genomic organization, differential expression, and ... · genomic organization, differential...

Genomic Organization, Differential Expression,and Interaction of SQUAMOSA Promoter-Binding-LikeTranscription Factors and microRNA156 in Rice1[W]

Kabin Xie, Congqing Wu, and Lizhong Xiong*

National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research(Wuhan), Huazhong Agricultural University, Wuhan 430070, China

Transcription factors play essential roles in the developmental processes of plants. Many such factors are regulated bymicroRNAs (miRNAs). SQUAMOSA (SQUA) promoter-binding-like (SPL) genes encode plant-specific transcription factors,some of which contain complementary sequences of miRNA156. In this study, 19 rice (Oryza sativa) SPL (OsSPL) genes and 12rice miRNA156 (OsmiR156) precursors were identified in the rice genome. Sequence and experimental analysis suggested that11 OsSPL genes were putative targets of OsmiR156. Plant SPL proteins were classified into six subgroups based on thephylogenetic analysis of SQUA promoter-binding protein domain. Diverse exon-intron structures and distinct organizations ofputative motifs beyond the SQUA promoter-binding protein domains were identified in the OsSPL gene family. Transcriptlevel analysis of OsSPL genes in various rice tissues and organs revealed different tempospatial expression patterns. More thanhalf of the OsSPL genes including most OsmiR156-targeted genes are predominantly expressed in the young panicles, whereasOsmiR156 genes are predominantly expressed in the young shoots and leaves of rice. Overexpression of two OsmiR156 genes(OsmiR156b and OsmiR156h) in rice resulted in severe dwarfism, strongly reduced panicle size, and delayed flowering,suggesting that OsmiR156 and OsSPL target genes are involved in various developmental processes, especially the flowerdevelopment of rice. Different patterns of transcript changes (decreased or unchanged) of different target genes in same tissueand of same target gene in different tissues detected in the OsmiR156-overexpressing plants suggested diverse interactionsbetween OsmiR156 and OsSPL target genes in a tissue-specific manner.

Transcription factors play essential roles in the reg-ulation networks of plant developmental processes. InArabidopsis (Arabidopsis thaliana), about 5.9% of esti-matedgenes encodeputative transcription factors; about45% of these are specific to plants (Riechmann et al.,2000). The temporal and spatial expressions of sometranscription factors can change the identity of cells ortissues by regulating the expression of specific down-stream genes.

SQUAMOSA (SQUA) promoter-binding-like (SPL)genes represent a family of plant-specific transcriptionfactors (Klein et al., 1996; Cardon et al., 1999). Thecommon feature of SPL genes is that SPL proteins con-tain a highly conserved DNA-binding domain (SQUApromoter-binding protein [SBP] domain). This domainfeatures a novel zinc finger motif that contains twozinc-binding sites assembled as Cys-Cys-His-Cys and

Cys-Cys-Cys-His, respectively (Yamasaki et al., 2004).SBP1 and SBP2 are the foremost SPL genes isolatedfrom Antirrhinum majus and were named based on thein vitro-binding activity of SBP1 and SBP2 to the cis-element upstream of the gene SQUA (Klein et al., 1996).SQUA is a member of the MIKC group of the MADS-box gene family in Arabidopsis that specifies flowermeristem identity (Huijser et al., 1992; Jack, 2004). Theexpression of SBP1 and SBP2 is developmentally reg-ulated and transcriptional activation of SBP1 and SBP2precedes that of SQUA (Klein et al., 1996). In Arabi-dopsis, AtSPL3 was identified as an ortholog of SBP1and SBP2 (Cardon et al., 1997, 1999). The AtSPL3 rec-ognized a conserved cis-element in the promoter re-gion of APETALA1 (AP1), an ortholog of SQUA inArabidopsis (Cardon et al., 1997). The sequence of thecis-element to which SPLs bind in vitro was predictedto be TNCGTACAA in Arabidopsis (Cardon et al.,1999). Another SPL protein (BpSPL1) isolated fromBetula pendula also showed binding ability to the cis-element of BpMADS5, a close homolog of ArabidopsisFRUITFULL (Lannenpaa et al., 2004). Recently, a SBPdomain protein Copper Response Regulator, whichregulates nutritional copper signal and recognizes aGTAC core cis-element, was identified in single-cellgreen plant Chlamydomonas (Kropat et al., 2005).

In the Arabidopsis genome, 16 putative SPL genesthat contained the SBP domain were predicted basedon sequence analysis, and several AtSPL genes werethought to have roles in the regulation of plant

1 This work was supported by grants from the National Programon High Technology Development, the National Program on theDevelopment of Basic Research, and the National Natural ScienceFoundation, China.

* Corresponding author; e-mail [email protected]; fax86–27–87287092.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Lizhong Xiong ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.106.084475

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development (Cardon et al., 1999). Constitutive ex-pression of AtSPL3 in Arabidopsis caused early flow-ering that is similar to the phenotype that resultedfrom the overexpression of AP1 (Cardon et al., 1997).However, constitutive expression of AtSPL3 in the ap1mutant showed that AP1 was not required for the earlyflowering phenotype of the AtSPL3 transgenic plants(Cardon et al., 1997). The analysis of three independenttransposon-tagged atspl8 mutants indicated thatAtSPL8 was involved in the regulation of microsporo-genesis, megasporogenesis, trichome formation onsepals, and stamen filament elongation (Unte et al.,2003). The T-DNA insertion mutant of atspl14 showedelongated petioles and enhanced leaf margin serration(Stone et al., 2005). In maize (Zea mays), the liguleless1mutant of an SPL gene showed defects in ligule andauricle formation and no blade-sheath boundary(Moreno et al., 1997).Discovery of microRNAs (miRNAs) has profoundly

enriched our understanding of gene regulation inanimals and plants. MiRNAs are small RNA mole-cules (20–24 nucleotides) that can bind to the mRNAsof target genes by imperfect base pairing and result incleavage ofmRNA or repression of translation througha RNA-induced silencing complex (Bartel, 2004). Numer-ous miRNAs have recently been identified (Reinhartet al., 2002; Bonnet et al., 2004; Sunkar and Zhu,2004; Sunkar et al., 2005). More than 2,900 miRNAentries have been registered in the miRNA database(miRbase; http://microrna.sanger.ac.uk/, release 7.0),including 117 from Arabidopsis and 173 from rice(Oryza sativa). Increasing evidence shows that plantmiRNAs constitute a substantial fraction of the generegulatory networks for diverse aspects of plant devel-opment such as leaf polarity (Kidner and Martienssen,2004), leaf morphogenesis (Palatnik et al., 2003), flow-ering development (Chen, 2004; Millar and Gubler,2005), root cap formation (Wang et al., 2005), auxinsignal regulation (Guo et al., 2005; Mallory et al., 2005),and stress response (Jones-Rhoades and Bartel,2004; Sunkar and Zhu, 2004). About half the targetgenes ofmiRNA are transcription factors (Bartel, 2004).Computational analysis indicated that some Arabi-dopsis SPL genes were also regulated by miRNAgenes of the miRNA156 family (Rhoades et al., 2002;Bonnet et al., 2004). ThemiR156 and its target genes arethought to be involved in some important develop-mental processes since overexpression of AtmiR156 inArabidopsis resulted in various phenotypic changessuch as increased number of leaves, delayed flowering,and decreased apical dominance (Schwab et al., 2005).As one of the most important crops worldwide, rice

has become a model plant of monocot species forfunctional genomics studies. Systematic analysis ofSPL and miR156 genes in rice will certainly improveour understanding of the complex regulatory networksin monocot species. Here we report on the analyses ofSPL and miR156 gene families in rice genome for theirgenomic organization, gene structures, motif compo-sition, and expression levels in various tissues and

organs of rice. Moreover, two OsmiR156 precursorswere overexpressed in rice to study the functionalrelationship of SPL and miR156 genes.

