ORIGINAL PAPER

Genome-wide analysis of intronless genes in riceand Arabidopsis

Mukesh Jain & Paramjit Khurana & Akhilesh K. Tyagi &Jitendra P. Khurana

Received: 3 February 2007 /Revised: 7 April 2007 /Accepted: 6 May 2007 / Published online: 20 June 2007# Springer-Verlag 2007

Abstract Intronless genes, a characteristic feature ofprokaryotes, constitute a significant portion of the eukary-otic genomes. Our analysis revealed the presence of 11,109(19.9%) and 5,846 (21.7%) intronless genes in rice andArabidopsis genomes, respectively, belonging to differentcellular role and gene ontology categories. The distributionand conservation of rice and Arabidopsis intronless genesamong different taxonomic groups have been analyzed. Atotal of 301 and 296 intronless genes from rice andArabidopsis, respectively, are conserved among organismsrepresenting the three major domains of life, i.e., archaea,bacteria, and eukaryotes. These evolutionarily conservedproteins are predicted to be involved in housekeepingcellular functions. Interestingly, among the 68% of rice and77% of Arabidopsis intronless genes present only ineukaryotic genomes, approximately 51% and 57% geneshave orthologs only in plants, and thus may represent theplant-specific genes. Furthermore, 831 and 144 intronlessgenes of rice and Arabidopsis, respectively, referred to asORFans, do not exhibit homology to any of the genes in thedatabase and may perform species-specific functions. Thesedata can serve as a resource for further comparative,evolutionary, and functional analysis of intronless genes inplants and other organisms.

Keywords Rice .Arabidopsis . Intronless genes . Evolution

Introduction

Most of the eukaryotic genes are interrupted by one or morenoncoding, intragenic sequences called introns. The avail-ability of complete genomic sequence of different organismsand the annotated and transcribed sequences help delineatethe structure of genes by resolving exons and introns. Thishas resulted in the identification of a number of singleexonic/intronless genes in eukaryotic genomes, althoughthey are considered to be a characteristic feature of pro-karyotes. Genome SEGE, a database on intronless genesfrom nine completely sequenced eukaryotic genomes, isavailable (Sakharkar and Kangueane 2004). The study ofintronless genes from different organisms is particularlyinteresting because it helps in understanding gene evolution.Recently, evolutionary analyses of intronless genes in humanand mouse has been reported (Agarwal and Gupta 2005;Sakharkar et al. 2006). Some large gene families, includingG-protein receptors and olfactory receptors in human andmouse, are intronless (Gentles and Karlin 1999; Takeda et al.2002).

The information on the occurrence and analysis ofintronless genes in plants is scanty. To our knowledge, nodetailed analysis of intronless genes at whole genome levelhas been performed in plants. However, several isolatedreports on the presence of intronless genes belonging tolarge gene families, such as F-box proteins, DEAD boxRNA helicases, pentatricopeptide repeat (PPR) containingproteins in Arabidopsis, are available (Aubourg et al. 1999;Gagne et al. 2002; Lecharny et al. 2003; Lurin et al. 2004).Recently, we reported all the 58 members of early auxin-

Funct Integr Genomics (2008) 8:69–78DOI 10.1007/s10142-007-0052-9

Electronic supplementary material The online version of this article(doi:10.1007/s10142-007-0052-9) contains supplementary material,which is available to authorized users.

M. Jain : P. Khurana :A. K. Tyagi : J. P. Khurana (*)Interdisciplinary Centre for Plant Genomicsand Department of Plant Molecular Biology,University of Delhi South Campus, Benito Juarez Road,New Delhi 110 021, Indiae-mail: khuranaj@genomeindia.org

responsive SAUR (small auxin-up RNAs) gene family inrice to be intronless (Jain et al. 2006b). The availability ofcomplete genome sequences of rice, the model monocotplant, and Arabidopsis, the model dicot plant, provides usnot only a genetic blueprint but also an opportunity forstudying functional and evolutionary genomics in plants(The Arabidopsis Genome Initiative 2000; Paterson et al.2004b; International Rice Genome Sequencing Project 2005;Vij et al. 2006). Furthermore, the comparative genomics ofrice and Arabidopsis can be used to gain knowledge of geneorganization and is particularly helpful in examininggenome evolution in both monocot and dicot plants.

