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Genome-wide targeted prediction of ABA responsive genes in rice based on over-represented cis-motif in co-expressed genes

Genome-wide targeted prediction of ABA responsive genes in rice based on over-represented cis-motif in co-expressed genes

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Published by Abhay Kumar

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

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Published by: Abhay Kumar on Jan 25, 2013
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Genome-wide targeted prediction of ABA responsive genes in ricebased on over-represented
cis-
motif in co-expressed genes
Sangram K. Lenka
Æ
Bikash Lohia
Æ
Abhay Kumar
Æ
Viswanathan Chinnusamy
Æ
Kailash C. Bansal
Received: 19 May 2008/Accepted: 16 October 2008/Published online: 8 November 2008
Ó
The Author(s) 2008. This article is published with open access at Springerlink.com
Abstract
Abscisic acid (ABA), the popular plant stresshormone, plays a key role in regulation of sub-set of stressresponsive genes. These genes respond to ABA throughspecific transcription factors which bind to
cis
-regulatoryelements present in their promoters. We discovered theABA Responsive Element (ABRE) core (ACGT) contain-ing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressedgenes in rice. Targeted gene prediction strategy using thismotif led to the identification of 402 protein coding genespotentially regulated by ABA-dependent molecular geneticnetwork. RT-PCR analysis of arbitrarily chosen 45 genesfrom the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABAresponsive genes showed enrichment of signal transductionand stress related genes among diverse functionalcategories.
Keywords
Abscisic acid (ABA)
Á
Genome-wide
Á
Rice
Á
Co-expressed genes
Á
Cis
-regulatory elements
Abbreviations
ABA Abscisic acidCRE
Cis
-regulatory elementABRE ABA responsive elementGO Gene ontologyCDS Coding DNA sequence
Introduction
Abiotic stresses like drought, salinity, cold and high tem-perature are the predominant environmental factorslimiting productivity of crop plants (Breshears et al.2005;Schroter et al.2005). Plants respond to these environ-mental cues at molecular level by altering expression of different sets of genes (Qureshi et al.2007; Tran et al.2007). Expression of such genes is mainly regulatedthrough transcriptional control process, while post-tran-scriptional and post-translational processes also play acrucial role. Transcriptional control machinery appears tobe conserved among plant species (Hakimi et al.2000; Hirtet al.1990). It is well established from different experi-ments over past decades that promoters containing aparticular
cis-
element respond to a specific trigger (Chin-nusamy et al.2003; Viswanathan and Zhu2002; Yamaguchi-Shinozaki and Shinozaki2005; Zhou et al.2007). Combinatorial interactions of 
cis-
acting DNA ele-ments in the promoters with
trans
-acting protein factors arekey processes governing spatio-temporal gene expression(Bustos et al.1991; Hartmann et al.2005; Hauffe et al. 1993). At an organism level, vast array of moleculargenetic networks are operational in a very complex anddynamic mode. Complete understanding of the moleculargenetic networks is a long cherished goal of system biol-ogists (Chinnusamy et al.2004; Li et al.2006a). Targeted modification of molecular genetic networks has a tremen-dous potential for engineering tailor made elite genotypes‘‘by
-
design.’’
Electronic supplementary material
The online version of thisarticle (doi:10.1007/s11103-008-9423-4) contains supplementarymaterial, which is available to authorized users.S. K. Lenka
Á
B. Lohia
Á
A. Kumar
Á
K. C. Bansal (
&
)National Research Centre on Plant Biotechnology, IndianAgricultural Research Institute, New Delhi 110012, Indiae-mail: kailashbansal@hotmail.comV. ChinnusamyWater Technology Center, Indian Agricultural ResearchInstitute, New Delhi 110012, India
 123
Plant Mol Biol (2009) 69:261–271DOI 10.1007/s11103-008-9423-4
 
