Dynamic DNA Cytosine Methylation And Their Responses To ?-Aminobutyric Acid Signals In Plant Cells

DNA cytosine methylation(5mC) plays an important role in plant development. A variety of environmental factors such as cellular endogenous and exogenous stress factors could trigger the dynamic changes of DNA methylation. The level of genomic DNA methylation will be decreased or increased when plant suffered from under abiotic stress. In order to adapt to the changes of environment, more genes are inducibly expressed. It has found that the accumulation of ?-aminobutyric acid(GABA) when the plant is subjected to abiotic stress. However, few data show the links of GABA signal and DNA methylation.In order to explore the role of ?-aminobutyric acid(GABA) on cytosine methylation(5mC) in genomic DNA and its possible regulatory mechanisms, we use Arabidopsis roots and root callus as the research material to analyze 5mC content and its response mechanism to GABA signal in the process of stem cell differentiation and de-differentiation; we also compared the content changes of 5mC and its response to GABA signal in the polarized growth of tobacco pollen tubes and the roots of Arabidopsis. And also we analyzed the influence of GABA treatment on the expression of PLETHORA1(PLT1) and WUSCHEL-RELATED HOMEOBOX 5(WOX5), which are the key genes to modulate root development in Arabidopsis. The results showed that:1. GABA(1.0 mmol/L) treatment significantly decreased 5mC content in Arabidopsis root, and concurrently increased 5-hydroxy cytosine(5hmC) content; However, GABA has increased the content of 5mC in the callus, and reduced the oxidation production of 5mC demethylation(via 5hmC);2. GABA(1.0 mmol/L) treatment increased the level of methylation in quiescent center(QC) cells and surrounding stem cells(SSCs) which are derived from callus;3. When the medium was supplemented with GABA(1.0 mmol/L), the level of genomic DNA 5hmC was reduced in root and QC callus cells, but increased in root and SSCs callus;4. During the process of Arabidopsis root growth, addition of GABA(1.0 mmol/L) in the medium increased the percentage of 5hmC in the genomic DNA methylation(P<0.05); however,the content of 5hmC was decreased in the root callus when GABA(1.0 mmol/L) addition(P<0.05); in QC cells, GABA treatment decreased the percentage of 5hmC, reaching highly significant differences(P<0.01); In SSCs callus, GABA treatment caused 5hmC decline(P<0.05);5. GABA(1.0 mmol/L) could significantly promote the growth of tobacco pollen tubes and Arabidopsis roots(P<0.05);6. GABA(1.0 mmol/L) treatment significantly decreased the content of 5mC in the tobacco pollen tubes and Arabidopsis roots genomic DNA, and increased the content of the 5hmC;7. GABA(1.0 mmol/L) treatment upregulated the expression of PLT1 and WOX5 in 7-d-old roots of Arabidopsis(P<0.05). This indicated that GABA could be a signaling molecule involving in the regulation of root development by modulation both the expression of PLT1 and WOX5.5hmC is an important intermediate of 5mC active demethylation via its oxidative metabolic pathway. Our results demonstrated for the first time that GABA, an important exogenous signal, could regulate the dynamic changes of DNA methylation. These dynamic changes have different regulatory patterns in Arabidopsis growing roots and its callus, this maybe linked with cell division, differentiation and the maintenance of stem cell fate, and may also related to the growth regulation of tobacco pollen tubes and Arabidopsis roots, which promoted by GABA treatment.