Dynamic Histone Acetylation of Late Embryonic Genes during Seed Germination

Dynamic histone acetylation of late embryonic genes during seed germination

Helen H.Tai1,*,George C.C.Tai2and Tannis Beardmore1

1Canadian Forest Service,Natural Resources Canada,P.O.Box4000,E3B5P7,Fredericton,NB,Canada (*author for correspondence;e-mail htai@nrcan.gc.ca);2Potato Research Centre,Agriculture and Agri-Food Canada,P.O.Box20280,E3B4Z7,Fredericton,NB,Canada

Received27April2005;accepted in revised form12August2005

Key words:chromatin,gene expression,histone acetylation,histone deacetylase,microarray,seed germination

Abstract

Histone acetylation is involved in the regulation of gene expression in plants and eukaryotes.Histone deacetylases(HDACs)are enzymes that catalyze the removal of acetyl groups from histones,which is associated with the repression of gene expression.To study the role of histone acetylation in the regulation of gene expression during seed germination,trichostatin A(TSA),a speci?c inhibitor of histone deacety-lase,was used to treat imbibing Arabidopsis thaliana seeds.GeneChip arrays were used to show that TSA induces up-regulation of45genes and down-regulation of27genes during seed germination.Eight TSA-up-regulated genes were selected for further analysis–RAB18,RD29B,ATEM1,HSP70and four late embryogenesis abundant protein genes(LEA).A gene expression time course shows that these eight genes are expressed at high levels in the dry seed and repressed upon seed imbibition at an exponential rate.In the presence of TSA,the onset of repression of the eight genes is not a?ected but the?nal level of repressed expression is elevated.Chromatin immunoprecipitation and HDAC assays show that there is a transient histone deacetylation event during seed germination at1day after imbibition,which serves as a key developmental signal that a?ects the repression of the eight genes.

Abbreviations:ABRE,abscisic acid response element;ANOVA,analysis of variance;ChIP,chromatin immunoprecipitation;DAI,days after imbibition;DNA,deoxyribonucleic acid;HAT,histone acetyl-transferase;HDAC(s),histone deacetylase(s);LIMMA,linear models for microarray analysis;MS, Murashige and Skoog;qPCR,quantitative PCR;RNA,ribonucleic acid;RTqPCR,reverse transcription quantitative PCR;TSA,Trichostatin A

Introduction

Typically,seed development proceeds through a series of con?uent stages and is terminated as the seed undergoes maturation drying,becoming qui-escent or dormant.Imbibition of a nondormant seed results in germination and subsequent seed-ling development.The transition from the devel-opmental to the germinative program is associated with changes in the pattern of gene expression (Comai and Harada,1990;Gallardo et al.,2001; Nakabayashi et al.,2005;Nambara et al.,2000; Soeda et al.,2005).Evidence suggests that seed germination marks the end of embryonic develop-ment and rapid repression of embryonic genes is observed with seed imbibition.The pkl mutant in Arabidopsis thaliana shows a loss of repression of embryonic traits in seedlings(Ogas et al.,1997;

Plant Molecular Biology(2005)59:909–925óSpringer2005 DOI10.1007/s11103-005-2081-x

Henderson et al.,2004)and a loss of repression of the LEAFY COTYLEDON embryonic identity genes,LEC1,LEC2,and FUS3(Rider et al., 2003).The PKL gene encodes a protein that is similar to CHD3,a mammalian chromatin protein (Ogas et al.,1999),indicating that chromatin may have a role in the repression of embryonic genes. In this study we investigate the role of covalent modi?cation of chromatin in the control of embryonic gene repression during seed germina-tion.

The basic unit of chromatin is the nucleosome, which consists of146bp of DNA wrapped around a tetramer of H3/H4and two dimers of H2A/H2B(Wolffe,1992).Histones can be chem-ically modi?ed through the action of enzymes that add or remove acetyl,methyl,phosphoryl,ubiqu-itin or sumo from the N-terminal tail of the proteins that are exposed on the surface of the nucleosome(Jenuwein and Allis,2001).The functions of these modi?cations in cellular and developmental processes are beginning to be elucidated.This study addresses histone acetyla-tion.Earlier studies show a correlation between histone acetylation and increased gene transcrip-tion(Allfrey et al.,1964;Hebbes et al.,1988).In addition,several transcriptional co-activators have intrinsic histone acetyltransferase(HAT) activity and transcriptional co-repressor com-plexes contain histone deacetylase(HDAC)(Ah-ringer,2000;Sterner and Berger,2000;Struhl, 1998).Furthermore,studies also show that active regions along chromosomes are enriched in acet-ylated histones whereas inactive regions are not (Turner,1993).

Transcription factor recruitment of HATs to promoter regions is associated with acetylation of histones and activation of gene expression(Kuo et al.,1998,2000),whereas the targeted recruit-ment of HDACs to promoters is associated with deacetylation of histones and gene repression (Kadosh and Struhl,1998a,b;Rundlett et al., 1998).However,histone acetylation and deacety-lation also occurs over larger chromosomal domains that extend beyond promoters and is not under the control of sequence speci?c tran-scription factors(Kurdistani and Grunstein, 2003).Histone acetylation changes not targeted to promoters are referred to as‘global acetylation and deacetylation’may function to control basal transcription or allow for reversal of targeted acetylation changes after removal of the recruited HATs or HDACs(Krebs et al.,2000;Kuo et al., 2000;Vogelauer et al.,2000;Katan-Khaykovich and Struhl,2002).

Histones are also covalently modi?ed in plants and plant HATs and HDACs have been identi?ed (Stockinger et al.,2001;Pandey et al.,2002;Loidl, 2004).Arabidopsis studies also demonstrate that HDAC functions as a repressor in plants(Wu et al.,2000a,b Zhou et al.,2004).Anti-sense expression knockouts of HDACs,ATHD1and ATHD2A(Wu et al.,2000a,b;Tian and Chen, 2001),an ATHD1T-DNA insertional mutant (Tian et al.,2004),ATHD2A transgenic Arabid-opsis thaliana(Zhou et al.,2004)and OSHDAC1 transgenic rice(Jang et al.,2003)were used to identify a role for HDACs in plant development. Recently ATHDI(also called HD19)was also shown to be involved in regulation of pathogen response genes(Zhou et al.,2005).Histone acet-ylation is also involved in the regulation of FLC,a ?owering control gene in Arabidopsis thaliana(He et al.,2003)and the pea plastocyanin gene(Chua et al.,2001,2003).

An advantage of using chemical inhibitors of HDAC is that the timing of the loss of HDAC activity can be controlled.Small molecules trap-oxin and trichostatin A(TSA)were used in mammalian cells to characterize the function of HDAC1(Taunton et al.,1996;Richon et al., 2000).Chemical inhibitors of HDACs are also effective in plants(Brosch et al.,1995;Lechner et al.,1996)and speci?c chemical inhibitors of TSA were used to identify a role for HDACs in plants in nucleolar dominance(Chen and Pikaard, 1997;Lawrence et al.,2004),root meristem pro-liferation(Murphy et al.,2000)and tobacco pro-toplast cell cycle progression(Li et al.,2005). Microarray analysis in tobacco and Arabidopsis thaliana seedlings also show that TSA induces changes in gene expression and a?ects histone acetylation at speci?c genes(Chua et al.,2004; Chang and Pikaard,2005).

Our study investigates the role of HDAC on the expression,acetylation and deacetylation of Arabidopsis thaliana genes during seed germina-tion through inhibition of HDAC with TSA. Arabidopsis thaliana has HDAC homologs that belong to three HDAC families,RPD3/HDA1, HD2,and SIR2(Pandey et al.,2002).TSA inhibits RPD3/HDA1-like HDACs and plant-speci?c

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HD2HDACs(Yoshida et al.,1990,1995;Lechner et al.,1996),but does not affect the SIR2class of HDACs(Imai et al.,2000).Out of the17members of the HDAC family in Arabidopsis thaliana there are14RPD3/HDA1-like and HD2genes(Pandey et al.,2002).An analysis of gene expression using GeneChip arrays and reverse transcription quan-titative polymerase chain reaction(RTqPCR) identi?ed eight genes whose repression during imbibition is altered with TSA treatment.These genes are expressed during late maturation in the embryo,involved in dehydration stress responses and contain an upstream abscisic acid response element(ABRE).The genes also show a deacety-lation pattern during germination that is corre-lated with HDAC activity levels providing evidence that they are controlled by HDAC during seed germination.

Materials and methods

Plant material and growth conditions

Seeds from Arabidopsis thaliana ecotype Columbia were surface sterilized and sown on nylon mem-branes on Murashige and Skoog(MS)media (Murashige and Skoog,1962)containing0.8% agar with and without10l M Trichostatin A (TSA)(Sigma Chemical Co.,USA)in10cm Petri dishes with10ml media/dish.Seeds were germi-nated under16h light conditions.One hundred milligrams of seeds were sown per10cm dish for samples used for RNA preparation or ChIP. GeneChip array expression analysis

Total RNA was prepared from500mg of plant tissue from dry seeds and seeds imbibed for3days in the presence and absence of10l M TSA using a LiCl extraction method described previously(Tai et al.,2004).RNA quality was assessed by agarose gel electrophoresis and spectrophotometry.The RNA was then processed for use on Affymetrix Arabidopsis GeneChip arrays(Santa Clara,USA), according to the manufacturer’s protocol.Brie?y, cDNA was synthesized from total RNA using reverse transcriptase followed by second strand synthesis to generate double-stranded cDNA.An in vitro transcription reaction was used to generate biotinylated cRNA.After puri?cation and fragmentation,cRNA was used in a hybridization mix containing added hybridization controls and arrays hybridized for16h at45°C.Standard post hybridization wash with protocol EukGE-WS2 and double-stain protocols were used on an A?ymetrix GeneChip Fluidics Station400.Arrays were scanned on an A?ymetrix GeneChip Scanner 2500.

Two replicates of cRNA samples from two different batches of3-day germinated seeds either treated with TSA or untreated were prepared.Two sets of replicate samples were hybridized to the Arabidopsis Genome array with8300gene se-quences and used for statistical analysis.Scanned arrays were normalized globally and analyzed with A?ymetrix MAS5.0software to obtain absent/ present calls and to assure that all quality param-eters were in the recommended range(Supplemen-tal material Table1).

Statistical analysis

Gene expression signal data obtained from the replicated8300gene Arabidopsis Genome arrays on the A?ymetrix Gene Chip Fluidics Station were statistically analyzed in two stages:

1.The affy module(Gautier et al.,2004)of the

Bioconductor open-source software(http:// https://www.sodocs.net/doc/f12997708.html,)was used to normalize and compute expression values using the robust multichip average(RMA)method (Irizarry et al.,2003).The linear model for microarray analysis(LIMMA)(Smyth,2004), implemented using the limma module of the Bioconductor software,was used for statistical analysis of gene expression data.P=0.025 was used in LIMMA analysis to obtain a list of di?erentially expressed genes.