RESULTS

Identification of OsSPL and OsmiR156 Genesin the Rice Genome

We used BLAST to search the GeneBank databasewith reported SPL protein sequences as queries, andonly 15 putative SPL genes were identified in the ricegenome. To identify all putative rice SPL genes, wesearched the annotation database of rice (data down-loaded fromThe Institute forGenomicResearch [TIGR],Beijing Genomic Institute, and Knowledge-BasedOryza Molecular Biological Encyclopedia [KOME]) witha profile Hidden Molkov model (pHMM) of the SBPdomain, since pHMM was considered to be a moreefficient approach than pairwise comparison (Eddy,1998). By removing the redundant sequences, weidentified 22 putative protein sequences by pHMMsearches with an E value less than 1E-5. However, twoof them (AK10915 and 11681.m02353 [gene modelnumber of TIGR rice pseudomolecular 3.0]) had nozinc finger motif that features the SBP domain andwere excluded from further analysis. Two other genemodels (11681.m02775 and 11681.m02776 that areclosely linked but interrupted by a 27.6 kb of genomicsequence) can form one complete SBP domain onlywhen the two gene models are fused. Therefore, atleast 19 putative SPL genes existed in the rice genome.For convenience of description, a systemic nomencla-ture of OsSPLxvn (x for the serial number of SPL genesbased on their order on chromosomes, vn for variantnumberof differentially spliced transcripts)was adoptedfor rice SPL genes (Table I). To date, 11OsSPL genes aresupported with full-length cDNAs in the KOME da-tabase (Table I). In this study, the complete openreading frames of all the OsSPL genes except OsSPL19were isolated from indica rice Minghui 63 with gene-specific primers (Supplemental Table I). The OsSPL19was excluded from the following analysis unless spe-cially pointed out, since it is likely a pseudogene.

Twelve putative members of the miR156 family inrice were predicted in themiRbase. Currently, only fiveOsmiR156 precursors from three loci are supported bycDNAs or expressed sequence tags in the publicdatabase. OsmiR156d matches the cDNA sequenceAK073452. OsmiR156b and OsmiR156c are mapped tothe same transcription unit (accession no. AK110797),which was similar to the phenomenon that manymiRNAs in animals are encoded by a polycistronictranscript through an initial phase of local duplication(Tanzer and Stadler, 2004). Precursors of OsmiR156hand OsmiR156j are from the same transcription unitAK103769 but are different in length.

A comparison of the OsmiR156 mature sequenceto the OsSPL sequences showed that 11 OsSPLscontained sequences that are complementary to the

SPL and miR156 Gene Families in Rice

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OsmiR156 mature sequence, with one mismatch at the14th nucleotide (for miR156a–j and miR156l), or onemismatch at the first (for OsmiR156l) or last nucleotide(Fig. 1A). Another search of all rice genomic sequenceswith the mature sequence of OsmiR156 resulted in noadditional significant matches except the 11 OsSPLand OsmiR156 genes, which suggests that OsmiR156may specially targetOsSPL genes in rice. The target sitesof OsmiR156 are located in the coding regions (down-stream of the SBP domain) exceptOsSPL4 andOsSPL13that contain the target sites in the 3#-untranslatedregion (UTR). Interestingly, the amino acid residuesencoded by the miR156 targeting sites are exactly thesame or highly conserved (for one residue) for the ninetargetOsSPL genes (Fig. 1A) and 10 targetAtSPL genesin Arabidopsis (Rhoades et al., 2002).

The chromosome locations of OsSPL genes andOsmiR156 precursors were determined by in siliconmapping of gene sequences to the rice pseudomole-cules build 4.0 (International Rice Genome SequencingProject, 2005). All OsSPL genes (including OsSPL19)and OsmiR156 precursors can be mapped to the ricegenome according to physical maps of bacterial arti-

ficial chromosome (BAC)/P1-derived artificial chro-mosome (PAC) clones. OsSPL genes and OsmiR156precursors are unevenly distributed in the rice genome(Fig. 1B). Chromosomal regions clustered with closelylinked OsSPL and/or OsmiR156 genes were found inchromosomes 2 (OsSPL4, OsSPL5, and OsmiR156d), 6(OsSPL10, OsSPL11, OsmiR156h, and OsmiR156j), 8(OsSPL14 and OsSPL15), and 9 (OsSPL17, OsSPL18,and OsmiR156k). In the regions clustered with OsSPLand OsmiR156 genes, at least one OsSPL gene containsthe sequence that is complementary to the maturesequence of miR156.

Phylogenetic Analysis of the OsSPL and OsmiR156Gene Families

To study the evolutional relationships of SPL genesin plants, we collected a data set of 48 putative SPLprotein sequences, including 18 from rice, 16 fromArabidopsis, and 14 fromother floweringplants (maize,A. majus, and B. pendula) for phylogenetic analysis(gene names and accession numbers are listed inSupplemental Table II). Alignment of the full-length

Table I. General information about OsSPL genes

Gene Namea Locusb BACc Spliced FormsdAccession No. of

cDNA in KOMEe

OsSPL1 LOC_Os01g18850 AP002482 OsSPL1v1 AK068471OsSPL1v2 AK072849

OsSPL2 LOC_Os01g69830 AP006167 AK062581OsSPL3 LOC_Os02g04680 AP005294OsSPL4 LOC_Os02g07780 AP005772 OsSPL4v1 AK062651

OsSPL4v2 AK066057OsSPL4v3 AK068702OsSPL4v4 AK061484

OsSPL5 LOC_Os02g08070 AP004838 OsSPL5v1OsSPL5v2

OsSPL6 LOC_Os03g61760 AC092557 OsSPL6v1 AK069847OsSPL6v2 AK072164

OsSPL7 LOC_Os04g46580 AL606633 AK068749OsSPL8 LOC_Os04g56170 AL606685 AK068104OsSPL9 LOC_Os05g33810 AC137620 AK100057OsSPL10 LOC_Os06g44860 AP005760OsSPL11 LOC_Os06g45310 AP004744OsSPL12 LOC_Os06g49010 AP004324 OsSPL12v1 AK061641

OsSPL12v2 AK073179OsSPL12v3 AK102136

OsSPL13 LOC_Os07g32170 AP005186OsSPL14 LOC_Os08g39890 AP006049 AK107191OsSPL15 LOC_Os08g40260 AP004765OsSPL16 LOC_Os08g41940 AP005529 AK109469OsSPL17 AC108762 AK063561OsSPL18 AC108759OsSPL19f LOC_Os11g30370,

LOC_Os11g30380AC133608

aSerial nomenclature of OsSPL genes based on the order of chromosomes. bTIGR rice pseudomol-ecule 3.0 locus identifier of each gene (no locus identifier numbers were found for OsSPL17 andOsSPL18). cAccession number of rice (O. sativa L. subsp. japonica) BAC clone. dAlternativespliced forms for OsSPL genes if available. eGenBank number of the full-length cDNA in KOMEdatabase if available. fAn insert of sequence (about 27.6 kb) is found in the two gene models(LOC_Os11g30370 and LOC_Os11g30380) and the SBP domain was separated by the insert.

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protein sequences showed no consensus sequenceswhen the SBP domains were masked. Therefore, onlythe protein sequences of SBP domain (SupplementalTable II) were used for phylogenetic analysis.The phylogenetic tree suggested that the plant SPL

family was evolutionally diversified (Fig. 2). The 48plant SPLs were classified into six subgroups (S1–S6)according to the unrooted phylogenetic tree. Gener-ally, SPLs from Arabidopsis and rice are almost evenlydistributed in the six subgroups (Fig. 2). Within spe-

cific subgroups (such as S5), however, rice SPLs hadcloser relationships to SPLs of maize than to SPLs ofArabidopsis. These results suggest that plant SPLgenes may be derived from common ancestors, butsome of which may have been differentiated sepa-rately in monocotyledon and dicotyledon plants. In-terestingly, the miR156-targeted SPLs (only SPLs inrice and Arabidopsis were analyzed) were distributedin four subgroups (S1, S2, S3, and S6) but not in theother two subgroups (S4 and S5).

The multiple alignments of the miR156 stem-loopsequences from rice and Arabidopsis revealed ex-tremely low sequence identity beyond the maturemiRNA sequence region (Supplemental Fig. 1). Phy-logenetic analysis of the OsmiR156 family with thealigned sequences suggested that the bootstrap valuesof most internodes were not significant enough to sup-port either Maxparisom or maximum-likelihood trees(data not shown). The second structures of OsmiR156family calculated with the RNAalifold program(Hofacker et al., 2002) revealed no consensus secondstructure beyond the mature miR156 encoding region(Supplemental Fig. 1). Variable insertions of nucleo-tides also existed in the complementary region of themature OsmiR156 sequence, which has made thestem-loop structure more diversified. These resultssuggest that the precursor sequences of the OsmiR156family have been extremely diversified beyond themature sequence region. Nevertheless, the divergentsecondary structures ofOsmiR156precursorspredictedby Vienna RNA package version 1.4 (Hofacker, 2003)are quite stable based on the free energy (Supplemen-tal Fig. 2). Phylogenetic tree of miR156 family wasderived from RNA-based phylogenetic inference anal-ysis (PHASE version 2.0), a method that specificallyconsiders the secondary structure and compensatoryvariations in paired nucleotides (Hudelot et al., 2003),although the postprobability for some node of thephylogenetic tree of miR156 family is relatively low.The phylogenetic tree suggested that some duplicatedOsmiR156 genes (such as OsmiR156h and OsmiR156j)had a distinct boundary to other miR156 genes fromArabidopsis and rice (Supplemental Fig. 3).