In this study, we have identified intronless genes in riceand Arabidopsis by alignments of their annotated gene andprotein sequences. Cellular role and gene ontology (GO)category of the intronless genes have been predicted. Thedistribution and conservation of rice and Arabidopsisintronless genes among different taxonomic groups havealso been analyzed. A large fraction of intronless geneswere found to be conserved in rice and Arabidopsis. Thesedata provide insights for understanding evolutionary mech-anisms underlying gene/genome evolution in plants.

Materials and methods

Identification of intronless genes

All the annotated gene and protein sequences of 12 ricechromosomes were downloaded from batch data downloadtool available on The Institute for Genomic Research (TIGR)Rice Genome Annotation website (Yuan et al. 2005; http://www.tigr.org/tdb/e2k1/osa1/). TIGR Osa1 version 4 of ricepseudomolecules includes 62,827 gene models; of which4,734 have alternative splicing isoforms resulting in non-redundant 55,890 genes (loci). For Arabidopsis, all theannotated gene and protein sequences were downloadedfrom The Arabidopsis Information Resource (TAIR) data-base. TAIR release 6 contains a total of 31,407 genemodels, of which 3,159 have splice variants resulting innon-redundant 26,973 genes (loci). To identify intronlessgenes, all the protein sequences of rice and Arabidopsiswere aligned to their gene sequences locally by TBLASTNprogram (Altschul et al. 1997) without any filter and theresults of only the top hit were extracted for each queryprotein sequence. The results were clustered in three steps.In the first step, the proteins aligned completely with thegene sequences in one block with 100% identity over theentire length were chosen. In the second step, the redundancy(different gene models representing same gene loci) amongthe selected genes was removed. As a last step, genesannotated as transposable elements (TE) were removed toget a final list of non-redundant intronless genes in rice andArabidopsis.

Analysis of intronless genes

Cellular role and GO category of all the intronless geneswere predicted by ProtFun 2.2 server. The prediction ofprotein function or GO category by ProtFun relies solely onthe protein sequence given as input; it does not rely on thesequence similarity, but instead on sequence-derived fea-tures such as predicted posttranslational modifications,protein sorting signals, and physical/chemical propertiescalculated from amino acid composition (Jensen et al. 2002,2003). Therefore, ProtFun allows the prediction of functionof even ORFan proteins where no homologs can be found.The functional categorization of some of the representativeproteins with known function has been confirmed manuallyalso. The presence or absence of these rice intronless genesin organisms belonging to different taxonomic groups wasdetermined by BLink (BLAST-Link) tool available atNCBI. Blink displays precomputed protein BLAST align-ments of each query protein sequence in the Entrez data-bases by taxonomic criteria (Wheeler et al. 2005). PERLscripts and JAVA parsing tools were used to automate thesesteps.

Paralog identification

To identify paralogs of rice and Arabidopsis intronlessgenes, their protein sequences were aligned with them-selves and protein sequences of intron-containing genes inan all-against-all BLASTP search. Two loci were defined asparalogs if they match the criteria of expect value cut-off ofE-10 or less with at least 20% identity over the 70% of theaverage length of two sequences.

Conserved intronless genes in rice and Arabidopsis

To identify conserved intronless genes between rice andArabidopsis, the protein sequences of all the predictedintronless genes of Arabidopsis were aligned against theprotein sequences of all the predicted intronless genes ofrice, and vice versa, by BLASTP. An expect value cut-offof E-6 or less was used to identify the conserved intronlessgenes in rice and Arabidopsis.

