Genome-wide in silico identification and experimental confirmation of abscisic acid-regulated genes

Genome-wide in silico identi?cation and experimental con?rmation of abscisic acid-regulated genes in Arabidopsis

Ming-Der Huang,Wen-Luan Wu*

Department of Life Sciences,National Cheng Kung University,Tainan701,Taiwan

Received25August2005;received in revised form10January2006;accepted10January2006

Available online30January2006

Abstract

The abscisic acid(ABA)hormone plays a major role in various aspects of plant growth and development,including seed maturation and germination,as well as adaptation to abiotic environmental stresses.With the purpose to identify novel ABA-regulated genes,we tried to genome-wide identi?cation of ABA-related genes using computational approach by searching ABA-responsive elements(ABREs,T/CACGTGT/GC)in entire Arabidopsis genome.137ABA-regulated candidate genes were found to possess two or more ABREs in the1200-bp upstream region of the translation start site.In an attempt to monitor the gene expression of these candidates in response to ABA,16genes were selected and examined by RT-PCR.Of these16,seven are unclassi?ed genes in Arabidopsis,12candidates were up-regulated by ABA and four had equal expression levels as control.In addition,Northern blot analysis indicated that some candidates were further shown to differentially respond to various stresses.The results suggested that these ABA-regulated genes may consist of other cis-acting elements responsive to abiotic stresses and con?rmed that in silico cloning of ABA-regulated genes through Arabidopsis genomic sequence database mining is a fast and reliable approach.

#2006Elsevier Ireland Ltd.All rights reserved.

Keywords:ABA-regulated gene;ABA-responsive element(ABRE);Abscisic acid(ABA);Arabidopsis thaliana;Genome-wide identi?cation;In silico

1.Introduction

Abscisic acid(ABA),a key phytohormone,plays a wide range of important roles in plant growth and development, including embryogenesis,seed dormancy,root and shoot growth,and transpiration[1,2].Furthermore,ABA plays a protective role in response to abiotic stress including cold, salinity and drought[1,3,4].These abiotic stresses generally result in an elevation of ABA levels,which lead to a number of physiological adaptations comprising stomata closure,growth inhibition and metabolism alternation.Studies have been focused on the physiological roles and its related signal transduction[5].At the molecular level,many of these changes are the result of alternations in gene expression.Therefore,it is important to isolate and identify the genes that are regulated in response to ABA.Genetic analysis based on the inhibitory effect of ABA on seed germination has yielded mutants with reduced ABA biosynthesis or altered ABA-responsiveness [1,6].Xiong and co-workers also isolated genes involving in ABA or stress signal transduction based on the screening of mutant reduced or enhanced RD29A::LUC expression[7,8]. However,the complication of ABA signal network made it relatively dif?cult to isolate and analyze ABA-regulated genes with traditional methods[9].With the completion of the Arabidopsis genome sequencing project,the recently devel-oped approaches such as microarray,SAGE(serial analysis of gene expression)and screening of T-DNA insertion database, had been used for the identi?cation of ABA-regulated genes in Arabidopsis.Several genes involved in ABA signal transduc-tion pathway were identi?ed by screening T-DNA insertion database[10,11].Microarray and SAGE technology are also useful tools for the analysis of genome-scale gene expression. Thousands of genes were examined and a number of genes have been reported to be expressed in response to ABA and various stresses[12,13].

ABA is involved in stress response(i.e.,drought and salinity) and has been shown to regulate the expression of various stress-responsive genes[14].Subsequent functional analysis of the ABA-regulated gene promoter region has led to the identi?cation of several ABA-responsive elements(ABREs)[15].Many ABA-

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Plant Science170(2006)986–993

*Corresponding author.Tel.:+88662757575x65523;fax:+88662742583.

E-mail address:wenluan2@https://www.360docs.net/doc/ca7187494.html,.tw(W.-L.Wu).