Availability of genome sequence of a crop plant like riceoffers a new challenge and opportunities to explore thegenetic mechanisms that regulate gene expression inresponse to various developmental and environmental cues.Rice is also closely related to other crop plants like wheat,maize, barley, sugarcane, oat, and sorghum, etc
.
A highdegree of genomic synteny is conserved across differentmember species in the family Gramineae (Buell et al.2005; Goff et al.2002). Hence, rice is an ideal model to study complex gene regulation data coupled with com-parative sequence information using computational tools.This type of study will give an insight to map, predict anddecipher gene regulation mechanisms and functional clas-sification of genes. Recently, a fare amount of large scalegene expression datasets have become available in rice inresponse to various stresses (Rabbani et al.2003; Yazakiet al.2003). However, the available data is not sufficient todo meta
-
analysis. Nevertheless, the rice gene expressiondata can be suitably integrated with promoter structure tofind out it’s possible correlations (Benedict et al.2006; Liet al.2006b).Roles of ABA in physiological, developmental andadaptive processes in plants are well known. Endogenouslevel of ABA is induced in response to various biotic(pathogen attack) and abiotic stresses (Fujita et al.2006;Verslues and Zhu2007)
.
Exogenous application of ABA toplants mimics various stresses in term of co-expression of different sets of genes (Destefano-Beltran et al.2006; Loik and Nobel1993). The gene regulation in response to theelevated levels of ABA is mainly modulated by transcrip-tional control. Various studies suggest that co-expressedgenes are likely involved in a common biological process.Integration of co-expression data with promoter structurein plants shows that promoters of co-expressed genes sharea common
cis-
regulatory element (Kim and Kim2006;Reiss et al.2006; Werner2001). Various algorithms such as expectation maximization (MEME) and Gibbs samplinghave been used to search motifs that are over-representedwithin the set of related biological sequences (Bailey andElkan1994; Bailey et al.2006; Thompson et al.2003). Evidences from the promoter dissection and transcriptionfactor binding experiments are the main references toevaluate the strength and confidence of computationalmethods. A handful of molecular dissection experimentlike deletion and linker scanning analysis have pinpointedthe ABA responsive elements (ABREs), also termed as G-box, C-box or G-box/C-box hybrids within promoters of the ABA responsive genes. ABRE contains ACGT as acore nucleotide sequence, which acts as a binding site forbZIP family transcription factors governing transcriptionalregulation of ABA responsive genes (Guiltinan et al.1990;Hattori et al.2002; Mundy et al.1990; Ross and Shen 2006; Shen and Ho1995). ABREs are also coupled to the non-ACGT coupling elements like CE1, CE3, DRE, O2S,motif III, or ACGT core containing ABRE itself (Hoboet al.1999; Shen et al.1996; Singh1998). As ABA plays crucial role in various signaling processes, it is logical toexpect that other stress responsive integration points withinABA responsive promoters also govern gene regulation. Incase of rice, genome-wide binding experiments like chro-matin-immunoprecipitation coupled with microarray(Chip–chip) are lacking. Several databases like PLACEand Plant-CARE among others provide experimental evi-dences regarding
cis-
elements and transcription factors inplants (Higo et al.1999; Lescot et al.2002). We present here, a targeted gene finding approach on agenome-wide scale in rice. Our prediction is based on over-represented ACGT core containing consensus motif foundin co-expressed ABA responsive genes in vegetative tis-sues. Experimental verification by RT-PCR proves highaccuracy (80%) of our integrated prediction method in therice genome. Database mining suggested expression of thepredicted genes in response to abiotic stresses as well.Among the diverse functional categories of genes, GOanalysis showed the enrichment of the stress related andABA signaling pathway genes among the genes predictedin this study.
Materials and methods
ABA responsive genes and random sequence datasetsFrom published microarray data, 105 genes showing twofold or more up-regulation in rice seedlings in response toABA treatment were identified (Rabbani et al.2003;Yazaki et al.2003). Sequences of these genes weredownloaded from NCBI database (http://www.ncbi.nlm.nih.gov/ ) and blasted with TIGR rice c-DNA sequencesand corresponding loci were listed. 1 kb upstreamsequences (promoters) from translational start site ATG,were downloaded from TIGR (http://www.tigr.org/plantProjects.shtml)
Oryza sativa
(Release 4.0; January 12,2006) (Ouyang et al.2007). Similarly other genomicsequences of rice and
Arabidopsis
used here were retrievedfrom the TIGR and TAIR databases, respectively. ABA up-regulated genes (692) in
Arabidopsis
identified by Li et al.(2006a,b), were considered in this study to search the presence of predicted CGMCACGTGB motif (Li et al.2006b). Scuffled sequences were generated by randomlytaking five ABA responsive promoters and scuffled100 times using ‘Sequence Manipulation Suite’(http:// www.bioinformatics.org/sms2/shuffle_dna.html). Otherrandom sequence data sets used here were also generatedby using ‘‘Sequence Manipulation Suite’’ (http://www.bioinformatics.org/sms2/random_dna.html).
262 Plant Mol Biol (2009) 69:261271
 123
 