Results and discussion

Identification of intronless genes in rice and Arabidopsis

The intronless genes in rice (Oryza sativa subsp. japonicacv Nipponbare) and Arabidopsis (Arabidopsis thaliana)were identified in three steps. The first step involved theTBLASTN alignment of all the annotated protein and genesequences. In the second step, redundant sequences repre-senting same gene loci were identified and removed to

70 Funct Integr Genomics (2008) 8:69–78

obtain a set of non-redundant intronless genes. Finally, as alast step, all the predicted intronless genes annotated astransposable elements (TEs) were removed. The overallanalysis revealed the presence of 11,109 and 5,846 intron-less genes in rice and Arabidopsis genomes, respectively.

The locus IDs and gene description of predicted intron-less genes in rice and Arabidopsis are provided in theSupplemental data files 1 and 2, respectively. The number ofpredicted intronless genes in Arabidopsis in our study islittle more than that predicted earlier (5,810) (Sakharkar andKangueane 2004). This slight increase in number may bebecause of the use of more recent version (TAIR6) ofArabidopsis genome annotation in this study. The BLASTsearch of the predicted protein sequences of 11,109intronless genes of japonica rice with the annotated proteinsof indica rice (cv 93-11) genome available at BGI RISe RiceGenome Database (http://rise.genomics.org.cn; Yu et al.2005) revealed that at least 68% of these genes areconserved (with ≥90% identity over the entire length) inboth subspecies (data not shown). This percentage mayincrease once a more exhaustive and accurate annotation ofthese two genomes becomes available.

The identified intronless genes are distributed on all therice (6–13%) and Arabidopsis (15–25%) chromosomes(Fig. 1). In rice, the highest number of intronless genes ispredicted on chromosome 1, the longest chromosome.Similarly, the longest chromosomes 1 and 5 of Arabidopsishave the highest number of intronless genes. Apparently, thedistribution of intronless genes in terms of number of genesper megabase (MB) among rice (26.6–32.8 genes per MB)and Arabidopsis (45.9–51.7 genes per MB) chromosomeslooks uniform. Interestingly, a strong bias towards theshorter length of proteins, which the intronless genesencode, was found. A strikingly large percentage of theseproteins (about 39% of rice and 29% of Arabidopsis) are of101 to 200 amino acids in length. While, the exact relevanceof this observation is not clear, this observation may havesome evolutionary significance. Although, the distributionof intronless genes on a rice or Arabidopsis chromosome israndom, several clusters of intronless genes are evident atsome rice and Arabidopsis chromosomes. Generally, theseclusters represent the members of multigene families, forexample, auxin-responsive genes and those encoding F-boxproteins, zinc finger proteins, cytochrome P450 proteins,AP2 domain proteins, and leucine-rich repeat proteins, thatare arranged in tandem repeats; however, some of thesetandemly repeated genes are interrupted with other genesand are not included in strictly defined tandem repeats.These results are consistent with the surprising outcome ofrice and Arabidopsis genome analysis that large percentages(14–17%) of their genes are arranged in tandem repeats(The Arabidopsis Genome Initiative 2000; InternationalRice Genome Sequencing Project 2005).

Recently, we found 17 (29%) members of intronlessSAUR gene family are clustered together in tandem on ricechromosome 9 (Jain et al. 2006b). It has been suggestedthat intronless gene families can evolve rapidly either bygene duplication or by reverse transcription/integration(Glusman et al. 2000; Lecharny et al. 2003; Lurin et al.2004; Jain et al. 2006b). Recently, Yu et al. (2005)presented evidence for the ongoing individual gene dupli-cations in rice, which provide a never-ending raw materialfor studying gene genesis and their functions.

Functional categorization

To learn about the functions of predicted rice andArabidopsis intronless genes, their annotations available atTIGR and TAIR, respectively, were explored. A significant-ly large percentage (60%) of rice intronless genes areannotated as hypothetical (39%) or expressed (21%)proteins. This indicates that functions of most of theseproteins are not known in rice or some of the predictedhypothetical proteins may be the result of incorrectannotations. However, only 37% of Arabidopsis intronless

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genes are annotated as hypothetical (6%) or expressed(31%) proteins. Putative functions have been assigned toother 40 and 63% of intronless genes in rice and Arabidopsis,respectively.