0168-9452/$–see front matter#2006Elsevier Ireland Ltd.All rights reserved.

doi:10.1016/j.plantsci.2006.01.004

regulated genes contain a conserved ABA-responsive element(T/ CACGTGGC)in their promoter regions.ABREs were?rst identi?ed in the promoter region of wheat Em gene(Em1a element;GGACACGTGGC)[16],contain the ACGT core sequence and can be considered a subset of a larger group of cis-elements known as‘‘G-box’’(CACGTG),which also is present in numerous genes regulated by other environmental cues[17]. Another group of ABREs,known as‘‘coupling element3’’(CE3) of barley HVA1gene or‘‘motif III’’of rice Rab16B share the CGCGTG core sequence[15].Other ABREs that do not belong to G-box-like or CGCGTG-containing ABREs have also been reported,such as Sph element-containing sequence of the maize C1gene[18],the MYB and the MYC binding sites of the Arabidopsis rd22gene[19].Recently,several transcription factors,which recognize ABREs and regulate ABA-regulated genes expression have been isolated.These ABREs binding proteins are families of basic leucine zipper(bZIP),basic helix–loop–helix(bHLH)or MYB-related transcription factor[20–22]. It is understood for the promoters of various stress response genes, a functional ABA-responsive promoter has been reported to contain multiple ABREs[23,24].In other words,a minimal sequence unit suf?cient for regulation of ABA is composed of various combinations of the ABREs.These studies indicated that stress-responsive genes expression are mediated by both ABA-dependent and-independent pathways,some of these stress-induced genes share similarities with genes of known function, but the roles of the majority remain unclear.A great deal of effort has been invested to study how ABA and stress regulated the expression of these genes,but little is known about their functions.

ABA-regulated and stress-inducible genes can be identi?ed by gene expression pro?ling via DNA microarray experiments. Although a large number of gene expression results have been reported,the number of identi?ed ABA-inducible genes is rather limited.This is partially due to the fact that gene expression pro?ling examines gene expressions at particular time points of cell cycles,in speci?c tissues,and under speci?c stress conditions, while some of the ABA-inducible genes may have never been triggered in such experiments.In addition,approaches employed for the identi?cation of ABA-regulated genes described as above are generally time-consuming,costly and labor intensive.To facilitate the genome-wide cloning of ABA-regulated genes from Arabidopsis,we identify novel ABA-regulated genes by using bioinformatics.In this study,we identi?ed ABA-regulated genes containing two or more ABREs in the promoter region through searching of ABREs within Arabidopsis genomic database and tested these candidate genes in response to ABA and various abiotic stresses by gene expression experimental analysis.We reported here that genome-wide identi?cation of ABA-regulated genes in silico is a fast and reliable method.

2.Materials and methods

https://www.360docs.net/doc/ca7187494.html,putational identi?cation of ABA-regulated candidate genes in Arabidopsis genome

The complete Arabidopsis genomic sequence database (release version5)was downloaded from the FTP site of National Center for Biotechnology Information(NCBI;ftp:// https://www.360docs.net/doc/ca7187494.html,/genomes/Arabidopsis_thaliana/).For in silico cloning the ABA-regulated genes,we developed a computer program(http://140.116.25.196/element.htm)to identify the genes possessing two or more ABREs(T/CACGTGT/GC). Since the prediction of promoter was dif?cult,we searched ABREs within the region upstream of the annotated translation start site rather than the sequence upstream of the predicted transcription start site.All promoter sequences1200-bp upstream of the annotated translation start site were scanned using the computer program described above for the presence of the ABREs.The functional categories of Arabidopsis ABA-regulated candidate genes were based on the de?nition of TAIR (The Arabidopsis Information Resource;https://www.360docs.net/doc/ca7187494.html,/).

2.2.Plant materials and stress treatment

Seeds of Arabidopsis thaliana(Columbia ecotype)were surface sterilized by a2min incubation in70%ethanol and 20min incubation in1%(w/v)sodium hypochlorite.After three washes with sterile water,seeds were sown in0.8% agarose plates containing half-strength Murashige and Skoog salt base[25]and grown in a controlled environment growth chamber under a16h light/8h dark cycle,a photo?uency rate of3000lux,and a temperature of228C.

Two-week-old seedlings grown on half-strength MS agar plates were subjected to ABA,NaCl,PEG(polyethylene glycol),or cold treatments.Seedlings were transferred to a?lter paper saturated with100m M ABA(mixed isomers,?cis/trans, Sigma,St.Louis,Missouri,USA),300mM NaCl or30%PEG (molecular weight,6000),respectively,and incubated at228C under dim light for3or4h.Control seedlings were transferred to water.For cold treatment,seedlings on half-strength MS agar plates were incubated on ice under dim light for24h.

