and the di-methylation level of histone H3K9 was decreased in the seedling leaves exposed to heat stress compared with the control group, accompanied by the increased nucleolus size indicative of chromatin decondensation. To research on the relationship of histone modification and ROS, we treated maize seedlings with different inhibitors of epigenetic modifications. Two histone phosphorylation inhibitors H89 and AT13148, two DNA methylation inhibitors 5-AC and RG108, two HDAC inhibitors TSA and CUDC-101 were included in our experiments. The inhibitors of different epigenetic modifications caused histone H4K5ac accumulation and cell cycle arrest at preprophase. The process was accompanied by ROS accumulation and antioxidant thiourea could release the preprophase arrest. Furthermore, treatment of seedlings with high-concentration Trichostatin A (TSA), which always results in genomic histone hyperacetylation, caused an increase in the O2- level within the cells. Moreover, the expression of PCD-related genes was regulated by histone modifications on gene promoters and exons.2. The chromatin regulation of 45S rDNA in heat stressWe found that nucleolus broke into several parts in heat stress and the time extension made the phenomenon more obvious. To study the function of nucleolus in heat stress, we used fluorescence in situ hybridization (FISH) detecting the variation of 45S rDNA in heat stress. The results revealed that proportion of cells with decondensed 45S rDNA was significantly increased in the first day and sixth day of heat treatment. The difference variation of nucleolus and 45S rDNA in heat stress represented the different functions in heat stress. To research on the function of 45S rDNA in heat stress, we used quantitative real-time PCR assay detecting the transcription of 45S rDNA. On the first day, the expression of ETS and 18S regions was activated after heat treatment. On the second and third days, the expression of ETS and 18S regions was a little activated after heat treatment. After the fourth day, the expression of ETS region was significantly activated whereas the expression of 18S region was only a little higher in heat stress, indicating the inhibition of transcriptional elongation in heat stress. We used mass spectrum and chromatin immunoprecipitation (ChIP) assays to research on the variation of DNA methylation and histone modifications on the 45S rDNA promoters in heat stress. The DNA methylation result suggested that DNA methylation on 45 S rDNA promoter was not changed after heat treatment. Furthermore, ChIP results revealed that histone modifications played a key role in the transcription initiation of 45S rDNA, but not the transcription elongation of 45S rDNA.