Several of these genes belonging to large gene familiesinvolved in various pathways, including protein synthesisand turnover (ribosomal proteins, F-box proteins), signaltransduction (auxin-responsive genes, protein kinases, pen-tatricopeptide repeat [PPR] proteins, leucine-rich repeat[LRR] proteins), DNA-binding (zinc finger proteins, AP2domain proteins), metabolism (cytochrome P450 proteins)and disease resistance proteins, are over represented in bothplants, indicating that intronless genes perform crucialfunctions in plant growth and development.

To further explore the functions of intronless genes,including those which are annotated as expressed orhypothetical proteins, their cellular role and GO categorywas determined using ProtFun (Fig. 2; Supplemental datafiles 3 and 4). This analysis revealed that the largest numberof rice and Arabidopsis intronless genes fall into thetranslation and energy metabolism functional categories,followed by cell envelope and amino acid biosynthesis.Metabolism represents the most abundant functional cate-gory in rice at the whole genome level also (Goff et al.2002; International Rice Genome Sequencing Project 2005).However, a significantly larger number of intronless genes

fall into translation, cell envelope, and amino acid biosyn-thesis functional categories compared to total genes,indicating their major role in basic cellular processes.Although the percentage of intronless genes predicted undervarious cellular role categories are similar for both rice andArabidopsis, the number vary significantly and in fact arehigher in rice, suggesting that the components of thesepathways have evolved more in rice. However, 11.14% ofArabidopsis intronless genes belong to amino acid biosyn-thesis category, as compared to only 6.74% in rice.Although there is a large difference in the percentage, thenumber of genes included in this category is nearly the samefor rice (748) and Arabidopsis (651), suggesting thatcomponents of some of the pathways are highly conservedand that the corresponding genes/gene families have notexpanded during evolution. Further, about 76% and 73%intronless genes of rice and Arabidopsis, respectively, couldbe associated with a GO category, with growth factor (morethan 30%) representing the most abundant category in bothplants. The proteins involved in transcription regulation,transport, immune response, and structural proteins are alsowell represented. The 14.41% (1215) of the rice intronlessgenes come under structural protein category; however, only4.77% (204) genes of this category are represented inArabidopsis, indicating a significantly large expansion ofthese genes in rice. The intronless genes involved in various

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Fig. 2 Functional categoriza-tion of rice and Arabidopsisintronless genes. Cellular role(a, c) and GO category (b, d)were determined for rice (a, b)and Arabidopsis (c, d)intronless genes by ProtFun andpercentage of genes included ineach category are given. Cellularrole categories are: AAB aminoacid biosynthesis,BC biosynthesis of cofactors,CE cell envelope, CP cellularprocesses, CIM centralintermediary metabolism, EMenergy metabolism, FAM fattyacid metabolism, PP purinesand pyrimidines, RF regulatoryfunctions, RT replication andtranscription, T translation,TB transport and binding.GO categories are: ST signaltransducer, R receptor,H hormone, SP structuralprotein, T transporter, IC ionchannel; VGIC voltage-gatedion channel, CC cation channel,TF transcription, TR transcrip-tion regulation, SR stressresponse, IR immune response,GF growth factor

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cellular processes are also predicted under immune responseGO category. However, the biological relevance of thisprediction is not clear. These results altogether shed somelight on the functional significance of intronless geneencoded proteins in plants.