2.3.Evaluation of ABA-regulated candidate gene transcript levels by RT-PCR and northern blot analysis Two-week-old seedlings on half-strength MS agar plates were treated with ABA,NaCl,PEG or cold as described above. Total RNA was extracted from whole seedlings of the control and stressed plants using RNeasy Plant Mini Kit(Qiagen, Germany).DNA contamination was removed with DNase (Qiagen)treatment for15min at room temperature.For preliminary evaluation of16candidates gene expression levels in response to ABA by reverse-transcription-PCR-coupled reactions(RT-PCR),the primer pairs for each gene(Table1) were designed from the full-length cDNA and EST sequence of genes that were available from SSP(Salk/Stanford/PGEC Consortium,https://www.360docs.net/doc/ca7187494.html,/SSP/index.html)or NCBI (https://www.360docs.net/doc/ca7187494.html,/).The primer speci?city for each given gene was con?rmed by sequencing of RT-PCR fragments. Total RNA(3m g)samples from stress treatment or control plants were reverse transcribed with200U of the SUPER-SCRIPT TM III RT(Invitrogen,CA,USA),500pmol dNTP and 5pmol oligo(dT)12–18primer at508C for60min.PCR was

M.-D.Huang,W.-L.Wu/Plant Science170(2006)986–993987

subsequently performed in 25m l of a reaction mix containing 2.0mM KCl,200pmol dNTP,50pmol of each gene-speci?c primer pair,one-tenth of ?rst strand synthesis cDNA and 1U of Taq DNA polymerase (Roche Diagnostics,Mannheim,Germany).The actin primers used in the control reaction were from the Arabidopsis Actin2gene (locus number At3g18780).A PTC-200Programmable Thermal Cycler (controller)(MJ Research,Watertown,MA,USA)was used with an initial denaturation step of 948C for 3min,followed by 25–35cycles of 948C for 60s,51or 538C 45s,728C for 45s,and a ?nal elongation step of 728C for 10min.The RT-PCR products were electrophoresed on a 1%agarose gel containing ethidium bromide and visualized under UV light.The intensity of each ampli?cation product was quanti?ed by scanning densitometry using an ImageQuant Software (Molecuar Dynamics,Sunny-vale,CA,USA).RT-PCR analysis was performed in duplicate using RNA from two different sets of plants.Results of the two replicates were similar,and only one replicate is depicted in

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988Table 1

Number of ABA-regulated candidate for genes for test

Gene

Code Protein a

ABREs b

Primer

c

a Code protein indicates the putative functions of gene products expected from sequence homologies.

b ABRE sequence observed in 1200-bp upstream region of the translation start site.c

Primer designed from full-length cDNA or EST sequence and used for RT-PCR.

Fig.2.The second RT-PCR was performed using the same reagents as those used for the ?rst PCR except that the RNAwas isolated from different plants.

Evaluation of the stress effects on the transcription of nine putative ABA-regulated genes by Northern blot analysis,samples of 10m g total RNA were denatured with a mixture of 18%glyoxal (v/v)and 10mM NaPO 4buffer (pH 7.0),fractionated by electrophoresis on a 1%agarose gel in 10mM NaPO 4buffer,pH 7.0,transferred onto Hybond-N +nylon membrane (Amersham Pharmacia Biotech)by the ‘‘downward capillary transfer’’method and ?xed using the Stratalinker 11800UV Crosslinker (Stratagene,La Jolla,CA,USA).The probes used for the Northern blotting assay were the cDNA fragments generated by RT-PCR with the primer pairs for each gene (Table 1).These cDNA fragments containing the 30-UTR and partial coding region of each given gene had been subjected to Southern analysis for con?rming their gene speci?cities.DNA probes were prepared from the fragment of RT-PCR ampli?cation and labelled with [a -32P]dCTP using Rediprime II DNA Labelling System (Amersham Pharmacia Biotech).Northern blots were prehybridized in 5%dextran sulfate (molecular weight,500,000),1?Denhardt’s solution,63%deionized formamide,100m g/ml of sonicated salmon sperm DNA,and 1?TPSE (50mM Tris–HCl pH 7.5,4mM Na 4P 2O 7,1%SDS,1mM EDTA)for 4–6h at 428C.Hybridization was carried out in the same solution at 428C with the addition of 32P-labelled probe for 12–16h.After hybridization,the membranes were washed twice with 2?SSC,0.1%SDS at room temperature for 30min,and once with 0.1?SSC,0.1%SDS at 428C for 30min.The ?lters were exposed to X-ray ?lms (X-Omat ?lm,Kodak,Japan)with intensifying screen at à808C.The ?nal hybridization of the Northern blot membranes was performed with a probe prepared from the Arabidopsis 18S rDNA,which was used as a control to show the amount of RNA loaded in each lane.For the removal of radioactive probes the following treatment was repeated at least twice:a solution of 0.5%SDS was boiled and poured over the membrane and allowed to cool to room temperature.The complete removal of the probes after each hybridization was veri?ed by exposure of the membrane to X-ray ?lm.3.Results and discussion