2.Genes identi?ed as differentially expressed

after stage1were candidates for second stage screening.The gene expression data normalized globally with Affymetrix MAS

5.0software was used.The data was sub-

jected to further discriminant analysis to identify those genes with large effects due to TSA treatment.Three gene expression vari-ables TSA,untreated and TSA/untreated ratio,over two replicates were subjected to canonical variate analysis(Bartlett,1947).

Canonical variate analysis was carried out

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using the software SYSTAT 10.2(Systat

Software Inc.,USA).

Functional classi?cation of genes

The TAIR database (https://www.sodocs.net/doc/f12997708.html,)was used to classify the predicted function of the genes and to identify upstream regions of the genes.Five hundred base pairs of DNA sequence upstream of the transcription start site was analyzed for con-served motifs using INCLUSive::Motif Sampler (Thijs et al.,2002a,b)and the PlantCare database (Rombauts et al.,1999,Lescot et al.,2002).Histone deacetylase assay

Plant material was ground in liquid nitrogen and lysed in 0.1M KCl,20mM HEPES/NaOH pH 7.9,0.2m EDTA,0.5mM DTT,and 0.5mM PMSF.The lysate was centrifuged at 15000rpm and the supernatant used in the colorimetric HDAC assay (BIOMOL Research Laboratories,Inc.,Plymouth Meeting,USA).Brie?y,the color-imetric acetylated lysine substrate was incubated with plant lysates.Deacetylation sensitizes sub-strate to the developer and causes an increase in absorption at 405nm,which is linearly correlated with HDAC activity.Protein concentrations were measured at A 280.The activity of HDAC is expressed at A 405/mg protein.

Reverse transcription quantitative polymerase chain reaction (RTqPCR)

Total RNA was isolated from dry seeds,and seeds one,three,and 6days after imbibition (DAI)on MS media containing 0.8%agar in the presence and absence of 10l M TSA.Total RNA was also isolated from seeds undergoing two additional treatments:(1)3days on media in absence TSA followed by 3days on media in the presence 10l M TSA (3d–3d TSA),(2)3days on media in the presence of 10l M TSA followed by 3days on media in the absence of TSA (3d TSA–3d).One microgram of RNA from each sample was used for cDNA synthesis using reverse transcriptase (Invitrogen,Burling-ton,Canada)following pre-treatment with RNase-free DNAse.RTqPCR was done using coding region-speci?c primers listed in Table 1.Preliminary experiments were run to ensure the

T a b l e 1.P r i m e r s u s e d f o r P C R .

G e n e f u n c t i o n

L o c u s P r o m o t e r r e g i o n

C o d i n g r e g i o n

U p p e r p r i m e r

L o w e r p r i m e r

U p p e r p r i m e r

L o w e r p r i m e r

r d 29B A T 5G 523005￠G A T T T T T T C T T T T G C C G T T T T G T T A T 5￠T C T G G C T T C T G T C T C T T T A C T T C T G 5￠C G A G A G C C C T G T A A A A G A T G A A A C T C C 5￠C T C C G C C A T G C C T C C C A A C G G A G G G T T G r a b 18

A T 5G 664005￠G T T A G T A C C G C C A C A A A G A A A A G G A T A G 5￠G T G A C G C G T G G C A G C A G C A G

5￠G C A C C A C G G C C A A G A G C A A C 5￠A T C G C A G G A C G T A C A T A C A T A A A A A G C A C

L E A A T 3G 175205￠A A T C G C T T G C C T C G T T G T T T G 5￠A C C T C T G T T T A C T G T G A T G T G T T C T C 5￠G C A C G A A G A C A A C A T C C A A C C C A C T C 5￠G G A G A T G C T A A A G A G A A G T T G G C G G A G L E A A T 3G 156705￠T C T C A T A A C A T G C G A C G A C G A T A C 5￠C G G C G G A G A A G A A G A C A C G 5￠C G C G A G G C C A A G G A C A A G G 5￠C C A G G A T C A C C G T C A C A A C A A G A A A C L E A A T 1G 526905￠G T A A A T A T C T A T G C A G T A A T G G C G G T 5￠A A C T T G G A G T T A T G T G C G A G T G T T A 5￠A G C C C A G T C A G C C C A A C A A A A G G 5￠A C G A A C G C A A C A A A C A C T A A T C A A A G C L E A A T 5G 067605￠T G T T T T A T C A A T T T G T T T T A T G C G A C T C 5￠T G A G A C G T G T C A T G A A A G A A G A G G 5￠C A A C G C G G C C A T G A A A G A A G C 5￠G A A C A C G A A A G T A A G A C A G A A A A C C A C G A A A t E m 1

A T 3G 518105￠T C T T G G G G G A A A C A G A A A A T G G 5￠A A A G G A A A C A A A A G G A G A G A A A A A G T G 5￠G T G G A G A G G C G A G G A A G G A G C A G T T A G 5￠T C C T T C C T C G C C T C T C C T C C T T T G T G T h s p 70A T 3G 125805￠C T T G T T T T C A A A A C G G G A G T T A C T A T T 5￠G G A A G A G A A G G C A G A G A G G T G T T T G 5￠G A G A G G G C A C G A A C A A A G G A C A A C 5￠G C A C C G C C G C T A C C A C C A G U

B Q 11A T 4G 050505￠T

C A G T A T A T G T C T C G C A G C A A A C T A T C

5￠G A C G A C T C G G T C G G T C A C G 5￠G A T C T T C G C C G G A A A G C A A C T T

5￠C C A C G G A G A C G G A G G A C C

T h e s e n s e (u p p e r )a n d a n t i s e n s e (l o w e r )p r i m e r s u s e d t o a m p l i f y t h e p r o m o t e r a n d c o d i n g r e g i o n s f o r e a c h g e n e a r e l i s t e d i n t h i s t a b l e i n t h e 5￠t o 3￠o r i e n t a t i o n .T h e p r e d i c t e d f u n c t i o n o f t h e p r o t e i n e n c o d e d b y t h e g e n e (g e n e f u n c t i o n ),a n d g e n e l o c u s (l o c u s )a r e l i s t e d .

912

ampli?cation of a single PCR product for each gene and the absence of any PCR products in the total RNA without reverse transcription (data not shown).A standard curve was gener-ated for each gene that consisted of serial dilutions of the dry seed cDNA(1,1/2,1/4,1/ 8,0).Template concentrations for each gene are given arbitrary values that are relative to the concentration of the template in the dry seed cDNA.RTqPCR was done with the DyNAmo SYBR green qPCR kit(Finnzymes Oy,Espoo, Finland)with the following cycling conditions: 2min initial denaturation at94°C;50cycles of 94°C30s,60°C30s,72°C30s;?nal elonga-tion72°C10min,4°C hold.Data presented is the average relative expression values for three replicates and the error bars represent the standard deviation.

Chromatin immunoprecipitation quantitative polymerase chain reaction(ChIP/qPCR)

ChIP was carried as described in Johnson et al. (Johnson et al.,2002)with the following mod-i?cation:DNA was puri?ed using the Qiaquick PCR puri?cation kit(Qiagen,Germany)instead of by phenol/chloroform/isoamyl alcohol extrac-tion and ethanol precipitation.Immunoprecipi-tation was done with an anti-acetyl histone H4 antibody speci?c for tetra-acetylated H4(Up-state,Charlottesville,USA).For qPCR serial dilution of leaf DNA was used to generate the standard curve.Leaf tissue was formaldehyde cross-linked,extracted and sonicated as for ChIP samples.Cross-links were reversed,resid-ual protein and DNA was puri?ed using the Qiaquick PCR puri?cation kit.Puri?ed DNA from the ChIP samples and the leaf standard curve samples were used for qPCR with the DyNAmo SYBR green qPCR kit(Finnzymes Oy,Espoo,Finland)with the following cycling conditions:2min initial denaturation at94°C; 50cycles of94°C30s,60°C30s,72°C30s;?nal elongation72°C10min,4°C hold.Dilu-tions of leaf template DNA for the standard curve were1,1/2,1/4,1/8,and0and a standard curve was generated for each promoter and coding region of each gene analyzed. Primers used for qPCR of promoter and coding regions are listed in Table1.The relative quantity of template DNA was calculated using the standard curve.Each sample was done in triplicate.Error bars represent the standard deviation.

Results

Identi?cation of genes a?ected by TSA treatment during seed germination

Three DAI was selected as the time point for gene chip expression analysis,since visible differences between untreated and TSA-treated seedlings could be observed.One function of HDAC is to repress genes;therefore,the following two char-acteristics were used to select genes for analysis:1. gene expression is up-regulated in the presence of TSA and 2.gene is repressed following seed imbibition.Affymetrix Arabidopsis GeneChip ar-rays were used to examine di?erences in gene expression between seeds imbibed for3days in the presence and absence of TSA(Supplemental material Table1).LIMMA analysis indicated that 72genes were a?ected by TSA treatment(Supple-mental material Table2).Genes with up-regulated expression are presented in Table2and genes with down-regulated expression presented in Table3.

It was highly desirable to validate the gene chip expression data using RTqPCR and to carry out chromatin analysis of TSA-affected genes;there-fore,further statistical analyses were applied to the data to narrow down the list of72genes to those that had the most highly signi?cant increases in expression in the presence of TSA.Three gene expression variables were used for multivariate statistical analysis of the72genes:1.mean signal when untreated(untreated),2.mean signal when treated with TSA(TSA),3.mean signal ratio of TSA treated over untreated(TSA/untreated) (Table4,Supplemental material Table3).Analy-ses of variances(ANOVA)were carried out for each of the three sets of gene expression data, TSA,untreated and TSA/untreated to evaluate differences between these three gene expression characteristics.Signi?cant differences between the three gene expression characteristics were found, which indicates that the variabilities in these three sets of data can be used to select genes.Canonical variate analysis(Bartlett,1947)was applied to examine the three gene expression variables together to identify TSA-responsive genes.The

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?rst two canonical variates account for97%of the variability of the data.Mean scores of the?rst (CAN1)and second(CAN2)canonical variates of the72genes are distributed in a two-dimension diagram(Figure1).CAN1mainly represents the variability due to gene expression levels,whereas CAN2represents mainly the variability due to TSA/untreated ratios.The majority of genes are

Table2.Genes up-regulated by TSA.

