Imprinting,methylation,and expression characterization of the maize ETHYLENEINSENSITIVE 2-like gene

Xiupeng Mei,Ping Li,Lu Wang,Chaoxian Liu,Lian Zhou,Chunyan Li,Yilin Cai*

Maize Research Institute,Key Laboratory of Biotechnology and Crop Quality Improvement,Ministry of Agriculture,Southwest University,Chongqing,China

Keywords:Maize Gene imprinting Methylation EIN2-like

ABSTRACT The endosperm plays essential roles in embryogenesis and seed germination and provides abundant resources for human food and industrial products.Identification of genes regulating the development of the endosperm and elucidation of their functions is of great importance for maize genetics and breeding.This study showed that the genespecific imprinted gene,ETHYLENE-INSENSITIVE 2-like(EIN2-like),is maternally expressed in both endosperm and embryo.The maternally expressed pattern was maintained throughout later seed developmental stages.Bisulfite sequencing using DNA obtained from hybrid endosperm tissues showed that the upstream regions of the alleles of EIN2-like were highly methylated at symmetrical sites(CG and CHG).A differentially methylated region in the upstream part of the maternal allele of EIN2-like was identified and found to be hypomethylated.Expression analysis showed that EIN2-like was highly expressed in the maize endosperm as well as at different stages of cell differentiation(8-12 days after pollination)in the hybrid endosperm.These results suggest that the maternally expressed gene EIN2-like may play crucial roles in the regulation of seed development.

1.Introduction

Cereal kernels,which provide a large proportion of the world's food supply,are composed mostly of a diploid embryo and a triploid endosperm[1].The endosperm supplies nutrients for embryogenesis and seed germination and also regulates the normal development of seeds[2,3].Manyunique transcriptional and epigenetic processes,including DNA methylation and genomic imprinting,occur primarily in the endosperm.Genomic imprinting is an epigenetic phenomenon in which genetically identical alleles are preferentially expressed depending on the parent of origin.In angiosperms,endosperm is the main tissue where imprinting occurs and only a small proportion of imprinted genes have been reported to be present in embryos[4-6].By genome-wide scanning of hybrid endosperms of Arabidopsis thaliana,rice(Oryza sativa),and maize(Zea mays L.),employing transcriptome sequencing technology,hundreds of imprinted genes were identified in single and multiple developmental stages[7-13].Many imprinted genes are crucial for the normal development of endosperm and seeds.For example,in A.thaliana,the loss of two maternally expressed genes,MEDEA(MEA)and FERTILIZATION-INDEPENDENT SEED2(FIS2),resulted in the fertilization-independent development of endosperm[14-19].A maternally expressed gene,FORMIN HOMOLOGY 5(FH5),was reported[20,21]to be responsible for endosperm cellularization depending on the activity of polycomb group proteins in seeds. The mutation of a maternally expressed gene,AGAMOUS-LIKE 62(AGL62),could cause precocious endosperm cellularization[8,11,22].In maize,several imprinted genes have been described in detail.Maternally expressed gene 1(Meg1),necessary and sufficient for the establishment and differentiation of endosperm nutrient transfercells,was alsofound[3,23]to play important roles in regulating maternal nutrient uptake,sucrose partitioning,and seed biomass.Mutation of a paternally expressed gene,Yucca1(Yuc1),resulted in small-sized kernels,retarded endosperm endoreduplication,and reduced dry matter in the endosperm[13,24].

Numerous findings suggest that DNA methylation and chromatin changes are important for regulating imprinting.Several previously identified candidate imprinted genes were found to be located near the endosperm differentially methylated region(DMR)in A.thaliana[8,11,25,26].For example,the expression of the maternally expressed gene,FLOWERING WAGENINGEN(FWA),was shown to be regulated by the DNA methylation status of its promoter[27].The expression of a maternally expressed gene,MATERNALLY EXPRESSED PABC-TERMINAL(MPC),was associated with its methylation status in the 5′upstream region and in the gene body[28].In maize,thousands of parent-of-origin-dependent DMRs(pDMRs)were revealed by genome-wide analysis of DNA methylation[29].The expression levels of several imprinted genes in maize,including Maternally expressed in embryo 1(Mee1)[4],Maize enhancer of zeste 1(Mez1)[30],and Fertilization independent endosperm 1(Fie1)[31],were identified as being associated with differences in DNA methylation and chromatin modification between parental alleles.Genome-wide analysis of DNA methylation in A.thaliana[25]suggested that genes methylated in transcribed regions were highly and constitutively expressed,whereas those methylated in promoter regions showed a greater degree of tissue-specific expression.