Diverse Exon-Intron Structure and Motif Compositionin OsSPL Family

Generally, the number of exons in the coding regionand the length of the coding sequences are similar forthe OsSPL genes within phylogenetic subgroups butquite different between phylogenetic subgroups (Sup-plemental Fig. 4). However, the exon-intron structuresof genes in S1 and S6 are less conserved than in theother four subgroups. An intron existed in the SBPdomains of all OsSPL genes except OsSPL4v1 andOsSPL2. The position of this intron is extremely con-served in rice (located in the codon of the 49th aminoacid of SBP domain) and in Arabidopsis (Cardon et al.,1999). Nevertheless, the length of this intron (fromnone to 27 kb) differs sharply among OsSPL genes

Figure 1. Sequence analysis of OsmiR156 genes. A, Sequence align-ment of OsmiR156 mature sequences with complementary sequencesof OsSPL genes. The conserved amino acid sequence encoded by thetarget sequences is shown at the bottom. The dots betweenmiR156 andtargeted OsSPL sequences indicate mismatches. The complementarysequences of OsmiR156 in OsSPL4 and OsSPL13 (indicated by anasterisk) are not in the coding regions. B, Distribution of OsSPL andOsmiR156 genes in rice genome. Locations of closely linked genes aremagnified. OsmiR156b and OsmiR156c are mapped to the sametranscript (corresponding to the cDNA AK110791). OsmiR156j andOsmiR156h are also mapped to same transcript (corresponding to thecDNA AK103769). OsSPL4 and OsSPL5 are closely linked withOsmiR156d, and OsSPL17 and OsSPL18 are closely linked withOsmiR156k. The putative target genes of OsmiR156 are underlined.





(Supplemental Fig. 4). Moreover, repetitive sequenceswere found in the intron for some OsSPL genes (datanot shown), which may indicate that the differentlength of this intron may be partially resulted from theexpansion of repetitive sequences. Based on the full-length cDNAs of OsSPL genes from the KOME data-base, at least eight OsSPLs (OsSPL1, OsSPL4, OsSPL5,OsSPL6, OsSPL10, OsSPL12, OsSPL13, and OsSPL17)have alternatively spliced forms (Supplemental Fig. 4).Except OsSPL4, all other three OsSPL genes have theiralternative splicing sites in the UTR regions.

According to the solution three-dimensional struc-ture of AtSPL4 and AtSPL7 proteins (Yamasaki et al.,2004), the SBP domain of OsSPLs can be divided intofour motifs (Fig. 3A): zinc finger 1 (Zn1), zinc finger 2(Zn2), joint peptide (JP) of Zn1 and Zn2, and nuclearlocation signal (NLS). Zn1 (CX4CX10–11HX5C) is aCCHC-type zinc finger and divergent in the SBP domain,especially for the S1 and S6 subgroups (SupplementalFig. 5); Zn2 (CX2CX15CX6H) is a CCCH-type zincfinger. JP is less conserved than the two zinc fingers(Fig. 3A) and was thought to have a crucial role in thecharacteristic packing of SBP domains and therebymodifies the protein-DNA interaction process (Yamasakiet al., 2004). The bipartite NLS motif is highly con-served not only in the SPL family but also in otherproteins (Dingwall and Laskey, 1991). Except for SBP

domain, no analysis of the remaining region of SPLproteins for conserved domains or motifs has beenreported. By using the software Multiple EM for MotifElicitation (MEME) version 3.0, 10 putative motifswere identified in OsSPL proteins with E values lessthan 1.00E-30 (Table II; Fig. 3B). A search of Inter-Prodatabase by Inter-ProScan (Mulder et al., 2005) withthese putative motifs from MEME showed that mostmotifs (except 4, 5, 7, and 8) have matches in the Inter-Pro database (Table II). Occasionally, one motif (suchas the JP in OsSPL14 and motif 4 in OsSPL1) appearedtwice in single protein sequence (Fig. 3B). The MEMEprogram considered the SBP domain as three motifs,Zn1, Zn2-NLS, and JP. Motif 7 is encoded by a con-served sequence that is complementary to the maturesequence of miR156 (Fig. 1). Although OsSPL6 con-tained a predicted motif 7 (Fig. 3B), it cannot beconsidered the target gene of miR156 since the DNAsequence of the predicted motif 7 has six mis-matches with the mature sequence of miR156. Notably,motif 6 is an ankyrin repeat (Interpro accession no.IPR002110), which is one of the most common motifsfor protein-protein interaction and has been found inproteins with diverse functions such as transcriptionalinitiators, cell-cycle regulators, cytoskeleton, ion trans-porters, and signal transducers (Lux et al., 1990; Gorinaand Pavletich, 1996; Luh et al., 1997; Batchelor et al.,

Figure 2. Unrooted tree of SPL family based on the protein sequences of SBP domains. The numbers at branching sites indicated theposterior probability values for nodal support. The SPL genes fromArabidopsis and rice that contained complementary sequence ofmiR156 were underlined. The names of Arabidopsis SPL genes were downloaded from the Arabidopsis Functional GenomicsNetwork (http://web.uni-frankfurt.de/fb15/botanik/mcb/AFGN/Huijser.htm); other plant SPL genes are from SwissProt. The SBPdomain sequencesof all these genesand theaccessionnumbersof genes fromplantsother than riceare listed inSupplemental Table I.The accession numbers of SPL genes of rice are listed in Table I. Am, A. majus; At, Arabidopsis; Zm, maize; Bp, Betu psy; Os, rice.

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1998; Mosavi et al., 2002). The putative functions forthe other motifs (4, 5, 8, 9, and 10) generated fromMEME are currently unknown.

Differential Expression Profiles of OsSPLand OsmiR156 Genes

Diverse gene structures of OsSPL and OsmiR156genes prompted us to investigate their expressionprofiles in various tissues or organs of rice. Preliminaryexperiments by RNA gel-blot analysis suggested thatmost of theOsSPL genes had very low expression levelsin most rice tissues and organs (data not shown). There-fore, semiquantitative reverse transcription (RT)-PCRwas used to detect the expression of OsSPL genes invarious rice tissues including panicles at differentdevelopmental stages since some SPL genes of dicot-yledon plants were thought to be involved in inflores-cence development (Klein et al., 1996; Cardon et al.,1997, 1999; Unte et al., 2003).Based on the RT-PCR of the non-target-site regions

in 3# UTR, the expression patterns of OsSPL genes canbe classified into three types according to their expres-sion patterns (Fig. 4A). The first type of genes (includ-ing OsSPL7, OsSPL12, OsSPL14, OsSPL16, OsSPL17,

and OsSPL18) expressed relatively stronger in youngpanicles than other tissues investigated. The secondtype of genes (OsSPL2, OsSPL4, OsSPL8, OsSPL10,OsSPL11, and OsSPL15) expressed in most of the tis-sues investigated, but had higher expression levels instem, leaf sheath, and young panicles than in othertissues. The third type of genes (OsSPL1, OsSPL3,OsSPL5, OsSPL6, OsSPL9, and OsSPL13) expressed inall the tissues investigated without obvious differenceof expression level. Interestingly, Most of the targetgenes showed higher expression levels in young pan-icle than in most other tissues. In addition, differen-tially spliced transcripts were detected in differenttissues for several OsSPL genes such as OsSPL4,OsSPL5, and OsSPL13 (Fig. 4A).

We also designed a pair of primers with amplifica-tion region covering the miR156 complementary site(Supplemental Table I) of each target OsSPL gene todetect the transcript level in the same set of rice tissues(Fig. 4A). The results showed that most of the 11 targetgenes had the same expression patterns as detected bythe primers from 3#-UTR regions. However, differentexpression in one or more tissues was detected by thetwo sets of primers for a few target genes includingOsSPL3 (in stem), OsSPL4 (in root and stem), OsSPL7

Figure 3. Organization of putative motifs in OsSPLs. A, Sequence LOGO view of the consensus SBP domain sequences based onthe 48 plant SPLs in Figure 2 (pHMM logo view of each SPL subgroup is shown in Supplemental Fig. 1). The height of the letter(amino acid) at each position represents the degree of conservation. The rectangles at the top of the plot indicate the motifs of theSBP domain. B, Putative motifs in OsSPLs identified by MEME. Numbered color boxes represent different putative motifs(annotations are listed in Table II). The expected values calculated by MEME are shown after gene names. S1 to S6 indicates thephylogenetic subgroups from Figure 2. The putative target genes of OsmiR156 are underlined.