Probe ID Locus identi?er Annotation log2-fold change 17407_s_at AT5G52300Low-temperature-responsive65kD protein(LTI65)

/desiccation-responsive protein29B(RD29B)

4.18

16038_s_at AT5G66400Dehydrin(RAB18) 3.93

18872_at AT3G17520Late embryogenesis abundant domain-containing protein 3.72

17310_at AT3G51810Em-like protein GEA1(EM1) 3.54

20641_at AT1G52690Late embryogenesis abundant protein 3.48

19918_at AT3G15670Late embryogenesis abundant protein 3.32

14097_at AT2G47770Benzodiazepine receptor-related 3.24

17282_s_at AT3G51810Em-like protein GEA1(EM1) 3.24

19186_s_at AT3G50970Dehydrin xero2(XERO2)/low-temperature-induced protein LTI30(LTI30) 3.12

20321_s_at AT1G73190Tonoplast intrinsic protein,alpha/alpha-TIP(TIP3.1) 3.05

19152_at AT5G06760Late embryogenesis abundant group1domain-containing protein 2.96

13645_at AT1G05340Expressed protein 2.88

13198_i_at AT4G2852012S seed storage protein 2.84

16450_s_at AT3G50980Dehydrin 2.65

13284_at AT3G12580Heat shock protein70 2.62

14420_at AT2G31980Cysteine proteinase inhibitor-related 2.48

19660_at AT2G40610Expansin 2.38

13199_r_at AT4G2852012S seed storage protein 2.31

15280_at AT2G19900Malate oxidoreductase 2.15

20220_at AT4G02280Sucrose synthase 2.11

16422_at AT2G33830Dormancy/auxin associated family protein 1.81

14077_at AT4G08950Phosphate-responsive protein 1.74

12072_at AT4G25170Expressed protein 1.70

18664_at AT2G17230Phosphate-responsive1family protein similar to phi-1

(phosphate-induced gene)[Nicotiana tabacum]

1.64

18876_at AT2G39980Transferase family protein 1.62

12802_at AT4G36040DNAJ heat shock N-terminal domain-containing protein(J11) 1.48

13488_at AT4G20070Peptidase M20/M25/M40family protein 1.46

16524_at AT1G54100Aldehyde dehydrogenase 1.44

19762_at AT4G21680Proton-dependent oligopeptide transport(POT)family protein 1.42

13514_s_at AT4G18210Purine permease family protein 1.41

20384_at AT4G36010Pathogenesis-related thaumatin family protein 1.39

19843_at AT1G61890MATE ef?ux family protein 1.26

13134_s_at AT2G47180Galactinol synthase 1.23

17388_at 1.21

16173_s_at AT4G37430Cytochrome P45081F1(CYP81F1)(CYP91A2) 1.21

12521_at AT3G51860Cation exchanger 1.20

19008_s_at AT2G28470Beta-galactosidase 1.16

20210_g_at AT4G39400Brassinosteroid insensitive1(BRI1) 1.12

16301_at AT4G22920Expressed protein 1.09

17464_at AT1G09970Leucine-rich repeat transmembrane protein kinase 1.08

12966_s_at AT4G34590bZIP transcription factor family protein 1.07

13099_s_at AT1G22710Sucrose transporter/sucrose-proton symporter(SUC2)0.97

17929_s_at AT5G57050Protein phosphatase2C ABI2/PP2C ABI2/abscisic acid-insensitive2(ABI2)0.93

19227_at AT4G13830DNAJ heat shock N-terminal domain-containing protein(J20)0.88

14659_s_at AT4G39090Cysteine proteinase RD19a(RD19A)0.86

A?ymetrix probe set identi?cation code(Probe ID),gene locus(Locus identi?er),functional annotation(Annotation)from TAIR (https://www.sodocs.net/doc/f12997708.html,)and log2-fold change in expression levels of TSA over untreated(log2-fold change)are listed.

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distributed together(group c)and six genes are distributed outside of the main area(groups a,b, and d).Four genes in groups a and b had high values for CAN2and two genes in group d had high CAN1values.The group d genes had very high expression levels in both TSA and untreated

Table3.Genes down-regulated by TSA.

Probe ID Locus

identi?er Annotation log2-fold

change

18571_at AT1G52070Jacalin lectin family protein similar to myrosinase-binding protein homolog)3.89 14076_at AT2G20520Fasciclin-like arabinogalactan-protein(FLA6))3.51 16150_at AT4G12480Protease inhibitor/seed storage/lipid transfer protein(LTP)family protein identical to

pEARLI1(Accession No.L43080)

)3.09

20367_s_at AT1G30870Cationic peroxidase)2.93 15985_at AT5G64100Peroxidase)2.61 20448_at AT4G00680Actin-depolymerizing factor)2.60 17832_at AT2G16060Nonsymbiotic hemoglobin1(HB1)(GLB1))2.51 16636_at AT5G44110ABC transporter family protein)2.32 16253_at AT2G17850Senescence-associated family protein contains similarity to ketoconazole resistant protein)2.21 17332_s_at AT4G22080;

AT4G22090

Pectate lyase family protein similar to pectate lyase2)2.20

19631_at AT2G46860Inorganic pyrophosphatase)2.18 14039_at AT2G195901-aminocyclopropane-1-carboxylate oxidase)2.11 15357_at AT4G33560Expressed protein)2.09 17953_at AT4G01480Inorganic pyrophosphatase)1.95 18970_at AT4G25790Allergen V5/Tpx-1-related family protein)1.74 19495_at AT5G19790;

AT5G19780

AP2domain-containing protein RAP2.11(RAP2.11))1.70

18998_at AT1G62440Leucine-rich repeat family protein/extensin family protein similar to extensin-like protein

[Lycopersicon esculentum]

)1.57

16014_at AT1G75750Gibberellin-regulated protein1(GASA1))1.57 13529_at AT2G25060Plastocyanin-like domain-containing protein)1.53 14031_at AT2G41970Protein kinase,putative similar to Pto kinase interactor1)1.41 15021_at AT4G25220Similar to glycerol-3-phosphate transporter(glycerol3-phosphate permease)[Homo sapiens])1.33 12472_s_at AT1G59960Aldo/keto reductase)1.12 17994_r_at AT3G01390Vacuolar ATP synthase subunit G1(VATG1))1.10 12330_at AT2G34080Cysteine proteinase)1.05 12352_at AT2G23630Multi-copper oxidase type I family protein)1.01 13680_at AT1G55020Lipoxygenase(LOX1))0.93 19999_s_at AT2G46490Expressed protein(APS2)identical to cDNA Aps2)0.90

A?ymetrix probe set identi?cation code(Probe ID),gene locus(Locus Identi?er),functional annotation(Annotation)from TAIR (https://www.sodocs.net/doc/f12997708.html,)and log2-fold change in expression levels of TSA over untreated are listed(log2-fold change)are listed.

Table4.Genes selected using canonical variate analysis.

Gene function Probe ID Locus Group Untreated TSA treated Ratio TSA/untreated rd29B17407_s_at AT5G52300a212.4511268.852.86

rab1816038_s_at AT5G66400b105029409.328.16

LEA18872_at AT3G17520b466.912882.7526.57

AtEm117282_s_at AT3G51810b156.953399.821.39

LEA20641_at AT3G15670c2342.732009.514.33

LEA19918_at AT1G52690c3292.9541896.211.75

LEA19152_at AT5G06760c3188.0534208.1510.03

hsp7013284_at AT3G12580c1706.3515981.19.34

The predicted function of the protein encoded by the gene(gene function),A?ymetrix probe set identi?cation code(probe ID),gene locus(locus)are listed.The group label from Figure1is indicated(group).Averaged gene expression signal values from the Affymetrix Arabidopsis Genome array for seeds three DAI in the presence of TSA(TSA treated)absence of TSA(untreated)and the ratio of the TSA treated signal divided by the untreated signal(ratio TSA/untreated)are presented as the mean of two replicate experiments.

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treatments,but did not show high TSA/untreated ratios and were not included in further analyses.Validation of the data was done using RTqPCR for the four genes in groups a and b distributed along CAN2.The four genes encode rab18,rd29B,AtEm1,and a LEA protein (Table 4).Four other genes within group c were selected,three of these encode LEA proteins and one encodes hsp 70(Table 4).All of the selected genes are among the genes showing up-regulation in the presence of TSA (Table 2).Gene expression was monitored in seeds one,three and six DAI in the presence and absence of TSA (Figure 2).Decreased expression of the eight selected genes during seed germination

All eight genes show exponential decreases in gene expression upon seed imbibition,which is plotted on a logarithmic axis in Figure 2.The genes show rapid decrease in transcript levels from seed imbibition to three DAI,when expression is near basal levels.By three DAI transcript levels are at less than 10%of dry seed levels.Steady-state basal transcript levels are reached by three DAI for the ATEM1gene (At3g51810)and for the other genes,transcript levels continue to decrease,but at a slower rate after 1DAI.Expression of the genes at three and six DAI is higher in the presence of TSA,but TSA does not a?ect onset of the decrease in

expression and it has no e?ect on gene expression at one DAI.

The up-stream regions of the eight genes were analyzed for conserved DNA regulatory motifs and all the genes except HSP70show the presence of the abscisic acid response element (ABRE),a cis-acting element that confers abscisic acid (ABA)inducible expression (Busk and Pages,1998;Rock,2000;Finkelstein et al.,2002)(Figure 3).Dynamic histone acetylation during seed imbibition An analysis of the acetylation and deacetylation of the eight genes during seed imbibition and seedling development was done using ChIP with the anti-acetylated H4antibody followed by quantitative PCR to quantify the amount DNA precipitated (ChIP/qPCR).Promoter regions targeted for ChIP analysis included the ?rst ABRE in all the genes except for HSP70,which did not have an ABRE.The coding regions targeted for analysis were selected from a single exon.The results in Figure 4show that acetylation of both the promoter and coding regions of the eight genes transiently decreases at one DAI and recovers by three DAI.In addition,acetylation levels of coding regions are either the same or higher than promoter regions.

The effects of TSA on histone H4acetylation during seed germination were also examined.Seeds were imbibed in the presence of TSA and used for ChIP/qPCR assays.Figure 4shows that the deacetylation event at one DAI is blocked by TSA for the eight genes.TSA blocks deacetylation in both promoter and coding regions,similarly.The UBQ 11gene expression levels are un-changed under a number of developmental and environmental conditions (Sun and Callis,1997)and it is used as an expression control gene on the Affymetrix gene chip.The gene chip data shows that UBQ 11gene did not undergo signi?cant changes in gene expression in the presence of TSA (Supplementary material Table 1).We also exam-ined the acetylation of the UBQ 11gene in both the promoter and coding regions.Acetylation of UBQ 11follows the same pattern as the other genes in the absence of TSA and is decreased at one DAI.In the presence of TSA,the acetylation of the coding region is blocked as observed for the other genes;however,the promoter region of UBQ 11is not a?ected by

TSA.

Distributions of genes based on scores of the ?rst

(CAN1)and second (CAN2)canonical scores.Groups of genes with similar scores are circled and labeled a–d.

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Figure2.TSA affects gene repression during germination.RTqPCR was used to monitor gene expression of the8genes identi?ed from the microarray analysis.Samples were taken at zero,one,three,and six DAI in the presence(dashed line)and absence(solid line)of TSA.qPCR was done in triplicate for each time point and relative expression values presented are the averages for each sample.Error bars represent standard deviation for the triplicate readings.The y-axis indicates relative expression values and the x-axis indicates the number of days after imbibition.