In maize,using the same reciprocal crosses from B73 and Mo17,hundreds of new imprinted genes were identified in one or multiple developmental stages by three research groups[10,12,13].However,different filtering strategies were applied by each group and a limited number of common genes were identified.In the present study,we cloned a candidate maternally expressed gene,ETHYLENE-INSENSITIVE2-like(EIN2-like,GRMZM2G370991),that was reported in all three reports.We also systematically analyzed its imprinting characteristics,stage of expression,and expression pattern in different organs as well as the DNA methylation status in the 5′upstream region of the gene.

2.Material and methods

2.1.Plant growth and tissue collection

The maize inbred lines B73,Mo17,Zheng 58,Chang7-2,Huang C,and 178 were grown at the Southwest University experiment station(Chongqing,China).Endosperm and embryo tissues were obtained from lines generated by reciprocal crossing or self-pollination of the inbred lines.Different organs of maize were collected from the inbred line B73.Endosperm and embryo tissues from three ears were collected on different days after pollination(DAP)by manual dissection.The embryo tissues were washed rapidly using RNase-free water and kept frozen in liquid nitrogen until use.

2.2.Gene cloning and bioinformatic analysis

The sequences of the gene and its predicted promoter region were downloaded from http://www.maizesequence.org/index.html.The gene was amplified from B73 using KOD FX DNA polymerase(Toyobo,Osaka,Japan).Rapid amplification of cDNA ends was used to determine the transcriptional start site(TSS)and 3′untranslated region(UTR)of EIN2-like.Cis-regulatory elements were predicted by Plant CARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)[32]and CpG islands were predicted by the EMBOSS Cpgplot program(http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/).Multiple sequence alignments were performed using Clustal X1.83,as implemented by MEGA5.0,on full-length proteins[33].Phylogenetic trees were constructed by neighbor joining with the JTT model and Complete Deletion[33].Reliability values at each branch point represent bootstrap cores(1000 replicates).Conserved motifs of the EIN2 protein sequences were identified by MEME(http://meme-suite.org/tools/meme).The sequences of all the primers used in the study are listed in Table S1.

2.3.Total RNA extraction and quantitative RT-PCR

Isolation of total RNA and assessment of its quality was performed as described previously[34].For quantitative RTPCR,cDNA was synthesized from 1 μg of total RNA using RevertAid First Strand cDNA Synthesis Kit(Thermo Scientific,Waltham,MA,USA),according to the manufacturer's protocol.The qRT-PCR was performed on a CFX96 Touch Cycler(Bio-Rad,Hercules,CA,USA)system and THUNDERBIRD SYBR qPCR Mix(Toyobo,Osaka,Japan)was used for the reaction.

2.4.Cleaved amplified polymorphic sequence(CAPS)analysis of allele-specific expression patterns

The EIN2-like genes from the inbred lines were sequenced for identifying single-nucleotide polymorphism (SNP)loci.SNP2CAPS software[35]was used to develop CAPS markers.The fragments contained the SNP loci were amplified and the PCR products were digested with selected Fast Digest enzyme(Thermo Scientific)and then separated by a garosegel electrophoresis.To exclude endosperm contamination,four developmental stages of the embryo were used to identify imprinting in the maize embryo.

Fig.1-Phylogenetic analysis of EIN2-like proteins.a.Genomic structure of EIN2-like.The EIN2-like coding region is represented by the blue boxes.CpG islands(represented by R1 and R2)were predicted with the EMBOSS Cpgplot program.TSS,transcriptional start site.b.Phylogenetic tree of multiple EIN2 proteins from different species.