3.1.In silico analysis of ABA-regulated candidate genes To identify ABA-regulated candidate genes in Arabidopsis ,we searched the genes containing ABREs within the promoter region in Arabidopsis genome.Although some computer programs for promoter prediction are available,i.e.Genscan (https://www.360docs.net/doc/ca7187494.html,/GENSCAN.html )and promoter predic-tion tool of BDGP (Berkeley Drosophila Genome project)(http://www.fruit?https://www.360docs.net/doc/ca7187494.html,/seq_tools/promoter.html ),the predic-tion of eukaryotic promoter has not been successful by using these programs due to variable essential cis -acting elements present in genes.It has been estimated that the 50-UTR of most Arabidopsis genes is less than 150-bp and the promoter region is about 1000-bp [26].Therefore,the searching region of

ABREs within the 1200-bp region upstream from the putative translational start site was set in this study.A preliminary search of ABA-regulated candidate genes containing one or more ABREs in the Arabidopsis genome using the ABRE sequence ‘‘T/CACGTGT/GC’’yielded several hundreds of hits.Because it had been reported that multiple copies of ABREs could confer a stronger ABA-responsiveness [27,28],we screened the genes possessing two or more ABREs to restrict the criteria in re?ned search.137of 26,207genes of Arabidopsis were found to possess two or more ABREs,and 10of 137genes containing three ABREs (as Supplemental Table 1).It is noted that the distribution of ABREs location sites are not random,69%ABREs being in the 600-bp upstream region of the translation start site (Fig.1A).The number of ABREs decreases when the distance is farther from the translation start site.Surprisingly,the distribution distance between ABREs,52and 71%genes are less than 100and 200-bp,respectively (Fig.1B).Only four

M.-D.Huang,W.-L.Wu /Plant Science 170(2006)986–993

989

Fig.1.Distribution of ABREs location sites.(A)Distribution of ABREs sites along the promoter regions of Arabidopsis ABA-regulated candidate genes.Location of ABRE site is de?ned as the position upstream putative translation start site.(B)Distribution of the distance between two ABREs of ABA-regulated candidate genes.

genes reveal the distance between two ABREs more than 900-bp.The putative ABA-regulated genes identi?ed here can be classi?ed according to the functional categories de?ned for the Arabidopsis genome (as Supplemental Table 1).While 82of the 137genes could be ascribed to one of the proposed functional categories for genes,the rest (40%)appear as unclassi?ed categories.Among these unclassi?ed genes,24(17.5%)and seven (5.1%)genes were unknown and hypothetical genes,respectively.The unclassi?ed genes of entire Arabidopsis genome are approximately 45%[29].This result indicates that the function of most putative ABA-regulated genes is presently unclear.The two largest amount of the genes identi?ed in this study belong to the metabolism (12.4%)and transcription (10.2%),respectively.However,these values are not signi?cantly different from the representa-tions of these categories in the entire genome.The rest other categories also show similar values as those in the entire genome.Since ABA plays a wide range of important roles in plant development and adaptive processes,it is not surprising that ABA regulated several categories of genes in Arabidopsis .Among these candidates,only three genes,AtECP63(locus number At2g36640)[30],AtPirin1(locus number At3g59220)[31]and RD26(locus number At4g27410)[32]have been proven to be ABA-regulated genes.On the other hand,Seki et al.monitored the expression pattern of 7000Arabidopsis genes under ABA and stress treatments using full-length cDNA microarray,and identi?ed 245ABA-regulated genes [12].Among those ABA-regulated genes found by Seki microarray approach,only eight genes (At1g32870,At2g22240,At2g22470,At3g10420,At3g29575,At4g27410,At4g27520