(in 10 cm long panicles), and OsSPL18 (in all the tis-sues except panicles).

The reliability of semiquantitative RT-PCR waschecked by real-time quantitative PCR analysis offour OsSPL genes (Fig. 4B). The results showed thatthe band intensity in semiquantitative RT-PCR analy-sis generally agreed well with the relative expressionlevel by real-time quantitative analysis, which wasespecially true for the samples with more than 2-folddifference of relative expression levels.

To evaluate the transcript level of miR156 precur-sors, gene-specific primers downstream of the hairpinstructure of miR156 were designed for RT-PCR withthe purpose of minimizing the inhibitive effect of sec-ondary structure on PCR reaction. Because of limitedlength of the regions for primer designation, only twopremature transcripts (OsmiR156d and OsmiR156h)could be amplified predominantly in young shoot,etiolated shoot, and seedling leaves (Fig. 4A). PAGERNA gel-blot analysis also showed that the matureOsmiR156 had strong expression in young shoot, etio-lated shoot, and seedling leaves, weak expression inroot, stamen, and pistil, and undetectable expressionlevel in stem and young panicles (Fig. 4C; Supple-mental Fig. 6). The PAGE RNA gel-blot analysis sug-gested that the expression profile of mature OsmiR156was essentially the same as of precursors. It was in-triguing to notice that the mature OsmiR156 had verylow level, if any, in young panicles whereas most of themiR156-targeted OsSPL genes had much stronger ex-pression in youngpanicles than other tissues. Such com-plementary expression patterns between OsmiR156and target genes suggested that the targetOsSPL genesmight be tempospatially regulated by OsmiR156.

Overexpression of OsmiR156 in Rice Resultedin Abnormal Growth and Development

The OsmiR156 family has at least five expressedmembers with the same matured miRNA sequence,

suggesting difficulties in obtaining the loss-of-functionmutant of individual OsmiRNA156 genes. Towardidentification of the functions of OmiR156 and its pu-tative target genes, two transcriptionally active (sup-ported by cDNA or expressed sequence tag sequences)OsmiR156 precursors (OsmiR156b and OsmiR156h)were transformed into rice under the control of maizeubiquitin promoter (Fig. 5A). PAGE RNA gel-blotanalysis showed that the matured transcript of Os-miR156 in the leaves of the transgenic plants was muchhigher than that in the wild type, suggesting thatOsmiR156b (Mb) and OsmiR156h (Mh) were overex-pressed (Fig. 5B). All the OsmiR156-overexpressingplants (T1 generation) showed dramatic morphologi-cal changes, including significantly (P , 0.001) in-creased number of tillers (10–15 times more than thewild type) and dwarfism (Fig. 5C), late flowering (7–10d delay of flowering), significantly (P , 0.01) reducednumber of spikelets and grains per panicle (Fig. 5D),and secondary branches of panicle (Fig. 5D). Eventhough the panicle size (length and number of spike-lets) of OsmiR156-overexpressing plants was severelyaffected, the fertility was not significantly differentfrom the wild type. All the phenotypes of OsmiR156-overexpressing plants were stably inherited in T2generation (Supplemental Fig. 7). Considering the factthat most of the OsmiR156-targeted OsSPLs, but notthe OsmiR156, were predominantly expressed in pan-icles, such severe morphological change related topanicles in all the OsmiR156-overexpressing plantssuggested that some, if not all, of the OsmiR156-targeted genes might be involved in the panicle de-velopment in rice.

Differential Interaction between OsmiR156 and TargetOsSPL Genes

We further checked the transcript levels of nineputative target genes of OsmiR156 in the panicles andleaves of OsmiR156-overexpression transgenic plants

Table II. Annotation of putative motifs of OsSPL proteins identified by MEME

Motif No.a E Valueb Sitesc Annotation of Motif Conserved Amino Acids of Each Motif

1 2.0E-1,142 44 Zn2 and NLS CQQCSRFHLLSEFDEGKRSCRRRLAGHNRRRRK2 5.1E-560 45 JP YYCRHKVCEMHSKEPRVVVNG3 1.2E-330 43 Zn1 GxQPPRCQVEGCKADLSSAKD4 1.00E-178 8 Unknown RTGKIVFKLFDKEPSDFPGRLRG-

QIFNWLSNMPSDMESYIRPGCIILTVY5 6.80E-130 21 Unknown GGRIGLNLGKRTYFEPAD6 1.80E-59 5 Ankyrin repeat TPLHIAAATDDAEDVLDALTDDPQQIGISAW-

KNARDATGFTPEDYARLRG7 2.00E-59 21 miR156 complementary sited DSSCALSLLSTQSW8 6.30E-33 6 Unknown KRGVEWDLNDWDWDSDLFLAT9 1.50E-32 5 Unknown MDCNMKSSFQWDWENLIMFGPST-

TENPKKQKPTE10 4.60E-27 4 Unknown EPGRFRSFLLDFSYPRVPSSVRDAWPGIQH

aThe motif numbers correspond to the numbers in Figure 3B. bThe expected value of each motif prediction was given in the MEMEprogram. cThe number of the 48 plant SPL genes in Figure 2 contained the motifs. dSome mRNA sequences in this motif are complementaryto miR156.

Xie et al.




by semiquantitative RT-PCR (Fig. 6A). The resultshowed that, compared to the wild type, three genes(OsSPL2, OsSPL12, and OsSPL13) had decreasedmRNA levels in the flag leaves of transgenic plants,two genes (OsSPL16 and OSPL18) had obviously de-creased mRNA levels in the panicles, one gene(OsSPL14) had decreased mRNA levels in both flagleaves and panicles, and the other three genes(OsSPL3, OsSPL7, and OsSPL11) had no change ofexpression level in either tissue. Real-time PCR anal-ysis of four genes (OsSPL3, OsSPL7, OsSPL11, andOsSPL12) suggested that, except OsSPL3, the results ofother three genes agreed well with the result by semi-quantitative RT-PCR (Fig. 6B). OsSPL3 had slightlydecreased transcript level in the flag leaves of trans-genic plants and such a minor change may not havebeen detected by semiquantitative RT-PCR analysis.

Significantly decreased mRNA levels in the trans-genic plants may suggest a cleavage of the targettranscripts by the OsmiR156. To further confirm this,target genes OsSPL12 and OsSPL14 were overex-pressed in rice and the transcript levels of the twogenes in the leaves of transgenic plants were checkedby RNA gel-blot analysis using probes downstream ofthe miR156 complementary sites (Fig. 6C). Among the20 independent transgenic plants checked for eachgene, more than half of the transgenic plants hadhigher transcript levels of target genes. A distinct band(indicated by an arrow in Fig. 6C) with the sizecorresponding to the cleaved product downstream ofthe cleavage site, was detected in the leaves of thetransgenic plants of OsSPL14 and the wild plants. Thisband, but not the band corresponding to the uncleavedtranscript, had much stronger intensity in the trans-genic plants than in the wild type, suggesting a cleav-age of the transgene by miRNA. A distinct band withthe size corresponding to the cleaved product down-stream of the cleavage site of OsSPL12 was also de-tected but the band intensity showed no differencebetween the transgenic plants ofOsSPL12 and the wildplants (Fig. 6C). Rather, the uncleaved transcript levelofOsSPL12was higher in most of the transgenic plants

Figure 4. Tissue-specific expression patterns of OsSPL genes andOsmiR156. A, Semiquantitative RT-PCR of OsSPLs and OsmiR156precursors in 13 different tissues or organs of rice. The rice Actin1 genewas used as the internal control. The miR156-targeted OsSPL genes

were underlined and two pairs of primers (Supplemental Table I) wereused for each gene, one located downstream of the miR156 comple-mentary site and the other (labeled with an asterisk) amplifying theregion covering the targeting site. PCR was failed forOsSPL17 with thetarget site-spanning primers. B, Real-time quantitative PCR analysis offour OsSPL genes. The relative expression levels were calibrated basedon sample 6 (0.5–1 cm panicle). C, PAGE RNA gel-blot analysisof mature miR156 using an end-labeled complementary sequence(5#-GTGCTCACTCTCTTCTGTCA-3#). The blotwas stripped and rehybri-dized with oligonucleotide probe complementary to U6 RNA as aloading control. Amount of RNA loaded in each lane was indicated byethidium bromide staining. Tissues or organs: 1, Root at tillering stage;2, stems at tillering stage; 3, leaf sheath at 4-leaf stage; 4, leaf lamina at4-leaf stage; 5, young panicle shorter than 0.2 cm; 6, 0.5 to 1 cm youngpanicle; 7, 3 to 5 cm young panicle; 8, longer than 10 cm young panicle;9, stamen; 10, pistil; 11, shoot at 1 d after germination; 12, 3-week-oldshoot; 13, 3-week-old etiolated shoot.





than in the wild type. Together, these results suggestthat different target OsSPL genes may be differentiallyregulated by OsmiR156.