Histone deacetylase activity during seed imbibition HDAC activity was examined in the presence and absence of TSA during seed imbibition (Figure 5).Seeds were imbibed on media for 6h,1d,and 3d.In the in vivo TSA treatment,seeds were imbibed on media containing 10l M TSA;hence dry seeds at 0DAI could not be treated with TSA in vivo .Lysates from imbibed seeds were prepared and assayed for HDAC activity.In the absence of TSA,HDAC activity decreases from slightly from dry seed to 6h following imbibition then sharply increases four fold at one DAI in untreated imbibed seeds.HDAC activity levels fall again at three DAI to levels that are approximately the same as dry seeds.The timing of the rise in HDAC activity at one DAI coincides with the decrease in histone H4acetylation of the eight genes observed in Figure 4.

The in vivo TSA treatment resulted in a partial inhibition of HDAC activity.Decreased HDAC activity was detected at one DAI and three DAI in TSA treated over untreated sam-ples with the largest inhibition (35%)induced one DAI.In vivo TSA treatment was compared with in vitro TSA treatment to check if the application of TSA in vivo was e?ective.In the in vitro TSA treatment,TSA was added to soluble lysates from seeds imbibed in the absence of TSA.By adding TSA to a soluble lysate,TSA does not have to penetrate plant tissues before binding to HDAC.The data in Figure 6shows

that TSA inhibits the histone deacetylase activity similarly with in vitro or in vivo TSA treatments indicating that treatment of live plants with TSA is e?ective.

TSA does not a?ect gene expression when added at three DAI

The transient deacetylation of the eight genes and the rise in HDAC activity at one DAI indicated that this may be a critical time point for HDAC regulation of gene expression.In addition,Fig-ure 5demonstrates that TSA can block the de-acetylation event at one DAI.We asked whether TSA would still be effective in up-regulating gene expression if it was added at three DAI instead 0DAI.To address this question,dry seeds were initially imbibed on media without TSA for 3days followed by transfer to TSA-containing media for an additional 3days (3d–3d TSA,Figure 6A).The expression of the eight genes was examined in this sample and compared to control samples imbibed on media in the presence (6d TSA)or absence (6d untreated)of TSA for 6days.We also asked whether up-regulation of gene expression in the presence of TSA is reversed after TSA is removed.For this experiment,dry seeds were imbibed on TSA-containing media for 3days,then transferred to media without TSA for an additional 3days and gene expression was examined (3d TSA–3d,Figure 6A).

Figure 6B shows that gene expression is in-creased in seeds imbibed on TSA-containing media continuously for 6days (6d TSA compared with 6d untreated).When TSA is added at three DAI (3d–3d TSA),expression of the eight genes is not increased,as the relative expression levels are the same as untreated imbibed seeds.Furthermore,withdrawal of TSA at three DAI (3d TSA–3d)returns expression levels for the eight genes 3days later to levels that are the same as untreated imbibed seeds.

Discussion

In our study we used TSA,an inhibitor of HDAC,to investigate the role of HDAC in controlling gene repression during seed germina-tion.Gene chip analysis indicated that 45genes are up-regulated in the presence of TSA and

27

Figure 3.Genes up-regulated by TSA share upstream ABRE.The ?gure shows a schematic diagram of the upstream region of each of the genes from )500to 0.The boxes indicate the locations of the abscisic acid response element (ABRE).The gene locus and predicted gene function are indicated on the right.

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genes are down-regulated(Tables2and3). Among the genes with the highest up-regulation in the presence of TSA are genes highly expressed in dry,mature seeds and repressed during seed germination.These genes include LEA proteins, 12S seed storage proteins,hsp70,and dehydrin (Galau and Hughes,1987;Hughes and Galau, 1991;Parcy et al.,1994;Rouse et al.,1996;Gallardo et al.,2001).The high expression of genes associated with dry,mature seeds in the presence of TSA correlates with the TSA-induced 1-day delay in germination observed(data not shown).However,expansin was also among the top TSA-up-regulated genes,but is associated with germination(Chen and Bradford,2000) indicating that not all the genes that are up-

Figure4.ChIP of promoter and coding regions show dynamic histone acetylation during seed germination.DNA associated with acetylated histone H4is immunoprecipitated with the anti-acetylated H4antibody and primers speci?c promoter or coding regions for each gene are used to amplify DNA for quantitative PCR(ChIP/qPCR).The y-axis values are the relative quantities of DNA template.x-axis labels indicate the following:seeds at6h of imbibition(6h),seeds one DAI(1d)and seeds three DAI(3d).The black bars are the amount of ChIP DNA from seeds that were imbibed in the absence of TSA and the gray bars are the amount of ChIP DNA from seeds that were imbibed in the presence of TSA.The DNA from seeds three DAI in the presence of TSA was used as for a control precipitation in the absence of antibody(No Ab).The locus and predicted function for each gene is located at the top of each pair of graphs.ChIP/qPCR data for the promoter region of each gene is in the graph on the left(promoter)and data for the coding region is in the graph on the right(coding).The average of amount of DNA for three replicates is presented and the error bars represent standard deviation.

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regulated by TSA are associated with expression in the dry,mature seed.

The TSA down-regulated genes include those encoding ACC oxidase,gibberellin-regulated pro-tein 1,extensin-like protein and peroxidases which are increased in expression during seed germina-tion (Aubert et al.,1998;Dubreucq et al.,2000;Petruzzelli et al.,2000;Bellani et al.,2002;Moroh-ashi,2002).Other genes encode proteins that are associated with growth such as actin-depolymer-izing factor (Chen et al.,2002).The association of TSA-down-regulated genes with germination and growth corresponds to the delay in germination and development with TSA treatment.

It was desirable to examine the expression of a selected number of genes up-regulated by TSA in more detail.To select genes,canonical variate analysis was used to identify genes showing highly signi?cant increases in expression in the presence of TSA.Four genes,RD29B ,RAB18,ATEM1and AT3G17520were selected.RAB18and RD29B are ABA responsive genes that are induced by desic-cation and cold temperature (Lang,1992,#291;Yamaguchi-Shinozaki,1993,#290),and ATEM1and AT3G17520encode LEA proteins.Previous studies by others have shown that RAB18and ATEM1are induced during embryo development and accumulate to high levels in the dry,mature seed (Gaubier et al.,1993;Parcy et al .,1994;Vicient et al.,2000).LEA proteins,in general,are highly expressed during late seed maturation and repressed during germination (Galau and

Hughes,1987;Hughes and Galau,1991)and are thought to be involved in maintaining protein structure in the during periods of dehydration (Wise,2003;Goyal et al.,2005).Our study focuses on genes showing repression during seed germina-tion,hence three other genes encoding LEA proteins (AT3G15670,AT1G52690,and AT5G06760)were selected for analysis.HSP70,which encodes a heat-shock protein that is repressed during seed germination (Sung et al.,2001),was also included in our study.The transcript levels of the eight genes were examined using RTqPCR to con?rm TSA effects on gene expression and to expand the analysis of gene expression over a time course.All eight genes were repressed exponentially during seed germination and TSA was found to inhibit repression for all genes examined.

RD29B ,RAB18,ATEM1,AT3G17520,AT3G15670,AT1G52690,and AT5G06760con-tain an ABRE in the upstream region indicating that ABA is involved in the induction of gene expression for these genes (Giraudat et al.,1992;Busk and Pages,1998).ABA plays an important role in embryo development,seed maturation and dormancy,and responses to environmental stres-ses such as cold,salt,and desiccation (Busk and Pages,1998;Finkelstein et al .,2002).Nakabayashi et al .(2005)also identi?ed many ABRE contain-ing genes strongly repressed upon imbibition including the eight genes from this study and shows that the ABRE is the most prominent upstream element in the dry seed transcriptome.The strong repression of late maturation genes during seed germination indicates a diversion of metabolic resources away from desiccation toler-ance and towards seedling growth.A subset of genes were found to be located in clusters and co-regulated during seed imbibition (Nakabayashi et al .,2005).We did not ?nd any evidence for gene clusters among the 72genes affected by TSA.In addition,the gene chip data indicates that the genes adjacent to the eight selected genes are not coordinately expressed.Therefore,the evidence indicates that TSA does not target localized regions in the chromosome.

The 72genes differentially expressed in the presence of TSA from this study were not affected by TSA in another study with Arabidopsis thaliana (Chang and Pikaard,2005).Many late maturation genes were up-regulated by TSA in our study,

but

Figure 5.HDAC activity increases at one DAI and inhibited by TSA.HDAC activity was measured using an assay with an acetylated substrate that absorbs A 405when deacetylated.HDAC activity was measure at 0,6h,1day and 3days after imbibition in the presence and absence of either in vivo or in vitro applied TSA.The bars represent HDAC activity mea-sured by the amount of product formed at A 405/mg protein.Values are the mean of six replicates and the error bars repre-sent the standard deviation.

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Chang and Pikaard (Chang and Pikaard,2005)did not ?nd up-regulation of this group of genes by TSA.A key difference between the two studies is the developmental stage of the plants –16-day-old seedlings as opposed to germinated seeds at 3DAI examined in this study.In addition,a lower concentration of TSA was used in the study by Chang and Pikaard (Chang and Pikaard,2005)(0.5l M),which was based on the concentration known to derepress silenced rRNA genes in Arabidopsis thaliana.Ten micromoles of TSA was used in this study since it was the

concentra-

Figure 6.Effects of TSA removal and addition after 3days.(A)TSA treatment of imbibing seeds was as follows:seeds imbibed for 6days on media without TSA (6d untreated),seeds imbibed for 6days on 10l M TSA containing media (6d TSA),seeds im-bibed for 3days on media without TSA then transferred to media containing 10l M TSA for 3days (3d–3d TSA),seeds imbibed for 3days on media containing10l M TSA then removed to media without TSA for 3days (3d TSA–3d).(B)RTqPCR was used to measure relative gene expression levels in seeds six DAI.The seeds were imbibed and treated as in ‘A’and relative expression for each treatment are presented as follows:6d untreated (dark gray bars),6d TSA (light gray bars),3d–3d TSA (white bars),and 3d TSA–3d (black bars).The locus and predicted protein function are indicated on the y -axis.The relative expression values repre-sent the average over three replicates and the errors bars represent the standard deviation.

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tion found to induce a delay in germination and development of Arabidopsis thaliana seedlings (data not shown).Genes a?ected by TSA in a study in tobacco(Chua et al.,2004)were also different from those identi?ed in Arabidopsis thaliana from our study and by Chang and Pikaard(Chang and Pikaard,2005);however,a custom array with88genes was used in the tobacco study that was missing many of the genes that were identi?ed as affected by TSA in Arabid-opsis thaliana.