M.-D.Huang,W.-L.Wu /Plant Science 170(2006)986–993

990Fig.2.Transcription level of ABA-regulated candidate genes.Fourteen-day-old seedlings grown on half-strength MS agar plates were treated with 100m M ABA for 4h.Total RNA from whole plant of either control or ABA-treated plants were used for gene expression assay in RT-PCR.Primers used for the ampli?cation were designed from full-length cDNA or EST sequence of these genes.Right,genes showed higher expression level than control.Left,genes revealed similar expression level as control.(C)Arabidopsis were treated with H 2O only;(A)ABA treated.Actin2was used as a positive control in the same reactions.

and At5g50360)identi?ed from this study included,suggesting that those ABA-regulated candidate genes which had not previously been identi?ed by other approaches,can be found through this in silico approach(as Supplemental Table1). 3.2.Experimental analysis of computational identi?ed

ABA-candidate genes in response to ABA

To con?rm that in silico search served to identify ABA-regulated genes,the RT-PCR was employed to monitor the gene expression.Promoter analysis had demonstrated that69% ABREs were located within the600-bp upstream region of the translation start site and the distance between two ABREs was most less than200-bp(Fig.1A and B).Based on these two criteria,16candidates were selected for further characteriza-tion of the gene expression by RT-PCR(Table1).Among these selected genes,three genes(At3g02140,At3g53040and At4g25570)consist of three ABREs and the rest contain two ABREs.Genes At3g10420and At4g27520had been previously identi?ed as ABA-regulated and stress-responsive genes by microarray[12],and used as positive controls in this experiment.For preliminary investigation of the effect of exogenous application of ABA on the expression of these genes in2-week-old Arabidopsis seedlings.Gene-speci?c primers used in RT-PCR were designed based on the cDNA full-length or EST sequence of genes(Table1).As shown in Fig.2,12(75%)of16test genes in ABA-treated Arabidopsis revealed much higher level of expression than those of control plants(1.5-fold increase in average),and four showed similar expression level to control.Both genes At3g53040and At4g33905were undetectable in control but expressed in ABA-treated plants.As a result to show that75%test genes revealed higher expression level than those of control(Fig.2), indicating that this in silico cloning is a useful and powerful approach.Furthermore,comparison of our results with available microarray data set from AtGenExpress(http:// https://www.360docs.net/doc/ca7187494.html,/info/expression/ATGenExpress.jsp),revealed that the expression of66(48%)candidate genes were signi?cantly increased(1.5-fold increase in average)after 10m M ABA treatment for3h.This suggested that using the computational approach for identi?cation of the ABA-regulated gene is quite reliable.Recently,Ramirez-Parra et al.identi?ed E2F-regulated genes by adopting the similar approach to search the E2F target site within Arabidopsis genomic sequence.Their results also showed that genes possessing transcription factor binding sites could be regulated by transcription factors[33].