DISCUSSION

Evolution of SPL and OsmiR156 Gene Families

SPL genes encode proteins that contain SBP do-mains. So far, no SPL homologous sequence has beenfound in animals, humans, or bacteria in the public da-tabases, which may suggest that SPL genes appearedafter the divergence of plants and animals and func-tion specifically in plants. In this study, all the cur-rently available SPL sequences (including all SPLsfrom rice and Arabidopsis) in the database werecollected and phylogenetically classified into six sub-groups (Fig. 2). Our results suggested that SPLs mightbe derived from several common ancestors before themonocot and dicot plants diverged and that the evo-lution of SPLs from same ancestor may be indepen-dent in monocots (such as rice and maize) and dicots(Arabidopsis and A. majus). Different subgroups di-verged quite differently. For example, S5 had relativelylower divergence than other subgroups, which is alsosupported by the fact that OsSPLs in S5 showed moreconserved exon-intron structure (Supplemental Fig. 4)and motif composition (Fig. 3) than did the SPLs inother subgroups. Most OsSPLs in the same phyloge-netic subgroup have similar exon-intron structuresand motif compositions, which suggests that the evo-

lution of SBP domain may be closely related tothe diversification of gene structure. Segmental du-plication of rice chromosomes has been reported(Vandepoele et al., 2003). At least four pairs of OsSPLgenes (OsSPL1 and 6,OsSPL3 and 12,OsSPL14 and 17,and OsSPL16 and 18) were located within such seg-mental duplication regions, which implies that dupli-cation of chromosomal segments has also contributedto the expansion of OsSPL gene family.

Most target genes of miRNAs identified in plants aretranscription factors. In this study, the miR156 familyin the rice genome was analyzed and the putativetarget genes of OsmiR156 were 11 OsSPL genes thatbelong to the plant-specific SBP transcription factorfamily. Although more and more miRNAs have beencloned, the origin of the miRNA remains as a puzzle.Amechanism of miRNA origination and evolution hasbeen proposed by analysis of two Arabidopsis miR161and miR163 families (Allen et al., 2004). In this model,miR161 and miR163 evolved relatively recently byduplication events and then adapted to the miRNAapparatus. The precursor sequences of miR161 andmiR163 have similarity with their target genes beyondthe complementary region of mature miRNA. For theOsmiR156 in this study, however, no similarity wasidentified out of the mature miRNA region comparedwith the target genes.Nevertheless,most of theOsmiR156and targeted OsSPL genes were located in clusters onrice chromosomes, which may suggest that OsmiR156is evolutionarily related to the targeted OsSPL genes.In addition, there is a distinct phylogenetic boundary

Figure 5. Overexpression ofOsmiR156in rice. A, Schematic diagram ofOsmiR156 overexpression construct.UBI, Maize ubiquitin gene promoter;Hpt, hygromycin resistance gene. B,PAGE RNA gel-blot analysis of miR156in the leaves of transgenic plants attillering stage. The exposure time ofblot was optimal for the signal of over-expressed transgene but not longenough to show the signal in wildtype (WT). Mb, OsmiR156b overex-pression; Mh, OsmiR156h overexpres-sion. C, Reduced plant height andincreased number of tillers in Mb andMh transgenic plants. The scale bar is10 cm. D, Number of spikelets andgrains per panicle in the transgenicplants and number of secondarybranches per panicle in the transgenicplants.

Xie et al.




betweenmiR156-targeted SPLs (S1–S3 and S6 subgroups)and non-miR156-targeted SPLs (S4 and S5 subgroups),suggesting that miR156 has been coexisted with a fewancestors of SPLs with or without miR156 target site.The evidence of miR156-mediated cleavage of SPLgenes was reported in moss (Arazi et al., 2005). Inter-estingly, SPL genes were found in single-cell plantChlamydomonas reinhardtii (Kropat et al., 2005). We alsosearched the draft genome sequence of C. reinhardtii(release 2.0, http://genome.jgi-psf.org/chlre2/chlre2.home.html) and no miR156 sequence was identified,which may suggest that miR156 evolved after theappearance of multicell plants.

Gene Structure Diversity of the OsSPL Family

By analyzing the entire complement of transcriptionfactors in model organisms, researchers have pro-posed that novel transcription factors may have beengenerated by the shuffling of DNA-binding domains(Morgenstern and Atchley, 1999; Riechmann et al.,2000). Shuffling of the zinc finger may also be anefficient approach to generate novel transcription fac-tors with DNA-binding activity (Bae et al., 2003). Inplants, zinc finger-contained transcription factors havebeen diversified in terms of the different constitutionsof zinc finger motifs such as the GATA, Dof (C-C-C-C

Figure 6. Transcript levels of OsmiR156 and target genes in the transgenic plants. A, Semiquantitative RT-PCR of nine putativetarget genes ofOsmiR156 in flag leaves and young panicles of theOsmiR156b (Mb)- andOsmiR156h (Mh)-overexpressing plants.The primers with amplicons spanning the complementary sites were used. The rice Actin1 gene was amplified as the internalcontrol. B, Real-time PCR analysis of four putative target genes of OsmiR156. The relative expression levelswere calibrated basedon the leaf sample of wild type. C, Transcript levels of two OsmiR156-targeted genes in the young leaves of transgenic plantsoverexpressing the target genes. DNA fragments downstream of the targeting sites were used as probes. The target gene-overexpressed plants are indicated by diamonds. The uncleaved and cleaved transcripts (bands) are indicated by arrows.





type), and WRKY families (C-C-H-H type; Takatsuji,1998). The SBP domain consists of two zinc fingers(Zn1 and Zn2) linked by a short JP and flanked by anNLS peptide. Such a combination of motifs featuresthe SPL transcription factor family and may provideflexibility in generating diverse SBP domains of SPLswith different functions.

Variable protein sequences beyond the SBP domainhave largely contributed to the diversification of theSPL gene family. Besides the SBP domain, seven otherputative motifs, including the site with a sequencecomplementary to miR156 were detected in OsSPLs,although most of these motifs await experimental datafor their functions. Different OsSPLs have distinct con-stitutions and organizations of these motifs. Somemotifs have duplicated in several SPL genes, whichfurther contributes to the diversity of the SPL genefamily. For example, OsSPL9 contains another Zn2 andNLS motif at the C terminus in the reverse directionand motif 4 (unknown function) was duplicated inOsSPL1 in the forward direction. The SPL gene familycan be phylogenetically classified into six subgroupsbased on the protein sequences of SBP domains,suggesting that the SBP domain may also contributeto the diversification of plant SPL genes.

The SBP domains of various SPL genes have con-served exon-intron structures, but intron length variessignificantly. Intron length is reported to be negativelycorrelated with the divergence and recombination ratein Drosophila melanogaster (Comeron and Kreitman,2000; Haddrill et al., 2005), and intron-specific selec-tive constraints have been maintained after the geneduplication that precedes the divergence of gene func-tions (Parsch, 2003). The high level of divergence in theintron sequences between OsSPL genes may also in-dicate that some of these introns have been involved inthe evolution and diversification of SPL proteins.

Diverse Tempospatial Expression Patternsof OsSPL Genes

Very low sequence similarity and different compo-sition of various motifs suggest that OsSPL genesmight have different expression patterns. To supportthis assumption, we investigated the transcript levelsof all OsSPL genes in 13 different tissues of rice. Ourdata clearly suggest that OsSPL genes exhibit distinctexpression patterns in terms of specificity and expres-sion level (Fig. 4). Even though the OsSPL genesshowed sequence diversity and different expressionprofiles, generally there is no obvious association be-tween gene structures and expression patterns.