Promoter-targeted acetylation and deacetyla-tion involve localized changes in histone acetyla-tion at promoter regions and are associated with increased and decreased gene expression,respec-tively(Struhl,1998;Kurdistani and Grunstein, 2003).The promoter regions of the eight genes and the control gene,UBQ11,are deacetylated at one DAI.TSA blocks the deacetylation of promoter regions for all eight genes but not UBQ11,whose expression is also not affected by TSA.These results indicate that TSA affects promoter acety-lation for genes whose expression is also affected by TSA.However,the timing of TSA effects on promoter acetylation and gene expression is not coordinated.TSA blocks deacetylation at one DAI,but the effect of TSA on gene expression is not observed until three DAI.One explanation is that transcript levels observed in the dry seed and at one DAI are due to high levels of stored mRNA (Nakabayashi et al.,2005).Therefore,TSA effects on de novo transcription will not be apparent until stored transcripts are further degraded at three DAI.Although,HDAC-mediated promoter-tar-geted deacetylation is a well characterized,HDAC may have other roles during seed germination.

Histone acetylation,is not limited to promoter regions and occurs globally throughout the genome(Krebs et al.,2000;Kuo et al.,2000; Vogelauer et al.,2000).The function of global acetylation is not as well understood as promoter-targeted acetylation.Studies in yeast show that global acetylation is involved in the regulation of basal transcription of the PHO5gene(Vogelauer et al.,2000).Global acetylation and deacetylation is also involved in the restoration of acetylation to steady-state levels after the removal of transcrip-tional activators or repressors(Katan-Khaykovich and Struhl,2002).The results presented here show that histone acetylation changes in the eight genes studied are not restricted to promoter regions indicating that HDAC effects are not limited to promoter-targeted events.In addition,studies in tobacco also demonstrate that TSA affects acety-lation at both upstream and coding regions(Chua et al.,2004).Therefore,HDAC may function during seed germination to control global histone acetylation levels and histone acetylation may be involved in restoring transcript levels after the removal of transcriptional activators such as ABI3 and ABI5,which are known to decrease after seed imbibition(Parcy et al.,1994;Lopez-Molina et al.,2001).

The effect of TSA on histone acetylation is greatest at one DAI where it is observed to block a deacetylation event.TSA is also observed to increase acetylation levels at three DAI for some genes.Histone acetylation changes are also asso-ciated with the cell cycle(Waterborg and Matthews,1983;Georgieva et al.,1991;Belyaev et al.,1997,1998;Lusser et al.,1997;Jasencakova et al.,2000,2001;Vogelauer et al.,2002;Wako et al.,2002l;Cimini et al.,2003;Li et al.,2005). During seed imbibition the cell cycle is reactivated after a period of arrest(Bewley and Black,1985; de Castro et al.,2000)and the observed dynamic histone acetylation during seed germination may be associated with progression of the cell cycle.In addition,TSA inhibits cell cycle progression in tobacco protoplasts and pea meristems(Murphy et al.,2000;Li et al.,2005),which correlates with our observation that TSA delays seed germination by1day.Hence,the deacetylation of the eight late maturation genes may be associated with a global deacetylation event during mitosis and the TSA effect on repression of the eight genes is associated with cell cycle inhibition that induces a develop-mental delay.

ChIP and HDAC assays demonstrate the occurrence of a transient deacetylation event during seed germination at one DAI,which can be blocked by TSA resulting in reduced repres-sion of the eight genes studied at three and six DAI.Removal of TSA at three DAI restores expression to the same level as untreated seeds, indicating that TSA effects on are reversible.TSA was also added at three DAI after the deacety-lation event,and was not found to affect the expression of the eight genes.Therefore,deacet-ylation at one DAI is a critical event in the regulation of the eight genes and may be a key process for seed germination.

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Acknowledgements

This work was supported by a grant from the Canadian Biotechnology Strategy,Regulatory Fund.The authors would like to thank Kathleen Forbes for her technical assistance and Dr.Xianzhou Nie for critically reviewing the manuscript.

References

Ahringer,J.2000.NuRD and SIN3histone deacetylase complexes in development.Trends Genet.16:351–356. Allfrey,V.G.,Faulkner,R.and Mirsky,A.E.1964.Acetylation and methylation of histones and their possible role in the regulation of RNA https://www.sodocs.net/doc/f12997708.html,A51: 786–794.

Aubert,D.,Chevillard,M.,Dorne,A.-M.,Arlaud,,G.r.and Herzog,M.1998.Expression patterns of GASA genes in Arabidopsis thaliana:the GASA4gene is up-regulated by gibberellins in meristematic regions.Plant Mol.Biol.36: 871–883.

Bartlett,M.S.1947.The general canonical correlation distri-bution.Ann.Math.Stat.18:1–17.

Bellani,L.M.,Guarnieri,M.and Scialabba,A.2002.Differ-ences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds.Physiol.Plant.114:102–108.

Belyaev,N.D.,Houben,A.,Baranczewski,P.and Schubert,I.

1997.Histone H4acetylation in plant heterochromatin is altered during the cell cycle.Chromosoma106:193–197. Belyaev,N.D.,Houben,A.,Baranczewski,P.and Schubert,I.

1998.The acetylation patterns of histones H3and H4along Vicia faba chromosomes are di?erent.Chromosome Res.6: 59–63.

Bewley,J.D.and Black,M.1985.Seeds Physiology of Development and Germination.Plenum Press,New York. Brosch,G.,Ransom,R.,Lechner,T.,Walton,J.D.and Loidl, P.1995.Inhibition of maize histone deacetylases by HC toxin,the host-selective toxin of Cochliobolus carbonum.

Plant Cell7:1941–1950.

Busk,P.K.and Pages,M.1998.Regulation of abscisic acid-induced transcription.Plant.Mol.Biol.37:425–435. Chang,S.and Pikaard, C.S.2005.Transcript pro?ling in Arabidopsis reveals complex responses to global inhibition of DNA methylation and histone deacetylation.J.Biol.

Chem.280:796–804.

Chen,C.Y.,Wong,E.I.,Vidali,L.,Estavillo,A.,Hepler,P.K., Wu,,H.-m.,and Cheung,A.Y.2002.The Regulation of actin organization by actin-depolymerizing factor in elon-gating pollen tubes.Plant Cell14:2175–2190.

Chen,F.and Bradford,K.J.2000.Expression of an expansin is associated with endosperm weakening during tomato seed germination.Plant Physiol.124:1265–1274.

Chen,Z.J.and Pikaard,C.S.1997.Epigenetic silencing of RNA polymerase I transcription:a role for DNA methylation and histone modi?cation in nucleolar dominance.Genes Dev.11: 2124–2136.

Chua,Y.L.,Brown,A.P.C.and Gray,J.C.2001.Targeted histone acetylation and altered nuclease accessibility over

short regions of the pea plastocyanin gene.Plant Cell13: 599–612.

Chua,Y.L.,Mott,E.,Brown,A.P.C.,MacLean,D.and Gray, J.C.2004.Microarray analysis of chromatin-immunoprecip-itated DNA identi?es speci?c regions of tobacco genes associated with acetylated histones.Plant J37:789–800. Chua,Y.L.,Watson,L.A.and Gray,J.C.2003.The transcrip-tional enhancer of the pea plastocyanin gene associates with the nuclear matrix and regulates gene expression through histone acetylation.Plant Cell15:1468–1479.

Cimini,D.,Mattiuzzo,M.,Torosantucci,L.and Degrassi,F.

2003.Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects.Mol.Biol.Cell14:3821–3833.

Comai,L.and Harada,J.1990.Transcriptional activities in dry seed nuclei indicate the timing of the transition from embryogeny to germination.PNAS87:2671–2674.

de Castro,R.D.,van Lammeren, A.A.M.,Groot,S.P.C., Bino,R.J.and Hilhorst,H.W.M.2000.Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not.Plant Physiol.122:327–336.

Dubreucq,B.,Berger,N.,Vincent,E.,Boisson,M.,Pelletier,

G.,Caboche,M.and Lepiniec,L.2000.The Arabidopsis

AtEPR1extensin-like gene is speci?cally expressed in endo-sperm during seed germination.Plant J.23:643–652. Finkelstein,R.R.,Gampala,S.S.L.and Rock, C.D.2002.

Abscisic Acid signaling in seeds and seedlings.Plant Cell14: S15–S45.

Galau,G.A.and Hughes,D.W.1987.Coordinate accumulation of homeologous transcripts of seven cotton Lea gene families during embryogenesis and germination.Dev.Biol.123: 213–221.

Gallardo,K.,Job,C.,Groot,S.P.C.,Puype,M.,Demol,H., Vandekerckhove,J.and Job,D.2001.Proteomic analysis of Arabidopsis seed germination and priming.Plant Physiol.

126:835–848.

Gaubier,P.,Raynal,M.,Hull,G.,Huestis,G.M.,Grellet,F., Arenas,C.,Pages,M.and Delseny,M.1993.Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation.Mol.Gen.Genet.238:409–418. Gautier,L.,Cope,L.,Bolstad,B.M.and Irizarry,R.A.2004.

affy–Analysis of affymetrix GeneChip data at the probe level.Bioinformatics20:307–315.

Georgieva,E.I.,Lopez-Rodas,G.,Sendra,R.,Grobner,P.and Loidl,P.1991.Histone acetylation in Zea mays.II.Biological signi?cance of post-translational histone acetylation during embryo germination.J.Biol.Chem.266:18751–18760. Giraudat,J.,Hauge,B.M.,Valon,C.,Smalle,J.,Parcy,F.and Goodman,H.M.1992.Isolation of the Arabidopsis ABI3 gene by positional cloning.Plant Cell4:1251–1261. Goyal,K.,Walton,L.J.and Tunnacliffe, A.2005.LEA proteins prevent protein aggregation due to water stress.

Biochem.J.388:151–157.

He,Y.,Michaels,S.D.and Amasino,R.M.2003.Regulation of ?owering time by histone acetylation in Arabidopsis.Science 302:1751–1754.

Hebbes,T.,Thorne,A.and Crane-Robinson,C.1988.A direct link between core histone acetylation and transcriptionally active chromatin.EMBO J.7:1395–1402.

Henderson,J.T.,Li,H.-C.,Rider,S.D.,Mordhorst, A.P., Romero-Severson,J.,Cheng,J.-C.,Robey,J.,Sung,Z.R.,de Vries,S.C.and Ogas,J.2004.PICKLE acts throughout the

923

plant to repress expression of embryonic traits and may play

a role in gibberellin-dependent responses.Plant Physiol.134:

995–1005.

Hughes, D.W.and Galau,G.A.1991.Developmental and environmental induction of Lea and LeaA mRNAs and the postabscission program during embryo culture.Plant Cell3: 605–618.

Imai,S.,Armstrong,C.M.,Kaeberlein,M.and Guarente,L.

2000.Transcriptional silencing and longevity protein Sir2is an NAD-dependent histone deacetylase.Nature403: 795–800.

