Histone Arginine Methylation Modulates de novo Shoot Regeneration in Arabidopsis thaliana

Author :HanHuaNan
Tutor:ZhangXianSheng
School :Shandong Agricultural University
CLC :Q946
TYPE :Masters thesis
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Year:2013
Abstract:
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.