Transcript levels of all OsSPL genes in the 13 differ-ent rice tissues or organs revealed that most of theputative OsmiR156-targeted genes expressed predom-inantly in the young panicles. More than half of theOsSPL genes expressed predominantly in the youngpanicles of rice, suggesting that some of these OsSPLgenes might be involved in the development of pan-icles in rice. Homologous SBP proteins in A. majus

(AmSBP1 and AmSBP2) and Arabidopsis (AtSPL3)have been reported with a binding ability to the cis-element in the promoter of floral organ identity genesSQUA andAP1, respectively (Klein et al., 1996; Cardonet al., 1997). However, phylogenetic analysis suggestedthat, except for OsSPL13, all the OsSPL genes withspecific or predominant expression in the young pan-icles were not in the subgroup (S1) that containedAmSBP1, AmSBP2, and AtSPL3 (Fig. 2).

Function and Interaction of OsmiR156 and OsSPLs

To further prove that OsSPL genes are regulated byOsmiR156, precursors of OsmiR156b and OsmiR156h(two representative OsmiR156 genes supported byexpressed sequences) were independently overex-pressed in rice. Transgenic plants of both OsmiR156genes exhibited similar phenotypic changes: a largenumber of tillers, dwarfism, small panicles, and de-layed flowering. These results may suggest that theexpression of endogenous OsmiR156 genes should beunder strict control to ensure the normal growth andde-velopment of rice. A similar phenotype was reportedin the miR156-overexpressing Arabidopsis plants thathad a moderate delay of flowering time under longdays, a large number of leaves, decrease of apicaldominant, and flowers from side shoots (Schwab et al.,2005).

To prove that such morphological changes resultedfrom suppression or loss of function of OsmiR156-targeted genes, we searched our Rice Mutant Database(Zhang et al., 2006) to findmutants ofOsSPL genes. Wehave thus far found two mutants for OsSPL12 andOsSPL14, respectively. The osspl12 mutant (ID no.03Z11AI07) exhibited delayed flowering time and theosspl14 mutant (ID no. 03Z11BJ86) showed dwarfism(Z. Chen, Q. Zhang, and C. Wu, unpublished data).Both OsSPL12 and OsSPL14 are putative targets ofOsmiR156 but differ in the mutant phenotypes. How-ever, the phenotypes of both mutants are included inthe phenotypes ofOsmiR156-overexpressed transgenicplants, suggesting thatOsSPL12 andOsSPL14might bethe targets of OsmiR156 and have different functions.

Most miRNAs regulate more than one target gene,and the target genes are often from one gene family.OsmiR156 control 11 target genes, and it might be oneof the largest number of genes by one miRNA. Thestudy of miR164 (interacting with five genes of NACfamily) in Arabidopsis shows that different membersof miR164 function slightly differently (Baker et al.,2005). In this study, overexpression of two OsmiR156members resulted in the same phenotype, but some ofthe target genes (such as OsSPL13 in leaves andOsSPL18 in panicles) showed differential cleavagebetween the two OsmiR156 overexpressors (Fig. 6A),suggesting differential interactions of a specific targetgene with different OsmiR156 members. Differentchanges of different target genes were also reportedin the miR172-overexpressing Arabidopsis plant inwhich the transcript level of the miR172 target gene

Xie et al.




TOE3 substantially increased; the transcript level ofanother target gene TOE2 substantially decreased(Schwab et al., 2005).To date, most of the identified plant miRNAs such as

miR164 (Guo et al., 2005), miR-JAW (Palatnik et al.,2003), and miR160 (Mallory et al., 2005) were reportedfor their regulation of target genes at transcriptionlevel by affecting the stability of target mRNA andthus degrading the target mRNA, whereas some plantmiRNAs such as miR172 (Chen, 2004) were proposedto regulate the target gene by translational repression.Recently, the effects on steady-state levels of targettranscripts were also suggested to be obscured by astrong feedback regulation (Schwab et al., 2005). In thisstudy, six target genes (OsSPL2, OsSPL12, OsSPL13,OsSPL14, OsSPL16, and OsSPL18) showed decreasedtranscript level in the leaves or the panicles of theOsmiR156-overexpressing rice plants compared to thewild-type plant. However, the other three target genes(OsSPL3, OsSPL7, and OsSPL11) showed no change oftranscript level in either tissue. Besides the possiblemechanisms suggested previously, it cannot be ex-cluded that these three genes are regulated by cleavagein other tissues not included in this study.

MATERIALS AND METHODS

Database Mining for OsSPL Genes in the Rice Genome

We established a local rice sequence database by downloading japonica

genomic sequence and annotation data from TIGR (http://rice.tigr.org; Yuan

et al., 2005), indica genomic sequence generated by whole genome shotgun

sequencing (Yu et al., 2002) from Beijing Genomic Institute, and japonica full-

length cDNA from KOME (Kikuchi et al., 2003). We performed a BLAST

search (Altschul et al., 1997) first in the local rice (Oryza sativa) sequence

database with the known SPL genes as queries. pHMM was used to identify

new SPL genes in the rice genome. The HMM profile of the SBP domain

(accession no. PF03110.17) was downloaded from the Pfam (http://

www.sanger.ac.uk/Software/Pfam/, release 17.0; Bateman et al., 2004), and

the HMMER package version 2.1 (Eddy, 1998) was used to search the local rice

protein database. All hits with expected values less than 1.0 were collected. To

exclude the redundant sequence of OsSPL genes, we aligned all sequences

with CLUASTALX (Thompson et al., 1997). We used a BLASTN search to

determine the chromosome locations of OsSPL genes by in silico mapping

their sequences to the rice BAC/PAC physical map. All nonredundant protein

sequences of putative OsSPL genes were manually checked for the SBP

domain. Arabidopsis (Arabidopsis thaliana) SPL genes were downloaded from

Munich Information Center for Protein Sequences Arabidopsis database

(http://mips.gsf.de/proj/thal/db/) and SPL genes from other species were

downloaded from Swiss-Prot (http://us.expasy.org/sprot/).

Bioinformatics Analysis of SPL and miR156Gene Families

Multiple alignments of SPL protein sequences were performed with

CLUSTALX (Thompson et al., 1997) and refinedmanually. Bayes phylogenetic

trees were reconstructed with the programMrBayes version 3.0 (Ronquist and

Huelsenbeck, 2003) under the JTT-f model of amino acid substitution and 4-g

category model. A total of 400,001 generations were performed with four

Markov chains with default heating values and tree sampling every 100

generations. The Markov chain converged after 9,000 generations and the first

100 sampled trees were discarded. The major consensus tree was deduced

from the remaining 901 sampled trees. The tree was edited with TreeView 1.5

(Page, 1996). We used the BLAST program to determine the intron-exon

structures of OsSPL genes by mapping cDNAs to genomic sequence. The

intron-exon structures of the OsSPL gene were illustrated proportionally to

the lengths of introns and exons. The HMM profile of the SBP domain was

reconstructed with the HMMbuild program in the HMMER package version

2.1 (Eddy, 1998), and alignment was viewed with sequence LOGO (Crooks

et al., 2004). MEME program (Bailey and Elkan, 1994) was used to predict the

potential motifs. All motifs discovered by MEME with expected values lower

than 2E-30 were searched in the Inter-Pro database with Inter-ProScan

(Mulder et al., 2005).

The stem-loop sequences of the plant miR156 family were downloaded

from the miRbase (Griffiths-Jones, 2004) to reconstruct the phylogenetic tree.

The sequences of stem regions (mature miRNA sequences and the comple-

mentary sequences) were aligned first by CLUSTALX. The sequences beyond

the stem regions were subsequently aligned with manual modification. The

secondary structures of OsmiR156 RNAs were generated with the RNAalifold

program (Hofacker et al., 2002), and sequences beyond the consensus sec-

ondary structure were masked for phylogenetic analysis. The Bayes tree was

constructed with a mixed nucleotide substitution model of REV and RNA7D

described in the PHASE program, which was specifically designed for RNA

phylogenetic inference according to the secondary structures (Hudelot et al.,

2003). The secondary structures of OsmiR156 RNAs and consensus secondary

structure of AtmiR156 and OsmiR156 were generated with the Vienna RNA

package version 1.4 (Hofacker, 2003). The target genes of OsmiR156 were

predicted by using themature sequences to search the rice genomic sequences.