2.5.Bisulfite sequencing

Endosperm tissue was dissected from B73×Mo17and Mo17×B73 kernels at 18 DAP.DNA extraction and bisulfite DNA conversion were performed as described previously[34].The sequences from B73 and Mo17 were analyzed for SNPs(excluding C-to-T and A-to-G loci),which were used to determine the methylation level of each allele.Regions containing at least five cytosines in both alleles were chosen and were assigned as differentially methylated regions(DMRs)if the methylation proportion of one allele was＜30%and that of the other allele was＞70%[12].

3.Results

3.1.Cloning and structural analysis of EIN2-like

EIN2-like was cloned from the endosperm of line B73.The fulllength genomic sequence of EIN2-like was 3.8 kb in length and contained two exons(2940 bp)and one intron(900 bp)(Fig.1-a).The protein encoded by the gene was predicted to contain～673 amino acids and its C-terminal domain was similar to that of the maize ETHYLENE-INSENSITIVE 2 protein(Fig.S1).The MaizeEIN2 protein consisted of at least 11 predicted transmembrane helices,a Nramp metal ion transporter domain,and a hydrophilic C-terminal domain identified in Arabidopsis EIN2[36,37].However,multiple sequence alignment showed that most of the transmembrane helices and the Nramp metal ion transporter domain were deleted in EIN2-like(Fig.S2).To analyze the evolutionary relationship of EIN2-like among A.thaliana,rice,maize,and Sorghum(Sorghum bicolor(L.)Moench),the full-length protein sequences of EIN2 from these species were aligned and a phylogenetic tree was constructed.As shown in Fig.1-b,EIN2-like did not cluster with known EIN2 proteins.These results suggest that neofunctionalization of EIN2-like may have occurred in the course of evolution.Further examination of the 2.5-kb EIN2-like fragment upstream of the ATG showed that EIN2-like contained a 238-bp repetitive sequence near the transcription start site and two predicted CpG islands in the upstream region(Fig.1-a,Fig.S3).Seven cis-regulatory elements(GCN4 and Skn-1)required for or involved in endosperm specific-expression(Table 1)[38-41]were predicted.

3.2.EIN2-like is a maternally expressed gene in maize endosperm and embryo

To determine the imprinted expression pattern of EIN2-like,we performed quantitative reverse transcription PCR(qRT-PCR)assay using endosperm tissues from different stages.We developed CAPS markers on the basis of the SNP loci of EIN2-like transcripts in the different maize inbred lines(Fig.S4-A,B).We chose the restriction enzyme Taq I for B73×Mo17 and Zheng58×Chang7-2.We detected no polymorphism in the Huang C×178 line for the EIN2-like alleles.

To validate the imprinted pattern of EIN2-like in maize endosperm,we first examined the expression pattern in the endosperms of the reciprocal cross of B73 and Mo17 at 14 DAP.We observed the clear presence of maternal EIN2-like alleles in the RT-PCR products digested with allele-specific restriction enzymes(Fig.2-a).These results indicated that EIN2-like is a maternally expressed gene(MEG)in the endosperm of lines obtained from the reciprocal crosses B73×Mo17(B×M)andMo17×B73(M×B)at 14 DAP.We also determined the expression pattern of EIN2-like in the embryo derived from reciprocal crosses of B73 and Mo17 at 14 DAP and found that EIN2-like was also expressed in the embryo in a maternal specific manner(Fig.2-c).Thus,we concluded that EIN2-like is regulated by genomic imprinting both in the maize endosperm and in embryo tissues derived from B73 and Mo17 reciprocal crosses at 14 DAP.We used another genetic background to determine whether EIN2-like has allele-specific or gene-specific imprinting in the endosperm.We detected the apparent presence of the maternal allele of EIN2-like in the endosperm from the reciprocal crosses Zheng58×Chang7-2(Z58×C7-2)and Chang7-2×Zheng58(C7-2×Z58)(Fig.2-b).

Table 1-cis-regulatory elements involved in endosperm expression.