Irizarry,R.A.,Bolstad,B.M.,Collin,F.,Cope,L.M.,Hobbs,

B.and Speed,T.P.2003.Summaries of affymetrix GeneChip

probe level data.Nucleic Acids Res.31:15.

Jang,I.C.,Pahk,Y.M.,Song,S.I.,Kwon,H.J.,Nahm,B.H.

and Kim,J.K.2003.Structure and expression of the rice class-I type histone deacetylase genes OsHDAC1-3:OsH-DAC1overexpression in transgenic plants leads to in-creased growth rate and altered architecture.Plant J.33: 531–541.

Jasencakova,Z.,Meister,A.and Schubert,I.2001.Chromatin organization and its relation to replication and histone acetylation during the cell cycle in barley.Chromosoma110: 83–92.

Jasencakova,Z.,Meister,A.,Walter,J.,Turner,B.M.and Schubert,I.2000.Histone H4acetylation of euchromatin and heterochromatin is cell cycle dependent and correlated with replication rather than with transcription.Plant Cell12: 2087–2100.

Jenuwein,T.and Allis,C.D.2001.Translating the histone code.

Science293:1074–1080.

Johnson,L.M.,Cao,X.and Jacobsen,S.E.2002.Interplay between two epigenetic marks:DNA methylation and histone H3lysine9methylation.Curr.Biol.12:1360–1367. Kadosh,D.and Struhl,K.1998a.Histone deacetylase activity of Rpd3is important for transcriptional repression in vivo.

Genes Dev.12:797–805.

Kadosh,D.and Struhl,K.1998b.Targeted recruitment of the Sin3-Rpd3histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo.Mol.Cell.

Biol.18:5121–5127.

Katan-Khaykovich,Y.and Struhl,K.2002.Dynamics of global histone acetylation and deacetylation in vivo:rapid restoration of normal histone acetylation status upon removal of activa-tors and repressors.Genes Dev.16:743–752.

Krebs,J.E.,Fry,C.J.,Samuels,M.L.and Peterson,C.L.2000.

Global role for chromatin remodeling enzymes in mitotic gene expression.Cell102:587–598.

Kuo,M.-H.,vom Baur,E.,Struhl,K.and Allis,C.D.2000.

Gcn4activator targets Gcn5histone acetyltransferase to speci?c promoters independently of transcription.Mol.Cell 6:1309–1320.

Kuo,M.-H.,Zhou,J.,Jambeck,P.,Churchill,M.E.A.and Allis,C.D.1998.Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo.

Genes Dev.12:627–639.

Kurdistani,S.K.and Grunstein,M.2003.Histone acetylation and deacetylation in yeast.Nat.Rev.Mol.Cell Biol.4: 276–284.

Lawrence,R.J.,Earley,K.,Pontes,O.,Silva,M.,Chen,Z.J., Neves,N.,Viegas,W.and Pikaard,C.S.2004.A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance.Mol.

Cell13:599–609.Lechner,T.,Lusser,A.,Brosch,G.,Eberharter,A.,Goralik-Schramel,M.and Loidl,P.1996.A comparative study of histone deacetylases of plant,fungal and vertebrate cells.

Biochim.Biophys.Acta1296:181–188.

Lescot,M.,Dehais,P.,Thijs,G.,Marchal,K.,Moreau,Y., Van de Peer,Y.,Rouze,P.and Rombauts,S.2002.

PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences.Nucleic Acids Res.30:325–327.

Li,Y.,Butenko,Y.and Gra?,G.2005.Histone deacetylation is required for progression through mitosis in tobacco cells.

Plant J.41:346–352.

Loidl,P.2004.A plant dialect of the histone language.Trends Plant Sci.9:84–90.

Lopez-Molina,L.,Mongrand,S.and Chua,N.-H.2001.A postgermination developmental arrest checkpoint is medi-ated by abscisic acid and requires the ABI5transcription factor in Arabidopsis.PNAS98:4782–4787.

Lusser,A.,Brosch,G.,Lopez-Rodas,G.and Loidl,P.1997.

Histone acetyltransferases during the cell cycle and differenti-ation of Physarum polycephalum.Eur.J.Cell Biol.74:102–110. Morohashi,Y.2002.Peroxidase activity develops in the micropylar endosperm of tomato seeds prior to radicle protrusion.J.Exp.Bot.53:1643–1650.

Murashige,T.and Skoog,F.1962.A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol.

Plant15:473–497.

Murphy,J.P.,McAleer,J.P.,Uglialoro,A.,Papile,J.,Weniger, J.,Bethelmie, F.and Tramontano,W.A.2000.Histone deacetylase inhibitors and cell proliferation in pea root meristems.Phytochemistry55:11–18.

Nakabayashi,K.,Okamoto,M.,Koshiba,T.,Kamiya,Y.and Nambara,E.2005.Genome-wide pro?ling of stored mRNA in Arabidopsis thaliana seed germination:epigenetic and genetic regulation of transcription in seed.Plant J.41:697–709. Nambara, E.,Hayama,R.,Tsuchiya,Y.,Nishimura,M., Kawaide,H.,Kamiya,Y.and Naito,S.2000.The role of ABI3and FUS3loci in Arabidopsis thaliana on phase transition from late embryo development to germination.

Dev.Biol.220:412–423.

Ogas,J.,Cheng,J.-C.,Sung,Z.R.and Somerville,C.1997.

Cellular differentiation regulated by gibberellin in the Arabidopsis thaliana pickle mutant.Science277:91–94. Ogas,J.,Kaufmann,S.,Henderson,J.and Somerville,C.1999.

PICKLE is a CHD3chromatin-remodeling factor that regulates the transition from embryonic to vegetative devel-opment in https://www.sodocs.net/doc/f12997708.html,A96: 13839–13844.

Pandey,R.,Muller,A.,Napoli,C.A.,Selinger,D.A.,Pikaard,

C.S.,Richards,E.J.,Bender,J.,Mount,

D.W.and Jorgen-

sen,R.A.2002.Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversi?cation of chromatin modi?cation among multicellular eukaryotes.Nucleic Acids Res.30:5036–5055. Parcy, F.,Valon, C.,Raynal,M.,Gaubier-Comella,P., Delseny,M.and Giraudat,J.1994.Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3locus and of endogenous abscisic acid.

Plant Cell6:1567–1582.

Petruzzelli,L.,Coraggio,I.and Leubner-Metzger,G.2000.

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of1-amino-cyclo-propane-1-carboxylic acid oxidase.Planta211: 144–149.

924

Richon,V.M.,Sandhoff,T.W.,Rifkind,R.A.and Marks,P.A.

2000.Histone deacetylase inhibitor selectively induces p21WAF1expression and gene-associated histone acetyla-tion.PNAS97:10014–10019.

Rider,D.S.,,Jr.,Henderson,J.T.,Jerome,R.E.,Edenberg,

H.J.,Romero-Severson,J.and Ogas,J.2003.Coordinate

repression of regulators of embryonic identity by PICKLE during germination in Arabidopsis.Plant J35: 33–43.

Rock, C.D.2000.Pathways to abscisic acid-regulated gene expression.New Phytol.148:357.

Rombauts,S.,Dehais,P.,Van Montagu,M.and Rouze,P.

1999.PlantCARE,a plant cis-acting regulatory element database.Nucleic Acids Res.27:295–296.

Rouse,D.T.,Marotta,R.and Parish,R.W.1996.Promoter and expression studies on an Arabidopsis thaliana dehydrin gene.FEBS Lett.381:252–256.

Rundlett,S.E.,Carmen,A.A.,Suka,N.,Turner,B.M.and Grunstein,M.1998.Transcriptional repression by UME6 involves deacetylation of lysine5of histone H4by RPD3.

Nature392:831–835.

Smyth,G.K.2004.Linear Models and Empirical Bayesmethods for Assessing Differential Expression in Microarray Exper-iments Statistical Applications in Genetics and Molecular Biology,No.1,Article3.Berkeley Electronic Press,Berkeley, CA,pp.1–25.

Soeda,Y.,Konings,M.C.J.M.,Vorst,O.,van Houwelingen,

A.M.M.L.,Stoopen,G.M.,Maliepaard,C.A.,Kodde,J.,

Bino,R.J.,Groot,S.P.C.and van der Geest,A.H.M.2005.

Gene expression programs during Brassica oleracea seed maturation,osmopriming,and germination are indicators of progression of the germination process and the stress tolerance level.Plant Physiol.137:354–368.

Sterner,D.E.and Berger,S.L.2000.Acetylation of histones and transcription-related factors.Microbiol.Mol.Biol.Rev.

64:435–459.

Stockinger,E.J.,Mao,Y.,Regier,M.K.,Triezenberg,S.J.and Thomashow,M.F.2001.Transcriptional adaptor and his-tone acetyltransferase proteins in Arabidopsis and their interactions with CBF1,a transcriptional activator involved in cold-regulated gene expression.Nucleic Acids Res.29: 1524–1533.

Struhl,K.1998.Histone acetylation and transcriptional regu-latory mechanisms.Genes Dev12:599–606.

Sun,C.-W.and Callis,J.1997.Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in di?erent organs and in response to environmental changes.Plant J 11:1017–1027.

Sung,D.Y.,Vierling,E.and Guy,https://www.sodocs.net/doc/f12997708.html,prehensive expression pro?le analysis of the Arabidopsis Hsp70gene family.Plant Physiol.126:789–800.

Tai,H.H.,Pelletier,C.and Beardmore,T.2004.Total RNA isolation from Picea marianna dry seed.Plant Mol.Biol.

Report.22:93.

Taunton,J.,Hassig,C.A.and Schreiber,S.L.1996.A mam-malian histone deacetylase related to the yeast transcrip-tional regulator Rpd3p.Science272:408–411.

Thijs,G.,Marchal,K.,Lescot,M.,Rombauts,S.,De Moor,B., Rouze,P.and Moreau,Y.2002a.A Gibbs sampling method to detect overrepresented motifs in the upstream regions of coexpressed https://www.sodocs.net/doc/f12997708.html,put.Biol.9:447–464.Thijs,G.,Moreau,Y.,De Smet,F.,Mathys,J.,Lescot,M., Rombauts,S.,Rouze,P.,De Moor,B.and Marchal,K.

2002b.INCLUSive:INtegrated Clustering,Upstream sequence retrieval and motif Sampling.Bioinformatics18: 331–332.

Tian,L.and Chen,Z.J.2001.Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regu-lation and https://www.sodocs.net/doc/f12997708.html,A98:200–205.

Tian,L.,Fong,M.P.,Wang,J.J.,Wei,N.E.,Jiang,H.,Doerge, R.W.and Chen,Z.J.2004.Reversible histone acetylation and deacetylation mediate genome-wide,promoter-depen-dent,and locus-speci?c changes in gene expression during plant development.Genetics169:337–345.