Taxonomic distribution

To investigate the evolutionary conservation of rice andArabidopsis intronless genes among different taxonomicgroups (archaea, bacteria, fungi, metazoan, plants, and othereukaryotes), the precomputed BLAST results of their proteinsequences were retrieved through Blink. Blink results for7,658 and 5,470 intronless genes in rice and Arabidopsis,respectively, could be retrieved. The results were analyzedin three steps. In the first step, genes conserved amongdifferent domains of life (viz. archaea, bacteria, andeukaryotes) were clustered for both rice and Arabidopsis(Fig. 3a,c; Supplemental data files 5 and 6). In the secondstep, the intronless genes limited specifically to a taxonomicgroup or in combination with another, were clustered(Fig. 3b,d; Supplemental data files 7 and 8). As a finalstep, the cellular role category of intronless genes present ineach taxonomic group or combination was determined(Tables 1 and 2). Such clustering provides important

information on the evolution and functional conservationof intronless genes. Interestingly, 301 of rice and 296 ofArabidopsis intronless genes are conserved across alldomains of life. These proteins are predicted to be involvedin basic cellular processes (e.g., translation and energymetabolism) and probably are essential for survival of alldomains of life. These intronless genes can be termed asslowly evolving genes. Consistent with this idea, it has beenproposed that essential genes evolve more slowly than non-essential genes (Wilson et al. 1977). Moreover, it has beendemonstrated that functionally important genes are moreevolutionarily conserved than less vital genes (Jordan et al.2002).

Furthermore, of the 343 and 350 intronless genes of riceand Arabidopsis, respectively, shared by archaea andeukaryotes, 42 and 54 are present only in these twokingdoms; the remaining 301 and 296 genes are presentin bacteria also. Similarly, 1,281 (16.7%) and 766 (14%)intronless genes of rice and Arabidopsis, respectively, areshared by bacteria and eukaryotes only. These groups ofproteins are well represented in the following functionalclasses: translation, cell envelope, energy metabolism,regulatory functions, and amino acid metabolism. None ofthe rice and Arabidopsis intronless genes has a homolog inarchaea or archaea and bacteria.

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Fig. 3 Distribution of rice andArabidopsis intronless genesamong different taxonomicgroups. a, c Venn diagramshowing the classification ofrice (a) and Arabidopsis (c)intronless genes into differentdomains (archaea, bacteria, andeukaryotes) of life.b, d Distribution of rice (b) andArabidopsis (d) intronless genesspecific to different taxonomicgroup combinations. A archaea,B bacteria, E eukaryotes, ABarchaea and bacteria, AE archaeaand eukaryotes, BE bacteria andeukaryotes, ABE archaea,bacteria, and eukaryotes,F fungi, M metazoan, P plants,OE other eukaryotes,AP archaea and plants, BP bac-teria and plants, MP metazoanand plants, FP fungi and plants,OEP other eukaryotes and plants

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Amazingly, 68% of rice and 77% of Arabidopsis intronlessgenes are present only in eukaryotic genomes. In addition,several rice and Arabidopsis intronless genes conserved ineukaryotic genomes are limited only to a taxonomic group(fungi, metazoan, plants, and other eukaryotes) or along withplants (metazoan and plants, fungi and plants, and othereukaryotes and plants). For example, 51% and 57% ofintronless genes of rice and Arabidopsis, respectively, haveorthologs only in plants and may represent the plant-specificproteins. These proteins are associated with diverse cellularrole categories, including translation, energy metabolism,cell envelope, amino acid biosynthesis, and transport andbinding. These proteins may have evolved only in plants orgot lost from other organisms during evolution. Interestingly,some of the rice and Arabidopsis intronless genes are present

only in archaea and bacteria other than the plants. Further-more, 30 rice and one Arabidopsis intronless genes havehomologs in bacteria only. These genes may represent theexamples of lateral gene transfer (LGT) events from archaeaor bacteria to plants. Although LGT has been acknowledgedas a major force in the evolution of prokaryotic genomes(Boucher et al. 2003), several recent evidences indicate thatthis process also occurs in the evolution of eukaryoticgenomes including plants (Rujan and Martin 2001; Copleyand Dhillon 2002; Andersson 2005).

