Effects Of DNA Methylation Level On The Nitrate Uptake Of Roots In Chrysanthemum

Chrysanthemum is one of the four major cut flowers in the world.Its cultivated areas and yields are in the front rank of flowers.The reasonable application of nitrogen fertilizer is very important for the growth and ornamental quality of chrysanthemum.Nitrate is the main form of nitrogen uptake in the roots of chrysanthemum.Some studies have found that there is a certain relationship between nitrogen uptake and methylation of plant roots.Therefore,we speculate that the absorption of nitrate in roots of chrysanthemum is related to DNA methylation.Although the research of the methylation of chrysanthemum has been reported at present,but there has been no report on the relationship between methylation level of chrysanthemum genome and NO₃^-absorption in roots.Therefore,it has a great significance to study the relationship between DNA methylation and NO₃^-absorption of chrysanthemum roots system for improving the use of NO₃^-and improving the yield and ornamental quality of chrysanthemum.In this experiment,the rooted cuttings of chrysanthemum'Jinba'was used as test materials.The chrysanthemum roots were treated with methylation inhibitor?5-azaC?and methyl donor?SAM?under the condition of nitorgen deficiency and suitable nitrogen hydroponics.After 0,5,10 and 15 days of treatment,the changes of DNA methylation level,root system configuration parameters,the contents of NO₃^-and the relative expression levels of NO₃^-transporter gene CmNRT1.1,CmNRT2.1 and CmNAR2.1 were measured.At the same time,NO₃^-contents in leaves,chlorophyll contents and net photosynthetic rate were detected.The relationship between DNA methylation level of roots and NO₃^-absorption in roots were further studied,which will provide theoretical basisfor improving the efficient use of nitrogen in chrysanthemums.The main results are as follows:1.After the treatment with 5-azaC and SAM,the methylation level of genomic DNA in the root system of chrysanthemum was observed.The results showed that,the number of methylated bands in the genomic DNA of the chrysanthemum root system was 119-202,with a ratio of 31.27%-49.55%.On the 15th day of the treatment,under nitrate deficient conditions,the DNA methylation rate of chrysanthemum under 5-azaC treatment was decreased by 8.06%,while the SAM treatment was increased by 8.65%compared with the control.under nitrate conditions,the DNA methylation rate of chrysanthemum under 5-azaC treatment was decreased by 6.27%,while the SAM treatment was increased by 6.94%compared with the control.2.After the treatment with 5-azaC and SAM,the root morphology and structure of chrysanthemum were observed.The results showed that,the morphological structure of the root of chrysanthemum was changed.On the 15th day,compared with the control,the average diameter,total surface area and total volume of chrysanthemum root under 5-azaC treatment increased significantly by 39.31%,12.60%and 27.12%.However,the average diameter,surface area and total volume under SAM treatment were significantly reduced by 17.93%,24.44%and 32.20%,respectively.3.After the treatment with 5-azaC and SAM,the NO₃^-contents in the roots of chrysanthemum were measured.The results showed that,under the condition of nitrate deficiency,the content of NO₃^-in the roots of chrysanthemum treated with 5-azaC and SAM was not significantly different from that of the control,but under nitrate conditions,on the 15th day of treatment,compared with the control,the contents of NO₃^-in roots treated with 5-azaC increased by 19.44%,while the contents of NO₃^-in the roots treated with SAM decreased by 13.89%.This indicates that the decrease of DNA methylation level in roots of chrysanthemum promotes the uptake of NO₃^-in roots,while the increase of DNA methylation level in chrysanthemum root inhibits the absorption of NO₃^-from roots.4.After the treatment with 5-azaC and SAM,the relative expression levels of the NO₃^-transporter genes CmNRT1.1,CmNRT2.1,and CmNAR2.1 in the roots of chrysanthemum were measured.The results showed that,under nitrate conditions,relative expression levels of CmNRT1.1,CmNRT2.1,and CmNAR2.1 genes in the roots were increased,and were higher than those in the absence of nitrate.Under nitrate conditions,the relative expression of these three genes peaked at the 15th day of treatment with 5-azaC.The results showed that,compared with the control,the relative expression levels of NO₃^-transporter genes CmNRT1.1,CmNRT2.1 and CmNAR2.1 in roots of chrysanthemum under 5-azaC treatment were increased by 14.29%,15.58%and 36.20%,but the relative expression levels of the genes were reduced by 17.86%,12.99%,and5.17%,respectively.5.After treatment with 5-azaC and SAM,the NO₃^-contents,chlorophyll contents,and net photosynthetic rates of the chrysanthemum leaves were measured.The results showed that,on the 15th day,under nitrate conditions,compared with the control,the contents of NO₃^-,chlorophyll contents and net photosynthetic rates of the leaves of chrysanthemum treated by 5-azaC increased by 6.50%,9.07%and 10.07%,but the contents of the three parameters treated by SAM decreased by 8.70%,3.69%and 5.38%,respectively.The results showed that 5-azaC treatment increased the NO₃^-contents,chlorophyll contents and net photosynthetic rates of chrysanthemum leaves.The above results suggested that the reduction of the methylation level of chrysanthemum genomic DNA leads to the up-regulation of the relative expression levels of the NO₃^-transporter genes CmNRT1.1,CmNRT2.1,and CmNAR2.1,thereby,promoting the absorption and utilization of NO₃^-from the roots of chrysanthemum.