Turner,B.M.1993.Decoding the nucleosome.Cell75:5–8. Vicient, C.M.,Hull,G.,Guilleminot,J.,Devic,M.and Delseny,M.2000.Differential expression of the Arabidopsis genes coding for Em-like proteins.J.Exp.Bot.51:1211–1220.

Vogelauer,M.,Rubbi,L.,Lucas,I.,Brewer,B.J.and Grun-stein,M.2002.Histone acetylation regulates the time of replication origin?ring.Mol.Cell10:1223–1233. Vogelauer,M.,Wu,J.,Suka,N.and Grunstein,M.2000.

Global histone acetylation and deacetylation in yeast.

Nature408:495–498.

Wako,T.,Fukuda,M.,Furushima-Shimogawara,R.,Belyaev, N.D.and Fukui,K.2002.Cell cycle-dependent and lysine residue-speci?c dynamic changes of histone H4acetylation in barley.Plant Mol.Biol.49:645–653.

Waterborg,J.H.and Matthews,H.R.1983.Patterns of histone acetylation in the cell cycle of Physarum polycephalum.

Biochemistry22:1489–1496.

Wise,M.2003.LEAping to conclusions:a computational reanalysis of late embryogenesis abundant proteins and their possible roles.BMC Bioinformat.4:52.

Wolffe,A.P.1992.Chromatin:Structure and Function.Aca-demic Press,London,England.

Wu,K.,Malik,K.,Tian,L.,Brown,D.and Miki,B.2000a.

Functional analysis of a RPD3histone deacetylase homo-logue in Arabidopsis thaliana.Plant Mol.Biol.44:167–176. Wu,K.,Tian,L.,Malik,K.,Brown,D.and Miki,B.2000b.

Functional analysis of HD2histone deacetylase homologues in Arabidopsis thaliana.Plant J.22:19–27.

Yoshida,M.,Horinouchi,S.and Beppu,T.1995.Trichostatin

A and trapoxin:novel chemical probes for the role of histone

acetylation in chromatin structure and function.Bioessays 17:423–430.

Yoshida,M.,Kijima,M.,Akita,M.and Beppu,T.1990.

Potent and speci?c inhibition of mammalian histone deacet-ylase both in vivo and in vitro by trichostatin A.J.Biol.

Chem.265:17174–17179.

Zhou,C.,Labbe,H.,Sridha,S.,Wang,L.,Tian,L.,Latoszek-Green,M.,Yang,Z.,Brown,D.,Miki,B.and Wu,K.2004.

Expression and function of HD2-type histone deacetylases in Arabidopsis development.Plant J.38:715–724.

Zhou,C.,Zhang,L.,Duan,J.,Miki,B.and Wu,K.2005.

HISTONE DEACETYLASE19is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.

Plant Cell17:1196–1204.

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4、（开头）人类不竭的欲望是推动历史车轮滚滚前进的原动力。欲望来自哪儿?来自人类生而拥有的——好奇心。（结尾）好奇心——它在推动世界进步的同时，也在积蓄着毁灭世界的力量。对待这一颗颗仍在膨胀的好奇心，我们应当何去何从?（《好奇心》） 5、（开头）为什么?两个人都上年纪了，相距不过几米的屋子，有什么必要隔几分钟就喊一下?每次去奶奶家，这件事总是会勾起我的好奇心。（结尾）我想，所谓爱，便是如此。就是我所爱的人，我惦念的人，必得在我看得见的地方，我手够得到的地方，我能够走到的地方，好好的存在着。 我庆幸我拥有好奇心，才得以知晓奶奶一辈的关心，温情与爱。我知道了，那声声呼唤是在说，有你在，整个世界，都在。自然收束朴素无华 6、生活是由苦乐、美丑交织而成的经纬网，它穿越时空而光芒犹在。需要我们不断探索和思考人生，只要我们能够在思考之中不断添加沙子、水和石头，人生就变得充实。让我们一起来做好和充实我们的人生之杯吧!（《杯中窥人生》） 7、人生，其实就是一次过程，很多事，很多人，失败过，经历过才会懂，才会成熟。当失败来临的时候，不要伤悲，而应该看作是一次成长的机会，一次锻炼的机会。冲过去，会更美好、更灿烂的生活等着你，更会有一番成就感；如果退而不前，那只能迎来更多的失败，更多人生的遗憾。当我们快要走完人生路时，回首这一生，特别是那些困难和失败时，会觉得，或许正是由于这些，丰富了我们的人生，

写人作文开头结尾大全

写人作文的开头结尾 开头： 1、爸爸长得真帅，一米八的个子，白净的脸，眼睛明亮，鼻梁挺直。就是有一点我不喜欢，他唇上、颌下、腮边总是密麻麻地长满了硬茬茬的胡子，把嘴也掩盖了。因为他工作忙，老是没时间刮。妈妈总是数落他只知道工作，不注重生活小节，爸爸总是“嘿嘿”一笑继续忙他的事。 2、爸爸今年四十一岁，一米八零的个头。他最爱穿一件深蓝色的西服，浅黑色 微微一笑 整齐洁白的牙齿，更显得可爱可敬。 3、一拍桌子，冲着我吼道：“跟你说过多少遍了？这已经是第五次了！你到底是忘了还是存心不想做？说！”“忘了……”眼泪在我的眼眶里打转。爸爸一气之下把那一页未完成的书页撕了。 4、我的妈妈今年已经39岁了。他中等身材，不胖不瘦，两道浓黑的眉毛下面长着一双炯炯有神的大眼睛。她的嘴角微微向上翘，好像总在微笑，给人一种和蔼可亲的感觉。最近我发现皱纹已经悄悄爬上了她的眼角。这些皱纹记载着妈妈的辛苦，记载着她对我无限的爱。 5、一阵咳嗽声把我从梦中惊醒，睁眼一看，啊，妈妈还没睡。我抬头看小闹钟已经11点了。我赶紧催促妈妈：“您怎么还不睡？医生不让您多注意身体吗？”妈妈回过头来笑着对我说：“你快睡吧！明天还要上学。”说完又改起作业来。 6.每当我遇到困难，每当我取得成绩，每当我心情烦躁，每当我满心欢喜时，我都想到我最爱的人——我的母亲那里倾诉我的心曲。 7.妈妈那张慈祥的脸浮现在我面前，岁月使她失去了青春的红润，生活的风霜在她脸上刻下了深深的皱纹，满头的乌发早已染上了寒霜，只有那紧紧抿着的双唇显示着坚毅、倔犟的性格。想着妈妈，我便有了信心和力量，永不放弃奋发向上。 8. 9.世间有真情，人间有真爱，而母爱就是永不凋零的花。我慢慢地介绍我的母亲吧！ 10.爷爷是我最爱的亲人，我的童年是在爷爷那边度过的。爷爷拉着我的手，教会我走路。是爷爷使我从小懂得了不少道理。我把爷爷看成自己幼年成长的拐杖。 11.奶奶的音容笑貌一直深深印在我的脑海。一幕幕回忆的画面时时温暖着我的心田。多少次梦中浮现出那张熟悉的脸，在我心中有个位置永远留给她——我的奶奶。 13.在记忆的长河里，有一块美丽的鹅卵石。当我高兴的时候，当我伤心的时候

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5、头：尘世间，事物的外表，仅凭这一点，足够断定一个人、一件事是否美与丑吗？何以见得？ 尾：可以见得的事是，一个人或一件事，是否美与丑，也许和天生具有的外表有关吧；但，一个人或一件事的美与丑更取决于内在的心性与品质吧……通过这两件事，我深信美与丑如此！ 6、头：任何一个大家，任何一件事，或许骄傲自豪来自于他们的成功；但喜悦的背后，更多的是他们，所付出的努力吧…… 尾：向往成功的我，更深信，这需要我的努力！而不是一味的徒手幻想…… 7、头：生命来之不易，正因如此，意义价值和难忘，才能给它图上绚丽多姿的色彩吧^^…… 尾：意义价值和难忘，让我的生命不再无味。我体会到了人生无味：酸甜苦辣01、站在历史的海岸漫溯那一道道历史沟渠：楚大夫沉吟泽畔，九死不悔；魏武帝扬鞭东指，壮心不已；陶渊明悠然南山，饮酒采菊……他们选择了永恒，纵然谄媚诬蔑视听，也不随其流扬其波，这是执着的选择；纵然马革裹尸，魂归狼烟，只是豪壮的选择；纵然一身清苦，终日难饱，也愿怡然自乐，躬耕陇亩，这是高雅的选择。在一番选择中，帝王将相成其盖世伟业，贤士迁客成其千古文章。 8、开头“ 每一次都在徘徊、孤单中坚强，每一次就算受伤也不闪泪光。” 今天我唱起了那首我从来不愿唱的隐形的《隐形的翅膀》。在以前，当别人唱起这首略带忧伤的歌时我总会搞破坏，专捡一些欢乐的歌曲来消减一下悲伤的气氛，因为我不想带着悲伤活在这

写事作文开头结尾大全

写事作文开头结尾大全文档编制序号：[KK8UY-LL9IO69-TTO6M3-MTOL89-

写事作文的开头结尾 开头： 1、在我脑海中有许多的事,不少已被流水般的时光冲得模糊了,可有一件事却使我怎么也忘不掉。 2、在我记忆的长河中,有高兴的事,有伤心的事,但最令我难忘的还是要算那次发生在……的事。 3.童年像一条河，朵朵浪花在朝阳照耀下充满欢乐。秋高气爽的季节，那次……真有趣。 4、往事总在我的脑海里流动，看似平静的心海总有波浪起伏，过去的日子，我忘不了，更忘不了那件令我感动的事。 5、感动如爱，时刻温暖着我的心，只要我们细细地发现，就能被感动，我就曾被这样的一件事所感动。 6、作家刘墉说：“成长史一种美丽的痛。”在我看来，这真是一句至理名言。你可能问我为什么，这还要从我记忆深处的一件事说起。 7、时间如流水一般匆匆而逝，很多的记忆随日子的消失而褪色。但有一件事，让我历历在目，感动不已。 8、在我的脑海里，有许多令我感动的事情，它们都像海边的贝壳一串串地在记忆里闪光。但其中有一件事令我最受感动。提起那件事，我至今记忆犹新。