ORFans

The species-specific unique sequences that share nosignificant sequence similarity with any ORFs outside the

Table 2 Distribution of Arabidopsis intronless genes in different taxonomic groups according to their functional category

Functional category B AE BE ABE E F M P AP BP MP FP OEP

AAB 0 6 48 38 511 0 0 423 0 6 18 9 2BC 0 2 14 16 93 0 0 79 0 2 7 0 1CE 1 1 193 39 672 0 0 550 0 30 37 17 12CP 0 0 0 0 0 0 0 0 0 0 0 0 0CIM 0 2 80 24 381 1 0 238 0 8 35 16 5EM 0 16 100 41 509 0 0 419 1 15 20 11 4FAM 0 0 7 0 34 0 1 22 0 1 1 1 1PP 0 0 28 28 220 0 0 148 0 5 26 6 0RF 0 4 41 7 294 0 0 182 0 3 33 3 11RT 0 0 23 5 87 0 0 60 0 2 5 1 1T 0 23 181 67 1,077 0 3 774 1 17 40 8 30TB 0 0 51 30 328 0 1 226 0 7 20 1 5Total 1 54 766 295 4,206 1 5 3,121 2 96 242 73 72

The description of functional categories and taxonomic groups is same as given for Table 1.

Table 1 Distribution of rice intronless genes in different taxonomic groups according to their functional category

Functional category B AE BE ABE E F M P OE AP BP MP FP OEP

AAB 0 10 83 25 406 0 0 349 0 0 14 23 9 3BC 0 0 25 9 106 0 0 81 0 0 4 6 4 1CE 4 1 221 43 808 1 1 638 0 0 48 56 28 8CP 0 0 0 0 1 0 0 0 0 0 0 0 0 0CIM 1 1 106 31 402 0 0 288 1 0 11 38 8 4EM 6 6 189 55 984 2 8 766 0 0 38 74 16 11FAM 0 1 12 1 42 0 3 36 0 1 2 0 0 0PP 1 2 69 21 329 0 0 216 0 0 14 37 17 4RF 3 3 92 12 259 0 5 165 0 0 14 35 4 2RT 1 0 39 3 86 0 1 59 1 0 5 4 4 1T 9 18 379 62 1323 7 12 984 0 1 89 94 24 10TB 5 0 66 39 425 0 0 321 0 0 17 33 5 2Total 30 42 1,281 301 5,172 10 30 3,903 2 2 256 400 119 46

AAB Amino acid biosynthesis, BC biosynthesis of cofactors, CE cell envelope, CP cellular processes, CIM central intermediary metabolism, EMenergy metabolism, FAM fatty acid metabolism, PP purines and pyrimidines, RF regulatory functions, RT replication and transcription, Ttranslation, TB transport and binding, B bacteria, AE archaea and eukaryotes, BE bacteria and eukaryotes, ABE archaea, bacteria, and eukaryotes,E eukaryotes, F fungi, M metazoan, P plants, OE other eukaryotes, AP archaea and plants, BP bacteria and plants, MP metazoan and plants, FPfungi and plants, OEP other eukaryotes and plants

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genome where they reside are referred to as ORFans, and,in particular, singleton ORFans (Fischer and Eisenberg1999; Siew and Fischer 2003a, b). The complete genomesequencing of several organisms has demonstrated thatORFans are integral components of most sequencedgenomes and the percentage of singleton ORFans can beas high as 60% as in the case of human malaria parasitePlasmodium falciparum (Gardner et al. 2002; Siew andFischer 2003a, b). Our study reveals 831 (7.48%) intronlessgenes represent ORFans in rice (Fig. 3a; Supplemental datafile 9). However, only 144 (2.46%) intronless genesrepresent ORFans in Arabidopsis (Fig. 3c; Supplementaldata file 9). We investigated these to learn about functionsthey encode.