Fig.2-Analysis of the expression pattern of EIN2-like gene.a,b The allele-specific expression of EIN2-like was investigated in the endosperm tissues obtained from reciprocal crosses at 14 DAP from different genetic backgrounds B73/Mo17 and Zheng58/Chang7-2.The first-named parent was the female parent.Act3 was used as a loading control.c.The allele-specific expression of EIN2-like was monitored in embryo tissues derived from B73/Mo17 reciprocal crosses at 14 DAP.Act3 was used as a loading control.

3.3.EIN2-like imprinted persistently throughout the later seed developmental stages

To determine whether the imprinted pattern of EIN2-like was persistently present at multiple time stages during endosperm development after pollination,we studied the allele specific expression of EIN2-like in the later seed developmental stages(10-28 DAP).We uniformly observed marked differences in the paternal and maternal EIN2-like alleles in endosperm tissue from the B73 and Mo17 reciprocal crosses from 10 to 28 DAP(Fig.2-a;Fig.3-a).In four stages during embryo development,we observed similar results(Fig.3-b).Collectively,these results show that EIN2-like imprinting persisted in later seed developmental stages.

3.4.EIN2-like alleles are highly methylated in their upstream region

To determine whether DNA methylation in the EIN2-like upstream region paralleled its maternal-specific expression pattern,we performed bisulfite sequencing using F1hybrid endosperm DNA extracted from B73 and Mo17 reciprocal crosses at 18 DAP and verified the reliability of our bisulfite converted DNA templates for the subsequent methylation analysis[34].We cloned a～2.5 kb fragment from the start cod on of EIN2-like and selected two regions(R1 and R2)for bisulfite sequencing based on a set of B73-and Mo17-specific primers(Fig.1-a,Fig.S3;Table S1).We found the R1 region to contain one CpG island and the R2 to contain one CpG island and a repetitive sequence(Fig.S3).We assigned the maternal and paternal alleles of EIN2-like based upon the B73/Mo17 polymorphisms.

At least 15 clones of the 5′upstream region of EIN2-like were sequenced for each fragment based on the SNPs in the inbred lines,B73 and Mo17.First,the hybridized combination B73×Mo17 was used for analyzing the methylation status of the maternal(Mo17)and paternal(B73)alleles.In region 1,76.6%,64.8%,and 4.1%methylated cytosines were observed at CG,CHG,and CHH sites,respectively,for the maternal alleles;and92.%,79.8%,and5.4% methylated cytosines were observed at CG,CHG,and CHH sites for the paternal alleles(Fig.4-a).The methylation status of parental alleles in the Mo17×B73 cross was also examined.The maternal alleles showed 51.3%,54.7%,and 5.0%methylated cytosines at CG,CHG,and CHH sites and the paternal alleles showed 95.7%,60.6%,and 2.1%methylated cytosines at CG,CHG,and CHH sites in region 1(Fig.4-c).In region 2,a similar high methylation status was detected in the parental alleles in B73 and Mo17 reciprocal crosses(Fig.4-b,d).Overall,＞50%methylated cytosines at CG,CHG sites in region 1(Fig.4-a,c;Table S2)and＞70%methylated cytosines at CG,CHG sites in region 2(Fig.4-b,d;Table S2)were observed in B73 and Mo17 reciprocal crosses.Collectively,these findings suggested that the EIN2-like alleles were highly methylated in symmetrical contexts(CG and CHG sites,H=A,T,C)but were less methylated in asymmetric contexts(CHH sites)in endosperm tissue from the B73 and Mo17 reciprocal crosses.

Fig.3-Allele-specific RT-PCR analysis of EIN2-like at different developmental stages of endosperm and embryo.Expression was monitored in endosperm tissue at multiple time points(10,12,16,18,20,22,24,26,and 28 days after pollination(DAP))and at three time points(18,22,and 26 DAP)in embryo tissue derived from B73/Mo17 reciprocal crosses.Act3 was used as a loading control.a.Imprinting pattern analysis of EIN2-like in endosperm tissue.b.Imprinting pattern analysis of EIN2-like in embryo tissue.