According to the characterized miRNAs in plants (Rhoades et al., 2002; Chen,

2004; Mallory et al., 2004; Guo et al., 2005; Mallory et al., 2005), rice genes

(except OsmiR156 precursors) that contained sequences with fewer than three

mismatches to mature miRNA156 sequence are considered to be putative

targets of OsmiR156.

Isolation of OsSPL and OsmiR156 Genesand Rice Transformation

For each OsSPL gene, a pair of primers (Supplemental Table I) was

designed to amplify the predicted full-length cDNA with cDNA templates

prepared from different tissues of Minghui 63 (rice L. subsp. indica), a parental

line for elite hybrid rice in China. ExTaq DNA polymerase (Takara) was used

to amplify the OsSPL genes with the following cycling profile: 94�C for 4 min;

25 to 30 cycles of 94�C for 40 s, 55�C for 40 s, and 72�C for 2 or 3 min; and

extension at 72�C for 10 min. The amplified products were cloned into pGEM-T

easy vector (Promega) and sequenced from both ends by using BigDye

Terminator Sequencing Ready kit (version 2.0 or 3.0) in an ABI PRISM 377 or

3730 sequencer.

Gene-specific primers (Supplemental Table I) were used to amplify the

OsmiR156b and OsmiR156h precursors from the rice genome. The genomic

fragments were cloned to pGEM-T Easy vector (Promega) and sequenced.

Then the fragments of two OsmiR156 precursors were cut by KpnI and BamHI

and ligated into the transformation vector pCAMBIA1301U under the control

of a maize (Zea mays) ubiquitin gene promoter. The full-length cDNA of

OsSPL12 and OsSPL14 were cloned into pCAMBIA1301 (provided by

CAMBIA) under the control of Cauliflower mosaic virus 35S promoter.

The Agrobacterium-mediated transformation method was used to intro-

duce constructs into Agrobacterium tumefaciens strain EHA105 by electropor-

ation and transformed into rice Zhonghua11 (rice L. subsp. japonica; Hiei et al.,

1994).

RT-PCR Analysis

The TRIZol reagent (Invitrogen) was used according the manufacturer’s

instructions to extract total RNAs of various tissues or organs from a life cycle

of rice Minghui63 (rice L. subsp. indica). Before RT, total RNAwas treated with

amplification-grade DNase I (Invitrogen) for 15 min to degrade possibly

contaminated residual genomic DNA. SuperScriptII reverse transcriptase

(Invitrogen) was used according to the manufacturer’s instructions to syn-

thesize first-strand cDNA from the DNase I-treated total RNA. About 1/20 of

the first-strand cDNA generated from 1 mg total RNAwas used as template for

PCR in a reaction volume of 50 mL with the rTaq DNA polymerase (Takara).

PCR was performed in an ABI 9700 Thermocycler (Applied System) with the

following cycling profile: 94�C for 3 min; 25 to 40 cycles at 94�C for 40 s, 55�Cor 60�C for 40 s, and 72�C for 1 min. Fifteen microliters of the PCR product was

separated in a 1.2% agarose gel and stained with ethidium bromide for

visualization. We used a pair of primers specific to rice Actin1 gene (accession

no. AK060893) for RT-PCR as internal control to compare the band intensity

between samples. For each OsSPL gene, a pair of primers with a 400 to 600 bp





amplicon was used for RT-PCR (Supplemental Table I) with 25, 30, 35, and 40

cycles, depending on the expression levels of different genes. All RT-PCRs

were repeated three times with independently reverse-transcribed templates.

Real-Time Quantitative RT-PCR

Relative quantification of gene expression by real-time PCRwas performed

on an ABI PRISM 7500 instrument (Applied Biosystems). The primers for real-

time PCR were designed by Primer Express Version 2.0 (Applied Biosystems;

Supplemental Table III). Rice Actin1 gene was used as endogenous control.

Real-time PCR was performed in an optical 96-well plate, including 12.5 mL

23 SYBR Green Master mix reagent (Applied Biosystems), 1 mL cDNA

samples, and 0.2 mM of each gene-specific primers, in final volume of 25 mL,

using the thermal cycles as follows: 50�C for 2 min, 95�C for 10 min; 40 or 45

cycles of 95�C for 30 s; 60�C for 30 s; and 72�C for 1 min. Disassociation curve

analysis was performed as follows: 95�C for 15 s; 60�C for 20 s; 95�C for 15min.

The relative expression levels were determined as described previously (Liang

et al., 2006).

RNA Gel-Blot Analysis of miRNA and SPL Genes

Mini-PROTEAN III system (Bio-Rad) was used to separate low-mass RNA

by electrophoresis in 20% urea-denatured polyacrylamide gel and blot it onto

nylon membranes. DNA Oligo (5#-GTGCTCACTCTCTTCTGTCA-3#) was

synthesized as a probe to detect the OsmiR156 level. The probes were labeled

and hybridized essentially according to Bartel’s description (Bartel, 2004). The

blot was washed four times (5, 10, 40, and 40 min, respectively) at 50�C with

washing buffer (33 SSC, 25 mM NaH2PO4, pH 7.5, 5% SDS, and 103

Denhardt’s solution). The FUJIFILM system (FJL5100, Fuji) was used to

quantify signal intensity. All the probes were stripped and then rehybridized

with DNA probe (5#-TGTATCGTTCCAATTTTATCGGATGT-3#) complemen-

tary to U6 RNA.

Sequence data of microRNA156 in rice from this article can be found in the

miRBase database under accession numbers MI0000653 to MI0000662,

MI0001090, and MI0001091.

ACKNOWLEDGMENTS

We thank Yinglong Cao and Meng Cai for kindly constructing and

providing the p1301U vector.

Received June 1, 2006; accepted July 13, 2006; published July 21, 2006.

LITERATURE CITED

Allen E, Xie Z, Gustafson AM, Sung GH, Spatafora JW, Carrington JC

(2004) Evolution of microRNA genes by inverted duplication of target

gene sequences in Arabidopsis thaliana. Nat Genet 36: 1282–1290

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,

Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of

protein database search programs. Nucleic Acids Res 25: 3389–3402

Arazi T, Talmor-Neiman M, Stav R, Riese M, Huijser P, Baulcombe DC

(2005) Cloning and characterization of micro-RNAs from moss. Plant J

43: 837–848

Bae KH, Kwon YD, Shin HC, Hwang MS, Ryu EH, Park KS, Yang HY, Lee

DK, Lee Y, Park J, et al (2003) Human zinc fingers as building blocks in

the construction of artificial transcription factors. Nat Biotechnol 21:

275–280

Bailey TL, Elkan C (1994) Fitting a mixture model by expectation maxi-

mization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol

Biol 2: 28–36

Baker CC, Sieber P, Wellmer F, Meyerowitz EM (2005) The early extra

petals1 mutant uncovers a role for microRNA miR164c in regulating

petal number in Arabidopsis. Curr Biol 15: 303–315

Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and

function. Cell 116: 281–297

Batchelor AH, Piper DE, de la Brousse FC, McKnight SL, Wolberger C

(1998) The structure of GABPalpha/beta: an ETS domain-ankyrin repeat

heterodimer bound to DNA. Science 279: 1037–1041

Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S,

Khanna A, Marshall M, Moxon S, Sonnhammer EL, et al (2004) The

Pfam protein families database. Nucleic Acids Res 32: D138–D141

Bonnet E, Wuyts J, Rouze P, Van de Peer Y (2004) Detection of 91 potential

conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa

identifies important target genes. Proc Natl Acad Sci USA 101: 11511–

11516

Cardon G, Hohmann S, Klein J, Nettesheim K, Saedler H, Huijser P

(1999) Molecular characterisation of the Arabidopsis SBP-box genes.

Gene 237: 91–104

Cardon GH, Hohmann S, Nettesheim K, Saedler H, Huijser P (1997)

Functional analysis of the Arabidopsis thaliana SBP-box gene SPL3: a

novel gene involved in the floral transition. Plant J 12: 367–377

Chen X (2004) A microRNA as a translational repressor of APETALA2 in

Arabidopsis flower development. Science 303: 2022–2025

Comeron JM, Kreitman M (2000) The correlation between intron length

and recombination in Drosophila: dynamic equilibrium between muta-

tional and selective forces. Genetics 156: 1175–1190

Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a

sequence logo generator. Genome Res 14: 1188–1190

Dingwall C, Laskey RA (1991) Nuclear targeting sequences—a consensus?