Most of rice intronless ORFans have been annotated ashypothetical and other few as expressed protein, suggestingthe possibility of mis-annotation. Therefore, to confirm thetranscription of rice ORFans, their expression was analyzedusing gene expression evidence search page (http://www.tigr.org/tdb/e2k1/osa1/locus_expression_evidence.shtml)available at TIGR rice genome annotation. This analysisrevealed that one or more massively parallel signaturesequencing (MPSS) tag, FL-cDNA, EST, and/or peptidesequence(s) were available for only 292 (35%) of 831 riceORFans, indicating that only some of these genes areexpressed and encode functional proteins. Conversely, mostof the Arabidopsis intronless ORFans represent expressedgenes. This can explain the large difference in the number ofpredicted intronless ORFans between rice and Arabidopsis.Putative function has been assigned to only three and ten ofintronless ORFans of rice and Arabidopsis, respectively.Taken together, it can be speculated that ORFans may haveevolved only recently in these organisms and performspecific functions that have not been characterized so far.The other possibility can be that these proteins might havebeen present in other organisms also but got lost duringevolution. However, the possibility of some of theseproteins moving from ORFan to non-ORFan categorycannot be ruled out as more and more of the genomesequences of different organisms will become available.Nevertheless, the functions of ORFans, the most interestinggenome content of these two groups of angiosperms, remainto be discovered.

Furthermore, because only a few ORFans have beenexperimentally characterized, it has been suggested thatmost ORFans are not likely to correspond to functionalproteins, but rather to rapidly evolving proteins with non-essential roles (Schmid and Aquadro 2001; Long 2001;Domazet-Loso and Tautz 2003). To investigate the functionsof rice and Arabidopsis intronless ORFans, their distributionamong different cellular role categories was determinedusing ProtFun (Fig. 4). Surprisingly, about 41% of rice and38% of Arabidopsis intronless ORFans belong to translation

category, a highly conserved protein machinery amongdifferent organisms. Another functional category wellrepresented among ORFans is energy metabolism. Theseresults indicate that even the components of basic cellularmachinery such as translation and metabolic processes arerapidly evolving, and several of these may perform species-specific functions.

Paralogs of intronless genes

Plant genomes seem to have evolved through polyploidiza-tion and subsequent gene loss (Bancroft 2002; Bowers et al.2003; Paterson et al. 2004a). Polyploidization gives rise togene duplications and some of these duplicated genes areretained as paralogs during evolution. The analysis ofancient polyploids shows that a much larger fraction offunctional paralogs is actually retained (Veitia 2005). Inpaleopolyploid yeast, Saccharomyces cerevisiae, about 12%of paralogs have retained functionality for 100 million years(Kellis et al. 2004). The fraction of retained paralogs rises to72% in maize, through 11 million years of evolution (Ahnand Tanksley 1993); however, their functionality is yet to beassessed.

Similarly, rice and Arabidopsis also provide manyexamples of functional paralogs retained over severalcycles of polyploidization (Veitia 2005; Chapman et al.2006). To identify the paralogs of rice and Arabidopsisintronless genes, BLAST search of their protein sequenceswere performed against themselves and other intron-containing genes. This analysis revealed that 1,709 (15%)and 1,359 (23%) of rice and Arabidopsis intronless genes,respectively, have at least one intronless or intron-containingparalog (Fig. 5). Many of these genes encode proteinscontaining conserved domains and are implicated in criticalprocesses of plant growth and development. The preferential

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retention of duplicated genes encoding key domains can beexplained partly due to gene homogenization processes(Chapman et al. 2006). The selective advantage of retentionof duplicated genes may help buffer crucial functions(Chapman et al. 2006). Among these, 121 of rice and 91of Arabidopsis intronless genes have both intronless andintron-containing paralogs. Another 127 and 82 intronlessgenes of rice and Arabidopsis, respectively, have onlyintron-containing paralogs. The genesis of intron-containingparalogs from intronless genes is explained by events ofrecombinations with reverse transcribed pre-mRNAs(Boudet et al. 2001; Lecharny et al. 2003). The examplesof evolution of gene families exhibiting diverse exon/intronstructures by duplication from a single ancestral gene arefound both in plants and animals (Gotoh 1998; Boudet et al.2001; Jain et al. 2006a). Moreover, the prevalence of introngain over intron loss in the evolution of paralogous genefamilies has been demonstrated in eukaryotes (Babenko etal. 2004), indicating the evolution of intron-containingparalogs from intronless genes.