In this study,no direct correlation between the number of ABRE present in a promoter and the fold transcriptional induction by ABA of the corresponding gene was observed. Genes At1g48300,At4g16490,At4g25570and At5g47180 contain two ABREs,their expression levels in ABA-treated plants were similar to those of control(Fig.2).One possible explanation for this is that the authentic promoter regions of these genes are unknown,the ABREs location sites might be not within the promoter regions proposed in this study.For instance,predicted promoter region of gene At5g47180by BDGP promoter prediction tool(http://www.fruit?https://www.360docs.net/doc/ca7187494.html,/seq_-tools/promoter.html)is approximate600bp upstream of the translation start site,while both ABREs of At5g47180are located in the410-and518-bp upstream of the putative translation start site,respectively,which are not within the predicted promoter region(Table1)and therefore this gene displayed non-ABA-regulated gene expression pattern.Alter-natively,other factors such as different stages and tissues of these genes expression,as well as experiment conditions may affect the expression data.For instance,gene Atpirin1have been identi?ed as an ABA-regulated gene[31],but the array data from AtGenExpress showed that the expression of Atpirin1 was down-regulated(0.72-fold decrease)in ABA-treated seedlings.The differences between the conditions used in these two experiments were the age of seedling,time and concentration of ABA treatment,7-day-old seedlings treated with10m M ABA for3h(microarray data from AtGenExpress) versus9-day-old seedlings treated with100m M ABA for4h [31].In this study,preliminary expression analysis by RT-PCR which was only carried out with2-week-old seedling treated with100m M ABA for4h(Fig.2).Thus,these four putative assigned as non-ABA-regulated genes showing similar expression level to control at4h after ABA treatment;these genes possibly responded late to ABA application at other time points or age of seedling.

3.3.ABA-regulated genes response to stress

To investigate whether the expression of ABA-regulated genes associated with abiotic stresses,eight ABA-regulated and one non-ABA-regulated genes were chosen for further North-ern blot analysis under osmotic,salt or cold stress.In total nine tested genes,seven(At1g07080,At1g51140,At3g02140, At3g10420,At4g21020,At4g27520and At4g33905)were detected to be induced in response to various stresses (Fig.3).Two genes At1g51140and At3g02140only responded to salt stress,and the transcript level of At4g02140was greatly induced.At3g10420showed high expression in either osmotic or salt stress.Under salt or cold stress,two genes At4g27520 and At4g33905showed higher expression level than those of control,whereas the level of At4g33905RNA was in such low abundance.The transcript level of At4g21020was also slightly induced by osmotic,salt or cold stress.Gene At3g53040 responded to ABA,but showed no inducible expression under these tested stress conditions.As a control for stress treatments, the transcript level of non-ABA-regulated gene At4g25570was also determined,whose expression did not change after all three stress treatments.

Several ABA-regulated genes showed higher expression levels in response to abiotic stress than those of control(Fig.3). This result suggested that these ABA-regulated genes may play important roles to enhance the stress tolerance.These ABA-regulated genes responded differentially to various abiotic stresses,suggesting that there are some other cis-acting elements within the promoter region of these genes,which are involved in response to stresses.Recently,ABREs interacting with DRE or DRE-core sequence were suf?cient

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for responding to ABA,cold and drought stresses had been reported by Narusaka et al.[34].Gene At4g33905contains two ABREs,one DRE-core and as-1sequence,and which responded to both salt and cold stresses;while gene At3g10420also possesses three ABREs and two DRE-core sequence,but which responded to both osmotic and salt stresses.Two ABREs and one LTRE-1(low-temperature-responsive element)sequence [35]are present within the promoter region of gene At4g27520,and its expression level was higher than that of control in response to both cold and salt stresses.These results further implicated that ABA-regulated genes,except for ABREs,associated with other cis -acting

elements,such as coupling or DRE element,could suf?ciently confer in response to ABA [15,34].In this work,we searched ABA-regulated candidate genes containing two ABREs,those containing a single ABRE and other coupling elements were not retrieved,such as RD29A and KIN2.Thus,the searching criteria set that ABRE combined with other cis -acting elements may result in further ?nding some more other ABA-regulated genes [36].Nevertheless,some ABA-regulated genes are post-transcriptional or -translational regulated,and these genes will not be identi?ed by this approach [37].

In conclusion,through this study we have developed a reliable and fast computational method to effectively identify ABA-regulated candidate genes based on the presence of their corresponding cis -acting elements in available Arabidopsis genomic sequence.Furthermore,results of experimental analysis clearly support the feasibility of such approaches.These approaches used the combination of molecular biology and bioinformatics made here will be useful to elucidate novel aspects of gene expression in model plants.Acknowledgments

We thank Dr.Chang-Hsien Yang,National Chung Hsing University,Taichung,Taiwan,for providing the Arabidopsis seeds.We also thank the anonymous reviewers for useful suggestions on the manuscript.Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at doi:10.1016/j.plantsci.2006.01.004.References

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