3.5.EIN2-like alleles are differentially methylated in symmetrical contexts

Based on the results of bisulfite sequencing,a higher level of methylation in symmetrical contexts(CG and CHG sites)was observed in the paternal alleles than in the maternal alleles in the 5′-upstream region of EIN2-like in the B73 and Mo17 reciprocal crosses(Fig.4-a,c)in region 1.However,the maternal alleles exhibited higher level of methylation than the paternal alleles in symmetrical contexts(CG and CHG sites)in region 2(Fig.4-b,d).In this region,a highly repetitive fragment(-300 to-60 bp)was detected,which was highly methylated at the CG sites in the reciprocal crosses of B73 and Mo17(Fig.S3-B;Fig.5-b).The EIN2-like alleles near the repetitive fragment were highly methylated in the symmetrical(CG and CHG)context(Fig.4-b,d;Fig.5-b,d).Further analysis revealed a DMR(red dotted line)in region 1 in the maternal alleles(hypomethylated,CG site)in B73×Mo17(Fig.5-a)and similar results were observed in the Mo17×B73 cross(Fig.5-c).Collectively,these results suggested that the parental alleles of EIN2-like were differentially methylated in symmetrical contexts and that a DMR was located in the 5′upstream region of EIN2-like.

3.6.EIN2-like is highly expressed in maize endosperm

To investigate the functions of EIN2-like in maize,we performed qRT-PCR to quantify its relative transcription levels int is sues of the inbred line B73.EIN2-like was highly expressed in maize kernel,especially in the endosperm,but less highly expressed in vegetative and reproductive organs(stem,root,leaf,ear,and tassel)of maize(Fig.6-a).We further analyzed the expression pattern of EIN2-like at the later developmental stages(10-28 DAP)of endosperm from B73×Mo17 and Mo17×B73.EIN2-like showed relatively high expression at 10 and 28 DAP in the endosperm(Fig.6-b).These results suggest that EIN2-like may function in seed development.

4.Discussion

Fig.4-Analysis of DNA methylation of EIN2-like.DNA was isolated from endosperm tissue obtained from B73(female)×Mo17(male)and Mo17(female)×B73(male).Three ears were collected at 18 days after pollination(DAP).After bisulfite conversion,specific primers were used to amplify different regions of EIN2-like.The products were cloned and sequenced to determine the methylation patterns of the forward strand of DNA,and B73/Mo17 sequence polymorphisms allowed exact assignment of the parental alleles for each clone.The label onthe X-axis indicates the methylationpattern and the text in the brackets(for example,-1380 to-820)indicates the distance relative to ATG.a,b Analysis of DNA methylation in the two 5′-upstream regions of EIN2-like in B73×Mo17.c,d Analysis of DNA methylation in the 5′upstream regions of EIN2-like in Mo17×B73.E achregion was cloned and at least 15 clones were sequenced.

In the present study,the maize maternally expressed gene EIN2-like was imprinted not only in the endosperm,but also in the embryo(Fig.2).These results are consistent with those previously[10]reported.Two types of imprinted expression patterns,namely allele-specific and gene-specific imprinting,have been described(Dzr1 and R)[42,43].In our study,EIN2-like showed the same imprinting pattern in the reciprocal crosses of Zheng58 and Chang7-2 as was observed in the crosses of B73 and Mo17.Thus,EIN2-like showed a gene-specific rather than an allele-specific imprinted pattern(Fig.2-b).Moreover,the parental allele of some imprinted genes appeared[44]to have a transient or delayed activation.For example,the maternal allele of maize Fie2 was specifically detected at 2 DAP,and the paternal allele was activated after 5 DAP,followed by the activation of biallelic expression after 9 DAP.In addition,the maternally expressed gene ZAG2 was consistently imprinted from 10 to 20 DAP in maize endosperm,but expressed in a binary manner at 22,24,and 28 DAP[5].Another maize imprinted gene,NUCLEAR FACTOR Y,SUBUNIT C 9(NF-YC9),was maternally expressed at 0-5 DAP,but paternally expressed at 7-15 DAP in the endosperm[13,34].These specific and interesting expression patterns were likely associated with specific stages of endosperm development.Unlike these previously reported imprinted genes,EIN2-like was expressed persistently throughout the seed developmental stages(Fig.3-a,b)[13].