Conserved intronless genes in rice and Arabidopsis

ProtFun analysis of functional category and GO annotationshowed consistency between rice and Arabidopsis intronlessproteins without strong biases, as described earlier. Further,by reciprocal BLASTP searches, 4,362 (74.6%) of theproteins encoded by intronless genes in Arabidopsis were

found to have orthologs in rice that are also intronless(Fig. 6a). This estimate is comparatively lower than that hasbeen estimated recently (about 90%) for all the Arabidopsisgenes (International Rice Genome Sequencing Project 2005).Nearly 16.6% (972) of these genes, are highly conserved(expect value <E-100) in rice and Arabidopsis and mayperform essentially similar functions in these plants. Theseproteins are involved in basic cellular processes such as cellenvelope, purines and pyrimidines, amino acid biosynthesisand transport and binding (Fig. 6b). Several gene families,including LRR repeat proteins, pentatricopeptide repeatproteins, protein kinases, disease resistance proteins, cyto-chrome P450 genes, are overrepresented in this list. Theother 1,484 Arabidopsis intronless genes lack significanthomology to rice intronless genes. Most of these areclassified as hypothetical or expressed proteins, whichsuggest that some of these may be inaccurately predictedand others could be dicot-specific. The comparative analysisof rice and Arabidopsis at whole genome level also showed

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Fig. 5 Venn diagram showing paralogs of rice and Arabidopsisintronless genes. The number of rice and Arabidopsis intronless geneswith intronless and/or intron-containing paralogs are given

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b

Nu

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er o

f g

enes

0

200

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<::::: High Homology Low ::::>

0

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AAB BC CE CP CIM EM FAM PP RF RT T TB

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Cellular roleFig. 6 Conservation among rice and Arabidopsis intronless genes.a Protein sequences of predicted Arabidopsis intronless genes werealigned with that of rice intronless genes (BLASTP E-value ≤−6). Thenumber of proteins with expect value <E-180 (high homology) to ≤E-6(low homology) are shown in intervals spanning ten exponents (forexample, <−180, −180 to −171, −170 to −161 and so on). b Distributionof highly conserved (<E-100) intronless genes in various cellular rolecategories. The description of cellular role categories is similar to thatgiven in legend to Fig. 2

76 Funct Integr Genomics (2008) 8:69–78

that several unknown and some of the well-characterizedArabidopsis genes do not have rice orthologs and vice versa(Goff et al. 2002; Delseny 2003; International Rice GenomeSequencing Project 2005). The basic difference betweenmonocots and dicots will become clear only when thefunctions of these largely unknown genes (both intronlessand intron-containing) will become clear.

In conclusion, based on our large-scale alignments ofannotated gene and protein sequences in rice and Arabi-dopsis, we estimate that about one-fifth of genes areintronless in these plants. The biological role of such alarge number of intronless genes in their genomes isperplexing. The functional characterization of intronlessORFans of rice and Arabidopsis can help understand thebasic difference between monocots and dicots. We believethat these results will help improve our understanding onthe differential selection (as a process or force) of intronlessgenes in plants and other eukaryotic genomes. Furthermore,the datasets provided can serve as source for comparative,evolutionary, and functional studies.

Acknowledgments We are thankful to Rashmi Jain for technicalassistance. This work was supported financially by the Department ofBiotechnology, Government of India, and the University GrantsCommission, New Delhi. MJ acknowledges the Council of Scientificand Industrial Research, New Delhi, for the award of Senior ResearchFellowship.

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