Histone Arginine Methylation Modulates De Novo Shoot Regeneration In Arabidopsis Thaliana

Mature plant tissues or organs can regenerate shoots through dedifferentiation anddifferentiation, which is known as de novo shoot regeneration. This capability of plants notonly makes asexual propagation and genetic transformation become possible, but alsoprovides us a useful system to study mechanism of plant organogenesis. Plant hormones,especially cytokinin and auxin, play a pivotal role in the process of shoot regeneration.Therefore, there is a great value for both research and application to study the mechanism ofhow plant hormone functions during de novo shoot regeneration.Epigenetic, mainly refers to the phenomenon that gene expression has a heritablechange but without any difference in nucleotide sequence. Epigenetic methods exist widely inplant development processes, mainly including DNA methylation, histone modifications andchromatin remodeling. Previous studies have shown that epigenetic factors, such as DNAmethylation and histone lysine modifications could regulate de novo shoot regeneration bymodulating the expression of WUS and ARF3, which were essential for shoot apical meristem.However, the mechanism of the shoot regeneration regulated by histone arginine methylationis still poorly understood.Here, we tried to determine how arginine methyltransferases (PRMT) regulates in vitroshoot regeneration in Arabidopsis. The main results are as follows:First, the phenotypes of in vitro shoot regeneration in prmt mutants were analyzed, inwhich we found that PRMTs, especially PRMT5, had an important effect on shootregeneration in Arabidopsis.Second, we analyzed the expression of genes that functioned in the shoot apicalmeristem in the mutant prmt5during shoot regeneration, and found the cytokinin oxidasegene CKX3was deregulated by PRMT5.Third, the expression levels of CKX3in different stages of de novo shoot regenerationwere analyzed by qRT-PCR. And we also observed the expression of CKX3response to different concentrations of exogenous cytokinin. All the results indicted that PRMT5inhibitedthe sensitivity of CKX3response to cytokinin.Fourth, the chromatin immunoprecipitation (ChIP) assay was designed and proved thatPRMT5could regulate the expression of CKX3negatively through H4R3sme2modification.Fifth, we analyzed the expression pattern of CKX3with its promoter driving GUSreporter and in situ hybridization experiment, and found that PRMT5had an important effecton the regional expression of CKX3in callus.Finally, through phenotype analysis in CKX3function deletion and overexpressionplants, we showed that CKX3played an important role in the process of in vitro shootregeneration.Taken together, our study revealed the molecular mechanism of histone argininemethylation modulating de novo shoot regeneration in Arabidopsis. Histone H4R3sme2modifications catalyzed by PRMT5, regulated the regional expression of CKX3, whichaffected the callus response to cytokinin signal, and finally decided the formation andmaintenance of SAM during de novo shoot regeneration.