Epigenetic Regulation Of Maize Cold Response

Due to the sessile developmental style, plants have developed unique mechanisms adopt the external stress. During acclimation, plants have many efficient short-term strategies to manipulate the gene expression and resist environmental stress. It is important to note that epigenetic mechanisms play a centrol role in the acclimation process. This flexible and reversible mechanism modifies the expression of stress-responsive gene and is an advantage in evolutionary terms.At the past decades, the regulation net work of plant acclimation gained a lot of interests, and was well studied. It has been demonstrated that plants resist cold stress through the ABA-independent pathway, which is also named the "ICE-DREB-COR" cascade (ICE:Inducer of CBF Expression; CBF/DREB:C-repeat Binding Factor/Dehydration Response Elements Binding protein; COR:Cold-regulated) Recently, the plant epigenetics involved in plant acclimation has become an important basic research topic, and became a possible new source of beneficial traits for plant breeding. It controls a vast array of biological functions not through gene mutations, but rather through reversible and heritable epigenetic mechanism ranging from hisone modifications (acetylation, methylation, phosphorylation, ubiqutination), DNA methylation (in plant, there are three motif:CpG, CpHpG and CpHpH, H=A, C or T) to miRNA pathway (relative to repeat sequence). As the carrier of genetic information, chromatin that is classified into euchromatin and heterochromatin according to the different structure plays a central role in nearly every aspect of the epigenetic regulation. In this research, we used maize as materials and investigated the epigenetic regulation of plant cold acclimation occurred in euchromatin and heterochromatin. The results are as follows:1. The global epigenetic change occurred during cold acclimation.The results of Western blot indicated that cold treatment reduced the level of H3K9ac, H4K5ac H44ac and H3K9me1. However the histone H3K9me2level was gradually increased and the histone H3S10p level kept steady. Immunostaining showed that major histone acetylation distributed in euchromatin. Although histone methylation and phosphorylation distributed throught the nuclei, they were rich in heterochromatin region. In addition, cold treatment induced a genomewide DNA demethylation and chromatin condensation.ChIP-Seq revealed that H3K9ac enriched in gene rich region and minor H3K9ac distributed in repeat sequence (include heterochromatin). Although cold treatment reduced the H3K9ac level both in gene rich region and some repeat sequence (Gypsy, Copia, Centr etc.), it was notable that a significant up-regulation of H3K9ac was occurred in Knob180-bp and TR-1repeat.2. Epigenetic regulation on euchromatin during cold acclimation.①HDACs positively regulate the expression of the cold-induced Zm DREB1gene. Q-PCR results indicated that HDACs and cold-responsive genes(ZmICEl, ZmDREB1and ZmCOR413) were up-regulated during the cold acclimation. But the expression of ZmDREB1and ZmCOR413were repressed in cold-TSA treated seedlings which is HDAC loss-fuction, while ZmICEl was not affected. Futher result demonstrated that TSA directly inhibited the expression of ZmDREB1rather than ZmCOR413.②HDACs regulate ZmDREBl through reversible epigenetic change that occurs in the special region of promoter. The results indicated that cold treatment increased histone acetylation (H3K9ac, H4K5ac and H44ac) in ICE1binding site which accompanied by down-regulation of histone H3K9me2and DNA methylation. In contrast, PRBS (Putative Repressor Binding Site) kept hypoacetylation (H3K9ac, H4K5ac and H44ac), hypermethylation (H3K9me2) and DNA hypomethylation during cold acclimation. In HDAC loss-function seedlings, cold treatment did not affect the epigenetic modification pattern in ICE1binding site, but changed the acetylation (up-regulated in H3K9ac, H4K5ac and H44ac) and methylation (down-regulated in H3K9me2and CpG) levels.In addition, CHART-PCR revealed that ICE1binding site maintained an "Open" state both in cold and cold-TSA treated seedlings. But TSA induced a chromatin remodeling ("Close" to "Open") in PRBS during cold acclimation. We speculated that HDACs maintained the hypoacetylation in PRBS and contributed to the chromatin condensation and inaccessibility of transcription repressor.③Pull down and Co-IP assay indicated that cold up-regulated HDACs (HDAC101, HDAC102, HDAC103, HDAC106, HDAC108, HDAC110) presented different interaction with HP1. In vitro, although three HDACs (HDAC106, HDAC108, HDAC110) can Co-IP with HP1, none can be detected in Pull down assay. This result indicated that HDACs did not combine to HP1directly; instead they maight cooperated with HP1in a large protein complex-"chromatin regulation machine".3. Cold-induced transcriptional activation of heterochromatin.Detailed analysis of two knob-associated repetitive sequences,180-bp and TR-1, demonstrated that cold activated their transcription and this cold-induced transcriptional activation of repetitive sequences is selective, dynamic, and associated with an increase in H3K9ac and a decrease in DNA methylation and H3K9me2. Furthermore, knob sequence expression is accompanied by localized chromatin remodeling and silencing is recovered upon prolonged treatment. These results suggest that cold-mediated transcription of heterochromatic knob sequences accompanied with epigenetic regulation might play an important role in the adaptation of plants to cold stimuli.Southern blot assay of cold-treated seedling revealed that Knob repeats copy number and forms did not significant change during the cold acclimation. Real time PCR results indicated that Knob180-bp repeat copy number maintain at6×105and Knob TR-1repeat copy number keep at3×105approximately. So far, we speculate that Knob repeat did not rearranged during cold treatment, and this cold-induced Konb repeat transcriptional activation may play a critical role through other RNA dependent epigenetic pathway.