DNA Methylation And Methylase Genes GmMET1 And GmROS1 Function Analysis Under Phosphorus Stress In Soybean

Phosphorus is one of the three essential nutrients for plant growth and development,which is limited and non-renewable.Soybean（Glycine max L.）is not only an important grain and oil crop but also a phosphorus loving crop.DNA methylation plays an important role in maintaining genomic stability,regulating gene expression,and responding to biological or abiotic stresses.So far,there is no studies report on DNA methylation in response to soybean phosphorus stress.In the present study,soybean material CP016 was selected and the seedlings were treated with phosphorus at different concentrations（0μmol/L,10μmol/L,100μmol/L,500μmol/L,and 1000μmol/L）by hydroponics.The botanical characters,reactive oxygen species（ROS）related（H₂O₂ and MDA） contents,and the activities of antioxidant enzyme（SOD,POD,and CAT）were detected,the DNA methylation was analyzed by methylation sensitive amplification polymorphism（MSAP）,qRT-PCR was used to study the expression of relevant genes,The function of Gm MET1 and Gm ROS1 genes was analyzed by virus induced gene silencing（VIGS）.The main results are as follows:1.With the increase of phosphorus concentration,the plant height,fresh weight,root length and root surface area of soybean seedlings increased first and then decreased.Both the phosphorus-free（0μmol/L） and high phosphorus（1000μmol/L） stresses significantly inhibited soybean growth,while low phosphorus stress（100μmol/L）promoted the growth of above ground and the extremely low phosphorus stress（10μmol/L） promoted root growth.2.The content of H₂O₂ in the roots of soybean seedlings was significantly increased under phosphorus-free and high phosphorus stress,and MDA content in roots of soybean seedlings was significantly increased by high phosphorus stress with the gradual increase of phosphorus concentration,the POD and CAT activities were decreased first and then increased in the root of soybean seedlings,while the SOD activities,starch,and sucrose contents increased first and then decreased.3.The results of MSAP analysis showed that the DNA methylation ratio and fully methylated ratio of soybean seedlings increased gradually with the increase of phosphorus concentration,while the hemi-methylated ratio decreased gradually.Specifically,the DNA methylation ratio of soybean seedling roots treated with phosphorus-free,normal phosphorus and high phosphorus supply were 43.04%（hemi-methylated ratiod was 17.72%,fully methylated ratio was 25.32%,the same as below）,48.52%（14.35%,34.18%） and 51.05%（13.08%,37.97%）,respectively.4.qRT-PCR analysis showed that under phosphorus-free stress,the expression level of POD related genes were significantly increased,while SOD activity related genes significantly decreased in the roots of soybean seedlings.The expression level of starch synthesis related genes were significantly increased,while sucrose synthesis related genes were significantly decreased.The expression level of methylase gene Gm DRM2 was significantly increased,while the methylase gene GmMET1 and demethylase gene GmROS1 were significantly decreased.5.VIGS analysis showed that the gene silencing success rate of needle-assisted vacuum osmotic method was the highest,followed by needle infiltration method,and root soaking assisted vacuum osmotic method was the lowest.Compared with empty vector pTRV2 plants,after Gm ROS1 gene silencing,the root growth of soybean under phosphorus free stress was significantly inhibited,the SOD activity was increased significantly,while POD and CAT activity were decreased significantly.Combined with these analyses results,this study showed that phosphorus-free（0 mol/L）and high phosphorus stress（1000 mol/L）significantly inhibited the growth of soybean seedlings,increased the ROS accumulation,disrupted antioxidant enzyme system,and significantly reduced the content of starch and sucrose.However,a certain degree of low phosphorus stress（100 mol/L）could promote the growth of soybean seedlings above ground,while extremely low phosphorus stress（10 mol/L） could promote the growth of seedling root.The level of DNA methylation in soybean seedling roots was decreased and increased by phosphorus deficiency and high phosphorus stress respectively,and the expression of physiological and biochemical related genes and methylase gene were also changed.The inhibition degree of soybean roots was increased under phosphorus stress when the demethylase GmROS1 gene was silenced.These results indicate that DNA methylation and GmROS1 gene plays an important role in response to phosphorus stress of soybean.