9、我经历的事情如天上的繁星，有的让我羞愧难当，有的让我愤怒无比，有是让我体验成功，有的让我感动不已。现在我就把令我感动的一件事讲给大家听。 10、成长是缓缓流淌的溪流，悄然的，我们就长大了，学会了坚强，也学会了坦然。很多的记忆随日子的消失而褪色，但有一件事让我历历在目，感动不已。 11.成长就好比我人生中的一条小船，行驶在波面上。有时风平浪静，有时会遇上汹涌澎湃的海浪。开启我记忆的大门，那件事依然让我记忆犹新。 12.岁月不会回头，抓住今天的每一秒，把希望系于明天的彩云，成长就是希望编织的彩带，串连回忆和向往。让时间回到两年前的那一刻，那件我至今难以忘怀的事又浮现在了我的脑海里。 13.日子一叶一叶地从生命之树飘落，不经意间，才蓦然发现岁月已给它套上了十二圈年轮。而我的年轮里最深的一圈，还要从那一年那件事说起。 14、生活是一张洁白的画纸，我们每个人都描绘着自己的色彩。生活是一杯香醇的美酒，我们每个人都品尝着自己的味道。生活是漫漫长路，我们每个人都是路上的远行者。打开记忆的大门，那件事深深感动着我。 15.生命只在年轮上转了十五圈，生活已告诉了我很多、很多……在我的心里深深埋藏挥之不去的，是那一年的，发生在我生命中的那件事。 16.在我成长的历程中，发生过很多的事情，就像在沙滩上走过一样，留下了一串串的脚印。

描写人物和事情的好开头和好结尾

好开头 ?1、往事总在我的脑海里流动，似平静的心海总有波浪起伏，过去的日子，我忘不了，更忘不了那件令我感动的事。 ?2、感动如爱，时刻温暖着我的心，只要我们细细地发现，就能被感动，我就曾被这样的一件事所感动。 ?3、在生活中，有千千万万件令我们感动的事，如：鸭子妈妈为了保护自己的蛋，寸步不离，鸟儿为了自己的孩子与猛兽做殊死搏斗，在这么多想不完、记不清、数不尽的令我感动的事情中，最令我难以忘怀的，还是*****的事。 ?4、时间过得真快，转眼间，****年过去了。但是不管过去了多少年，我却不会忘记这件令我感动的事情。 ?5、人们常说：母爱是伟大的，父爱是崇高的，可是我觉得师爱比母爱更伟大，比父爱更崇高，你若不信，听我说说我和我班李老师之间发生的一件令我感动的事吧。 ?6、爷爷的疼爱令我感动，爸爸的严中带爱要我感动，妈妈的温柔令我感动，但是，最令我感动的是发生在*****的一件事。?7、从小到大，能感动我的人和事不少，但大多数印象中已经模糊了。唯独一件事还很清晰。 ?8、时间如流水一般匆匆而逝，很多的记忆随日子的消失而退色，但有一件事，让我历历在目，感动不已。 ?9、在我的脑海里，有许多令我感动的事情，它们都像海边的贝壳一串串地但其中有一件事，令我最受感动，提起那件事，我至今记忆犹新。

好结尾： ?1、日月如梭，虽然这件事已过去很久了。但它依然在我的脑海里挥之不去，难以忘怀。 ?2、这件事过去很久了。却一直留在我的记忆中，感动着我，温暖着我。 ?3、直到现在，我对那件事还记忆犹新，学会感动吧！它会令你时时感动到温暖。 ?4、啊！母爱多么可敬，多么伟大。我要发自内心大声呐喊：“妈妈，我爱你！” ? 写人习作的精彩开头 1、“爸爸，爸爸”，家里又响起我对爸爸地呼唤。在我心中爸爸的爱是那么深沉，那么让人回味…… 2、咦？题目中的老王是谁？那就是我以前的同桌。他的眼睛小小的，戴着一副眼睛，很受人家欢迎呢！我们是常常吵架的同桌，虽然我们常常吵架，但是他在我眼里却是一个善良、爱开玩笑的人。 3、高高的身材，瘦瘦的胳膊，可却是“瘦是瘦，有肌肉！”他，可是我校的体育健将！要不是必须带书包，他还什么都不带，除了作业以外，任何的物品都放在学校的抽屉里，学习成绩那可是飞机上挂暖壶——高水平！他是谁呢？嘿嘿，我先不告诉你听，你听我细细道来。 4、有人说，母爱是永恒的，母爱是伟大的。母亲是多么的辛苦啊，是用她们的乳汁把我们养育成长。

写事作文开头结尾大全 (好)

写事作文的开头结尾（选一个喜欢的开头和结尾，会背会写） 开头： 1、在我的脑海里，有许多令我感动的事情，它们都像海边的贝壳一串串地在记忆里闪光。但其中有一件事令我最受感动。提起那件事，我至今记忆犹新。 2、在我记忆的长河中,有高兴的事,有伤心的事,但最令我难忘的还是要算那次发生在……的事。 3.童年像一条河，朵朵浪花在朝阳照耀下充满欢乐。秋高气爽的季节，那次……真有趣。 4、往事总在我的脑海里流动，看似平静的心海总有波浪起伏，过去的日子，我忘不了，更忘不了那件令我感动的事。 5、感动如爱，时刻温暖着我的心，只要我们细细地发现，就能被感动，我就曾被这样的一件事所感动。 6、作家刘墉说：“成长是一种美丽的痛。”在我看来，这真是一句至理名言。你可能问我为什么，这还要从我记忆深处的一件事说起。 7、时间如流水一般匆匆而逝，很多的记忆随日子的消失而褪色 有一件事，让我历历在目，感动不已。

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小学生经典作文开头结尾大全 开头：每个人都有一个童年,而童年趣事也就像海边那各色各样的贝壳,在海水的陪伴下闪闪发光,散发着五颜六色的光彩,数都数不完.而如今我的手上还握着那一颗最闪光的贝壳,那也正是我最难忘的一件童年趣事. 开头：“童年的梦，七彩的梦；童年的歌，欢乐的歌；童年的脚印一串串；童年的故事一摞摞。”这首歌是否能让你回想起美好的童年生活？在那五彩缤纷的岁月中，发生过许多事情，不像星星一样的明亮。我的童年是美好的，有许多事值得回忆…… 开头：童年中留下许许多多美好的回忆，这些回忆像一块块美味的甜点，值得我们去慢慢地品尝，细细地体会童年中的酸甜苦辣。 开头：生活中有着很多美好的记忆，这些回忆就像一首首美妙的音乐，需要我们去慢慢地感受，静静地回想生活中的喜怒哀乐。 开头：童年是一串七彩的珍珠，里面充满了童真与童趣；童年是一道无彩缤纷的彩虹，编织着无数的天真与快乐；童年是一曲动听的歌，跳动着一个个生动的音符。童年趣事百无禁忌，常常荡漾在童年梦中的我真的好想像睡美人那样一睡不醒，如果让我形容童年趣事的话，我会把它比喻一朵花，一朵芳香四溢的花；一个盒子，一个装着许多秘密的盒子；一个葡萄架，一个长满了数不清葡萄的葡萄架……，就让我们一起来享受那童话般的故事吧！ 开头：童年，就像一个五彩斑斓的盒子，里面有许多发光有趣的回忆。随着时间的流逝，有的回忆已被尘封了，而有的回忆却被时间的流水冲刷的更加璀璨。 开头：童年是一条五彩的河，童年是一道五彩的路，童年是一座闪亮的桥，童年是一本精彩的书。当我翻开它的时候，感到十分快乐。却又带着点留恋和向往。 开头：童年像一个五彩斑斓的盒子，里面装着许多闪光有趣的回忆．其中的一件小事，每当想起来，总要勾起我欢悦的回忆。 开头：童年是一杯水，清清的，淡淡的；童年是一道彩虹，充满色彩，五彩缤纷，让人回味无穷；童年是一个锦囊袋，装满幻想，装满理想，装满让人回味无穷的酸甜苦乐的事…… 结尾：童年趣事是在童年生活中积聚的宝贵财富，请记住它，它会让你的生活更加丰富多彩，会让你的生活充满欢声笑语。 开头：童年就像一条金色河流，它的每一朵浪花都装着我们幼儿时的往事，这一朵浪花有惊无险，那一朵浪花快乐无比，总之，这条河流里承载着我的美丽梦想，飞翔着我幼稚的童真，但有一件事我至今仍记忆犹新。 开头：童年，是欢乐的海洋。在回忆的海边，有无数的贝壳，有灰暗的，勾起一段伤心的往事；有灿烂的，使人想起童年趣事。我在那回忆的海岸，寻觅着最美丽的贝壳，啊，找到了…… 一、场景渲染式开头 1、“铃——铃——铃”下课铃响了，同学们一窝蜂似地涌出教室，到办公室去拿数学测验卷。 2、考试又到了，当各科老师公布了考试的日期后，同学们脸上都浮现出一种忧郁的情绪。 3、深夜，我又被父亲那苍老而沙哑的干咳声惊醒了。我半睁着朦胧的睡眼瞥了一眼窗户，凭我的直觉，大概是凌 晨3点左右。我隐约听见了父母的谈话声。 二、愿望实践式开头 1、眼看着桌子上的台历，被撕得只剩下最后一张了，我盼望已久的元旦就要来临了。

好开头好结尾摘抄大全

好开头好结尾摘抄大全 【篇一：好开头好结尾摘抄大全】 友谊 开头：现代人的友谊,很坚固又很脆弱.它是人间的宝藏,需我们珍爱.友谊的不可传递性,决定了它是一部孤本的书.我们可以和不同的人有不同的友谊,但我们不会和同一个人有不同的友谊.友谊是一条越掘越深的巷道,没有回头路可以走的,刻骨铭心的友谊也如仇恨一样,没齿难忘. 结尾：长留史册的,不是锱铢必较的利益,而是肝胆相照的情分,和朋友坦诚的交往,会使我们留存着对真情的敏感,会使我们的眼睛抹去云翳,心境重新开朗. 团结 开头：每次听到这首歌“团结就是力量”这首歌我总是感慨万千,特别是在xx事情后,体会更是深刻 结尾：一滴水是微不足道的,渗入泥土,便会消失不见,可汇聚成河却是川流不息.如同我们,在许多的困难面前,一个人总是凸显着单薄,而无法作为,可当我们团结起来,却可以创造无数的奇迹. 宽容 开头：宽容是人类生活中至高无尚的美德.因为宽容包含着人的心灵,因为宽容可以超越一切,因为宽容需要一颗博大的心.因为宽容是人类情感中最重要的一部分,这种情感能融化心头的冰霜.而缺乏宽容,将使个性从伟大堕落成连平凡都不如. 结尾：生活,往往纷繁,又常常平淡.正因为宽容如水,使纷繁经过过滤变得纯净；正因为宽容似火,使平淡通过锻烧日趋鲜明；更因为有这诗般的宽容,才赋予人生以艺术,赋予生命以永恒. 谅解 开头：谅解如一杯清茶,冲淡彼此之间的误会；谅解像一缕春风,吹化人间隔膜的冰层；谅解像一只寒梅,预示心灵来春的温情；谅解是一架彩虹,让就不相逢的情感放射光彩. 结尾：逝者如斯,历史的浪花淘尽了王朝的恩怨情仇,当今既没有了一方霸主,也没有了雄风王者；我们在追求团结、奋进、和谐、平等,我们同样应该独守那份恬然与空明,用每一句话没一种表情向人们昭示谅解,协同人们用谅解交往. 把握谅解,社会便会进步；