Motif discovery and motif searchFrom severalalgorithmsavailable,we chosethe expectationmaximization method MEME (Version 3.5.7) (http://meme.nbcr.net/meme/intro.html) for motif discovery using itsdefault setting for a minimum and maximum width of asingle motif as 10. The relevance of discovered motifs wasanalyzed using PLACE (http://www.dna.affrc.go.jp/ PLACE/ ) (Bailey and Elkan1994; Higo et al.1999). The motifs obtained from analyzed sequences were plottedaccording to their positions within the regions and theirconsensussequencesweregraphedusingWebLogo3:PublicBeta (http://weblogo.berkeley.edu/logo.cgi) (Crooks et al.2004). Perl and JavaScript were used to search the perfectmatch of the target motifs within the rice,
Arabidopsis
,random and scuffled sequences. We have considered onlyone orientation while searching motifs here.Analysis of gene ontology of predicted genesTo determine whether the occurrence of the discoveredmotifs were associated with specific gene functions, weretrieved the Plant GOSlim Assignment of rice Proteinsfrom TIGR Database (http://www.tigr.org/tdb/e2k1/osa1/ GO.retrieval.shtml) and correlated the annotated molecularfunction, biological process or cellular component of pre-dicted and control data sets of rice genes.Plant growth conditions, RNA sampling and RT-PCRanalysisRicecultivarNagina-22(
Oryzasativa
)seedsweregrownfor14 days at 28
°
C, 80% RH, and 12/12 h light/dark period inphytotron glass house. Sterile absorbent cotton soaked withHoagland’s solution was used as seed bed for growing riceseedlings. Plants were irrigated with Hoagland’s solution at3 days interval as supplemental irrigation.Leaf and root samples from seedling were collected after3, 5, and 24 h of 100
l
M ABA treatment. ABA was alsosprayed at every 2 h intervals from the first spraying. Fortreating roots, plants were submerged up to 1 cm aboveseed bed level in 100
l
M ABA for 3, 5, and 24 h. RNAwas extracted using TRI Reagent (Ambion, Inc. USA) andpooled from at least 20 independent controls and treatedplant samples, respectively and was treated with DNase-I(QIAGEN GmbH, Germany). Subsequently RNA cleanupwas carried out using RNeasy Plant Mini Kit (QIAGENGmbH, Germany).For RT-PCR analysis first strand c-DNA was synthe-sized by using 2
l
g of total RNA using Superscript-IIIreverse transcriptase (Invitrogen, USA) with oligo(dT)
20
primer following manufacturer’s instructions. Two micro-liter of c-DNA was used in 25
l
l of reaction volume withthe following PCR conditions to study the gene expression:30 cycles of 94
°
C for 1 min, annealing temperatureaccording to melting temperature of primers for 1 min, and72
°
C for 1 min, and then final extension at 72
°
C for10 min. List of primer sets used in the study are given inthe supplemental table (Additional Table 1). Out of thepredicted 402 genes, randomly selected 45 genes testedhere for RT-PCR analysis were not from the list of initialexpression datasets (except LOC_Os01g02120 andLOC_Os02g43330 which are used as test control); hencetheir responsiveness to ABA is virtually un-known. Simi-larly 15 genes were used as negative control withouthaving CGMCACGTGB motif. Quantitative estimation of RT-PCR amplicon on the gel was calculated as integrateddensity value (IDV) using AlphaEaseFC
TM
software.Accuracy percentage of our prediction was calculatedusing the conversion:Accuracy (%)
=
(Number of genes responsive to ABAdetected through RT-PCR/total number of genes tested forRT-PCR)
9
100.Stress related expression data mining and phenotypesearchingTo analyze the expression of predicted genes in response tocold, drought and salt stresses, the physical position of these loci in the rice pseudomolecules were retrieved andsearched against rice in the PlantQTL-GE database (http:// www.scbit.org/qtl2gene/new/plantqtl-ge.html) (Zeng et al.2007). All the above genes were searched for availablephenotype in the Rice
Tos17 
Insertion Mutant Database, if there is an insertion mutation (http://tos.nias.affrc.go.jp/ )(Miyao et al.2007).
Results
ABRE consensus motif discovery and gene predictionResponse of plants to a particular trigger might be medi-ated by a common transcriptional regulatory mechanism,hardwired by
cis-
acting elements as proven in other modelspecies (GuhaThakurta et al.2002; Wolfsberg et al.1999; Zhang et al.2005). The
cis-
acting DNA elements aregenerally degenerative in nature and difficult to discoverfrom the background but, ACGT-core containing ABREwas defined asACGTGKC, which matched very well withthe consensus derived from sequence comparison of ABA-responsive promoters in rice (Hattori et al.2002). A gen-ome wide computational prediction has successfullyclassified ABA responsive genes in
Arabidopsis
(Zhanget al.2005). The modular arrangement of ABRE with itsone of the coupling elements CE3 shows a clear divergence
Plant Mol Biol (2009) 69:261271 263
 123

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