The Role Of DNA Demethylase ROS1 In Maize Kernel Development

Maize is important grain and fodder crop,which has the highest crop area and total yield in China.The development of maize kernel is associated with the yield of maize and DNA demethylation has important regulation in plant development,but it is not clear for regulation in maize kernel development.Gene imprinting expression is a phenomenon where the allele-specific expression of genes is dependent on parent-of-origin.It speculated that the differential DNA methylation modifications between parental alleles is a reason for gene imprinting expression.However,DNA demethylase mediated methylation change,but it is not clear which whether regulates gene imprinting expression.As for the problems,this study firstly identified maize DNA demethylase and obtained their mutants.Analyzing the DNA methylation and gene expression between wild type and mutants and investigating the role of DNA demethylase in regulating maize kernel gene expression(including imprinting expression)and kernel development.The main results are as follows:1、The Arabidopsis genes encoding DNA demethylase were used to blast against gene annotations from Zea mays and four DNA demethylase genes were identified,named Zm ROS1a-1d Expression analysis showed Zm ROS1 a and Zm ROS1 b are main copies,which are respectively expressed highly in 14 days after pollination(14DAP)embryo and endosperm.2、EMS mutants of Zm ROS1 a and Zm ROS1 b were obtained,and the double mutant(zmros1ab)was obtained by crossing these two single mutants.Phenotypic analysis showed it is not significant between zmros1 a or zmros1 b and wild-type in growth and kernel development,but zmros1 ab has reduced height and flowered later in growth and reduced weight and reduced fertility in kernel development.zmros1a/+ 、 zmros1b/+ and zmros1a/+;zmros1b/+ were self-crossed and showed the percentage of single and double mutant alleles are expected,suggesting both single and double mutant gametophyte can be transmitted to next generation by paternal or maternal ways,and mutation of the two genes does not affect gamete viability.3、Performing whole-genome bisulfite sequencing using 14 DAP embryo and endosperm in wild-type and zmros1 ab.A comparison between DNA methylation of endosperm and embryo showed hypomethylation in endosperm.Consistently,more hypomethylated regions were identified in endosperm compare to embryo.Furtherly,approximately 40%hypomethylated regions in endosperm were only identified in wild type but showed no reduction of DNA methylation in zmros1 ab endosperm.This suggests Zm ROS1 abmediated DNA demethylation regulates hypomethylation of the regions(named“Zm ROS1ab-sensitive DMRs”)in endosperm.The expression of genes targeted by Zm ROS1ab-sensitive DMRs was analyzed and found these genes were preferentially down-regulated expressed in zmros1 ab related to wild-type.Associated with analysis of tissues expression pattern,found these genes were preferentially expressed in endosperm.Consistently,we identified all endosperm-specific expressed genes and found these genes showed DNA hypermethylation and preferentially down-regulation in zmros1 ab related to wild-type.These results indicate Zm ROS1ab-mediated DNA demethylation regulates expression of endosperm genes.4、Identifying the binding sites of Zm O2 that is a major regulator of gene expression in endosperm in wild-type and zmros1 ab by using DAP-seq.The result showed most binding sites(73%)were identified in wild-type were not identified in zmros1 ab.Differential binding sites between wild type and zmros1 ab were identified by using Diff Bind and found DNA methylation of the differential binding sites was hypermethylated in zmros1 ab related wild type.The difference of DNA methylation between wild type and zmros1 ab was removed by PCR amplification and the differential binding of the sites targeted by Zm O2 was disappeared.Moreover,the differential binding sites were associated with 107 expressed genes,of which 16 are differentially expressed between WT and zmros1 ab.These results suggest Zm ROS1ab-mediated DNA demethylation can regulate expression of some genes possibly by affecting the binding strength of transcriptional factor to its targets.5、To explore the role of Zm ROS1 ab in regulating gene imprinting expression,using either the wild type or the zmros1 ab mutant(both in B73 background)reciprocally crossed with Mo17,identifying the imprinted genes in 14 DAP and analyzing the expression and DNA methylation of imprinted genes alleles between wild type and zmros1 ab.The result showed zmros1 ab as female was transmitted,some endosperm-specific expressed MEGs(maternally expressed genes)were no longer the imprinted genes because of maternal allele expression was repressed,some endosperm-constitutively expressed PEGs(paternally expressed genes)were no longer the imprinted genes because of maternal allele expression was activated.The DNA methylation detection of parental alleles showed hypermethylation of maternal alleles in zmros1 ab related to wild type as female crossed with Mo17.These results determined Zm ROS1ab-mediated DNA demethylation regulates DNA methylation of maternal alleles in endosperm-specific expressed MEGs and endosperm-constitutively expressed PEGs and affects the formation of imprinted genes.