Trends Biochem Sci 16: 478–481

Eddy SR (1998) Profile hidden Markov models. Bioinformatics 14: 755–763

Gorina S, Pavletich NP (1996) Structure of the p53 tumor suppressor

bound to the ankyrin and SH3 domains of 53BP2. Science 274:

1001–1005

Griffiths-Jones S (2004) The microRNA registry. Nucleic Acids Res 32:

D109–D111

Guo H-S, Xie Q, Fei J-F, Chua N-H (2005) MicroRNA directs mRNA

cleavage of the transcription factor NAC1 to downregulate auxin signals

for Arabidopsis lateral root development. Plant Cell 17: 1376–1386

Haddrill PR, Charlesworth B, Halligan DL, Andolfatto P (2005) Patterns

of intron sequence evolution in Drosophila are dependent upon length

and GC content. Genome Biol 6: R67

Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of

rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis

of the boundaries of the T-DNA. Plant J 6: 271–282

Hofacker IL (2003) Vienna RNA secondary structure server. Nucleic Acids

Res 31: 3429–3431

Hofacker IL, Fekete M, Stadler PF (2002) Secondary structure prediction

for aligned RNA sequences. J Mol Biol 319: 1059–1066

Hudelot C, Gowri-Shankar V, Jow H, Rattray M, Higgs PG (2003) RNA-

based phylogenetic methods: application to mammalian mitochondrial

RNA sequences. Mol Phylogenet Evol 28: 241–252

Huijser P, Klein J, Lonnig WE, Meijer H, Saedler H, Sommer H (1992)

Bracteomania, an inflorescence anomaly, is caused by the loss of func-

tion of the MADS-box gene squamosa in Antirrhinummajus. EMBO J 11:

1239–1249

International Rice Genome Sequencing Project (2005) The map-based

sequence of the rice genome. Nature 436: 793–800

Jack T (2004) Molecular and genetic mechanisms of floral control. Plant

Cell 16: S1–S17

Jones-Rhoades MW, Bartel DP (2004) Computational identification of

plant microRNAs and their targets, including a stress-induced miRNA.

Mol Cell 14: 787–799

Kidner CA, Martienssen RA (2004) Spatially restricted microRNA directs

leaf polarity through ARGONAUTE1. Nature 428: 81–84

Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K, Kishimoto N,

Yazaki J, Ishikawa M, Yamada H, Ooka H, et al (2003) Collection,

mapping, and annotation of over 28,000 cDNA clones from japonica

rice. Science 301: 376–379

Klein J, Saedler H, Huijser P (1996) A new family of DNA binding proteins

includes putative transcriptional regulators of the Antirrhinum majus

floral meristem identity gene SQUAMOSA. Mol Gen Genet 250: 7–16

Kropat J, Tottey S, Birkenbihl RP, Depege N, Huijser P, Merchant S (2005)

A regulator of nutritional copper signaling in Chlamydomonas is an SBP

domain protein that recognizes the GTAC core of copper response

element. Proc Natl Acad Sci USA 102: 18730–18735

Lannenpaa M, Janonen I, Holtta-Vuori M, Gardemeister M, Porali I,

Sopanen T (2004) A new SBP-box gene BpSPL1 in silver birch (Betula

pendula). Physiol Plant 120: 491–500

Liang D, Wu C, Li C, Xu C, Zhang J, Kilian A, Li X, Zhang Q, Xiong L

(2006) Establishment of a patterned GAL4-VP16 transactivation system

for discovering gene function in rice. Plant J 46: 1059–1072

Xie et al.




Luh FY, Archer SJ, Domaille PJ, Smith BO, Owen D, Brotherton DH,

Raine AR, Xu X, Brizuela L, Brenner SL, et al (1997) Structure of the

cyclin-dependent kinase inhibitor p19Ink4d. Nature 389: 999–1003

Lux SE, Tse WT, Menninger JC, John KM, Harris P, Shalev O, Chilcote

RR, Marchesi SL, Watkins PC, Bennett V, et al (1990) Hereditary

spherocytosis associated with deletion of human erythrocyte ankyrin

gene on chromosome 8. Nature 345: 736–739

Mallory AC, Bartel DP, Bartel B (2005) MicroRNA-directed regulation of

Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper

development and modulates expression of early auxin response genes.

Plant Cell 17: 1360–1375

Mallory AC, Dugas DV, Bartel DP, Bartel B (2004) MicroRNA regulation of

NAC-domain targets is required for proper formation and separa-

tion of adjacent embryonic, vegetative, and floral organs. Curr Biol 14:

1035–1046

Millar AA, Gubler F (2005) The Arabidopsis GAMYB-Like genes, MYB33

and MYB65, are microRNA-regulated genes that redundantly facilitate

anther development. Plant Cell 17: 705–721

Moreno MA, Harper LC, Krueger RW, Dellaporta SL, Freeling M (1997)

liguleless1 encodes a nuclear-localized protein required for induction of

ligules and auricles during maize leaf organogenesis. Genes Dev 11:

616–628

Morgenstern B, Atchley WR (1999) Evolution of bHLH transcription

factors: modular evolution by domain shuffling? Mol Biol Evol 16:

1654–1663

Mosavi LK, Minor DL Jr, Peng ZY (2002) Consensus-derived structural

determinants of the ankyrin repeat motif. Proc Natl Acad Sci USA 99:

16029–16034

Mulder NJ, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D,

Bradley P, Bork P, Bucher P, Cerutti L, et al (2005) InterPro, progress

and status in 2005. Nucleic Acids Res 33: D201–D205

Page RD (1996) TreeView: an application to display phylogenetic trees on

personal computers. Comput Appl Biosci 12: 357–358

Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC,

Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature

425: 257–263

Parsch J (2003) Selective constraints on intron evolution in Drosophila.

Genetics 165: 1843–1851

Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002)

MicroRNAs in plants. Genes Dev 16: 1616–1626

Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002)

Prediction of plant microRNA targets. Cell 110: 513–520

Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L,

Pineda O, Ratcliffe OJ, Samaha RR, et al (2000) Arabidopsis transcrip-

tion factors: genome-wide comparative analysis among eukaryotes.

Science 290: 2105–2110

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic

inference under mixed models. Bioinformatics 19: 1572–1574

Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D

(2005) Specific effects of microRNAs on the plant transcriptome. Dev

Cell 8: 517–527

Stone JM, Liang X, Nekl ER, Stiers JJ (2005) Arabidopsis AtSPL14, a plant-

specific SBP-domain transcription factor, participates in plant develop-

ment and sensitivity to fumonisin B1. Plant J 41: 744–754

Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of

microRNAs from rice. Plant Cell 17: 1397–1411

Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other

small RNAs from Arabidopsis. Plant Cell 16: 2001–2019

Takatsuji H (1998) Zinc-finger transcription factors in plants. Cell Mol Life

Sci 54: 582–596

Tanzer A, Stadler PF (2004) Molecular evolution of a microRNA cluster.

J Mol Biol 339: 327–335

Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D (1997) The

CLUSTAL_X windows interface: flexible strategies for multiple se-

quence alignment aided by quality analysis tools. Nucleic Acids Res

25: 4876–4882

Unte US, Sorensen AM, Pesaresi P, Gandikota M, Leister D, Saedler H,

Huijser P (2003) SPL8, an SBP-box gene that affects pollen sac devel-

opment in Arabidopsis. Plant Cell 15: 1009–1019

Vandepoele K, Simillion C, Van de Peer Y (2003) Evidence that rice and

other cereals are ancient aneuploids. Plant Cell 15: 2192–2202

Wang J-W, Wang L-J, Mao Y-B, Cai W-J, Xue H-W, Chen X-Y (2005) Control

of root cap formation by microRNA-targeted auxin response factors in

Arabidopsis. Plant Cell 17: 2204–2216

Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T,

Aoki M, Seki E, Matsuda T, Nunokawa E, et al (2004) A novel zinc-

binding motif revealed by solution structures of DNA-binding do-

mains of Arabidopsis SBP-family transcription factors. J Mol Biol 337:

49–63

Yu J, Hu S, Wang J, Wong GK, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X,

et al (2002) A draft sequence of the rice genome (Oryza sativa L. ssp.

indica). Science 296: 79–92

Yuan Q, Ouyang S, Wang A, Zhu W, Maiti R, Lin H, Hamilton J, Haas B,

Sultana R, Cheung F, et al (2005) The institute for genomic research

Osa1 rice genome annotation database. Plant Physiol 138: 18–26

Zhang J, Li C, Wu C, Xiong L, Chen G, Zhang Q, Wang S (2006) RMD: a

rice mutant database for functional analysis of the rice genome. Nucleic

Acids Res 34: D745–D748





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