Despite great progress achieved in identifying new imprinted genes in plants,only a few have been functionally characterized in seed development.The evidence from study of several imprinted genes indicates that they are involved in the regulation of transition from endosperm cell division to cellularization[3,8,14,22,45].Other studies have indicated that imprinted genes also control nutrient allocation in plant seeds.The maize maternally expressed gene Meg1,specifically expressed in the basal endosperm transfer layer,was necessary and sufficient for the establishment and differentiation of the endosperm nutrient transfer cell[23].Meg1 was also reported to regulate maternal nutrient uptake,sucrose partitioning,and seed biomass yield[3].The paternally expressed gene Yuc1 was found to play vital roles in the indole-3-acetic acid(IAA)biosynthesis,which was essential for normal endosperm development in maize[13,24].In Arabidopsis,AtEIN2,the homolog of maize EIN2-like,was also imprinted,and positively regulated ethylene response[7,46];however,their mechanisms and regulation may differ.In our study,EIN2-like was preferentially expressed in maize endosperm and showed relatively high expression at the cell differentiation stage[47](8-12 DAP)in the endosperm(Fig.6-a,b).The high level of methylation of parental alleles in the 5′upstream region may be associated with the tissue-specific expression(Fig.4-a-d,Fig.6-a,b).Moreover,in the 5′upstream region of EIN2-like,we predicted seven cis-regulatory elements(GCN4 and Skn-1)that were required for or were involved in the expression in endosperm(Table 1).These results suggest that EIN2-like may play important roles in regulating endosperm development.

Fig.5-Analysis of methylation in the 5′-upstream regions of EIN2-like.a,b Analysis of methylation sites in endosperm tissues derived from B73×Mo17 crosses at 18 days after pollination(DAP).c,d Analysis of methylation sites in endosperm tissues derived from Mo17×B73 and at 18 DAP.The X-axis indicates the distance relative to ATG.The differentially methylated region is marked by a red dotted outline.

DNA methylation is thought to play an essential role in epigenetic regulation.Several findings[26,48]indicate that the targets of DNA methylation in the imprinting control region are mostly transposable elements and CpG-enriched regions.Many previously identified imprinted genes,such as Fie1,Mez1,Mee1,and Meg1,in plants support the notion that DMRs or hypomethylation of maternal alleles play a crucial role in regulating their expression pattern[4,23,30,31,49].In the present study,the high methylation status of EIN2-like alleles was observed in the 5′upstream region,including the repetitive sequence(Fig.4;Fig.5;Table S2).A previous study[10]revealed the presence of DMRs in EIN2-like and At EIN2.Notably,a maternally hypomethylated DMR was identified in region 1 in the reciprocal crosses of B73 and Mo17(Fig.5-a,c).However,a higher methylated level of the maternal alleles was observed in region 2(Fig.4-b,d;Fig.5-b,d).Thus,variations in the methylation pattern between the two parental alleles might explain the imprinted expression of EIN2-like.

This report has systematically described the imprinted pattern,characteristics,and stages of expression of a maternally expressed gene,EIN2-like,in maize.The upstream region of the parental allele of EIN2-like was highly and differentially methylated in symmetrical contexts(CG and CHG).The expression pattern in maize organs was highly tissuespecific,suggesting that EIN2-like may play crucial roles in endosperm development.These results provide a reference for further studies of EIN2-like and may be useful for exploiting favorable genes for maize breeding.

Fig.6-Expression analysis of EIN2-like in maize tissues.a.Root,stem,mature leaf,immature tassel(3-4 cm),immature ear(3-4 cm),endosperm 18 days after pollination,and embryo were isolated from the inbred line B73.b.The endosperm tissue at different developmental stages was isolated from the reciprocal crosses B73×Mo17(B×M)and Mo17×B73(M×B).The expression level was normalized to Act3 expression and data are means±SD of three biological replicates.

Acknowledgments

The authors thank the Major Research Projects of Chongqing(CSTC2016shms-ztzx80013,CSTC2016shms-ztzx80016)for financial support.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.08.001.