Mechanism Of Pollen And Seed Development Regulated By AtMBD3 In Arabidopsis

DNA cytosine methylation is an ancient epigenetic modification in evolution that plays important roles in regulating epigenetics and gene expression in different development stages in animals and plants.Many protein complexes are involved in writing,reading and erasing DNA methylation.It is a dynamic process established and maintained by methylation and demethylation.The de novo DNA methylation in Arabidopsis is established by RNA-mediated Rd DM pathway.Three DNA methylation modifications are found in plants,including symmetrically methylated CG,CHG and asymmetric methylated CHH(H stands for A,T,C).The downstream recognition proteins are required for DNA methylation to play roles in readers.Two types of methylation binding domain(MBD)proteins in Arabidopsis have been found that can recognize and bind methylated DNA,namely SRA(SET and RING finger associated domain)and MBD domain.The MBD protein family in Arabidopsis has 13 members that can recruit chromatin remodeling complexes,histone deacetylase and histone methyltransferase to methylated sites,thereby regulating gene expression.It has been found that AtMBD5,AtMBD6 and AtMBD7 can bind symmetrically methylated CG.However,little is known about how other proteins bind sequences and their functions involved in epigenetic modifications.Both bioinformatics and gene expression were performed to screen specific proteins to obtain mutants from all those AtMBD members that lacked research.After obtaining 5mutant lines,many abiotic stress were adopted to identify their phenotypes.AtMBD3 was chosen as the main study object to explore its effect on pollen and embryo development by using Atmbd3 mutants.The expression of those genes involved in pollen and embryo development was also carried out.The possible mechanism of AtMBD3 regulating pollen and embryo development was explored by screening interaction protein and binding methylated DNA sequences.The main results were listed as follows:1.Gene expression pattern of MBD proteins.Higher similarity was found among MBD sequences of 13 AtMBD protein members,while alignment of the residual sequences had lower similarity.When the 13 AtMBD protein members were expressed in nine tissues,including seedling,root,rhizome,rosette,cauline leaf,flower,silique,pollen and seed,AtMBD1,AtMBD2,AtMBD10 and AtMBD13 were found to highly expressed in seed.AtMBD3 was highly expressed in pollen and seed.AtMBD1,AtMBD2,AtMBD3,AtMBD4,AtMBD8,AtMBD12 were located in nucleus after analysis.2.Screening mutants of AtMBDs.The mutants of mbd1-1,mbd 3-1,mbd 3-2,mbd 4-1and mbd 13-1 were obtained by using CRISPR/Cas9 technique.After treating with ABA,Me JA,mannitol,Na Cl,37℃ and 44℃,both mbd3-1 and mbd3-2 mutants were found more tolerant to 0.5 μM ABA.Only mbd1-1 mutant showed dcp5-like phenotype after 5-d 37℃treatment,followed 5-d 22℃ culture.3.Confirming the effects of AtMBD3 on pollen and embryo development.GUS staining of different tissues from p MBD3:GUS/Col-0 transgenic plant was performed,and AtMBD3 was found to highly expressed in 14-d seedling cotyledon,4-d and 14-d seedling leaf vein and root,as well as in stigma and embryo after pollination.The mbd3 mutants showed shorter silique and seed abortion,containing early abortion of undeveloped embryo and late abortion of embryo development defect.Despite that both pollen morphology and its viability are normal,the exception of karyotype occurred.Compared with 92.17% normal trinuclear pollen produced in wild type,only 80% normal pollen was produced in mbd3 mutants with another 5% non-nucleus,3% one vegetative nucleus and 12% one vegetative and spermatocyte nucleus pollen.5.71% and 5.78% early aborted seeds were found in mbd3-1 and mbd3-2 mutants,respectively.10.86% and 18.88% late aborted seeds were found mbd3-1 and mbd3-2 mutants,respectively.43.75% late aborted seeds were found to cease at heart stage,and 31.25% at spherical to heart stage when normal seeds were fully developed.4.The function of MBD3 gene in the development of pollen and embryo was determined.The 32 genes,including sperm specific,pollen tube guiding and explosion genes,were expressed after screening.The sperm specific genes of DUO1,DAZ1,DAZ2,pollen tube guiding genes of PRK3 and PRK6,and pollen tube explosion genes of MYB97 and MYB120 were all down-regulated expression except PRK3.Both 49 up-regulated and 2down-regulated expression genes were found after RNA sequencing of mutant.The TT(TRANSPARENT TESTA)were the main proteins in up-regulated expression genes.They were enriched in secondary metabolic pathway and seeds development.The up-regulated genes of PME58,SAG12,FT and AEP4 shown by RNA sequencing were chosen to express,and they were found to be significantly up-regulated.A total of 950 up-regulated and 1128down-regulated genes were identified after transciptome analysis of wild type and mbd3-2mutant.The 11 genes with more studies were chosen to express,AIB4,AGL67,WOX2,WOX3,PID,KAN2,RGL3 and WOX1 were found to be significantly up-regulated,while ZOU and WOX8 were down-regulated.5.Screening the interaction protein of MBD3 and verifying its binding potential with methylated DNA.The in vitro interaction between MBD3 and PBL6 was found after screening 12 candidate proteins by using Y2 H after searching.PBL6 was a specifically expressed gene in pollen that also could interact with AtMBD3,AtMBD5 and AtMBD6 in vivo.The binding ability of AtMBD1,AtMBD3 and AtMBD4 to methylated DNA was evaluated by using electrophoretic mobility.The best condition for MBD1 induction was final concentration of 0.1 m M IPTG with 220 rpm at 37℃ for 6 h through constructing full length fusion proteins of MBD1-GST,MBD3-GST and MBD4-GST.The best condition for MBD3 and MBD4 induction was final concentration of 0.1 m M IPTG with 120 rpm at 16℃overnight.Both AtMBD1 and AtMBD3 bound symmetrically methylated CG and CHG sequences except nonsymmetric CHH.AtMBD4 could bind three type of sequences.AtMBD3 could bind nonmethylated DNA sequences,but MBD3 could only bind nonmethylated sequences in animals.A total of 711 changing methylated domains was found in Atmbd3 mutant by using Ch IP-seq,among which 687 domains had higher methylation than that of wild type.320 domains were overlapped with m CG methylation in wild type,218 with m CHG and 149 with m CHH.AtMBD3 preferred to bind symmetric DNA and nonsymmetric CHH sequences in vivo,which was corresponding to EMSA result.In summary,those MBD protein members with less study in Arabidopsis were chosen to analyze their bioinformatics and gene expression.Some of them were edited to obtain mutants,and their phenotypes were observed after treating with different abiotic stresses.Both pollen and embryo were used to sequence and some genes were expressed due to high expression of AtMBD3 d in pollen and embryo,and abortion of pollen and embryo in Atmbd3 mutants.The mechanism regulating pollen and embryo development of AtMBD3 was explored through screening its interaction proteins in vitro and in vivo,and analyzing its binding ability to methylated DNA.This study provides basis to further investigate molecular mechanism of MBD3 regulating pollen and embryo development,and effects of MBD proteins on plant growth.