The SLEEPER genes: a transposase-derived angiosperm-specific gene

15 Pages·2012·3.98 MB·English

Knipetal.BMCPlantBiology2012,12:192
http://www.biomedcentral.com/1471-2229/12/192
RESEARCH ARTICLE Open Access
The SLEEPER genes: a transposase-derived
angiosperm-specific gene family
Marijn Knip, Sylvia de Pater and Paul JJ Hooykaas*
Abstract
Background: DAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for
development inArabidopsis thaliana.Little is known about thepresence ofDAYSLEEPER orthologs inother species,
or how and when it was domesticated.We studied thepresence of DAYSLEEPER orthologs in plants and propose a
model for the domestication ofthe ancestral DAYSLEEPER gene inangiosperms.
Results: Using specific BLAST searches ingenomic and ESTlibraries, wefound thatDAYSLEEPER-like genes
(hereafter called SLEEPER genes)are unique to angiosperms. Basal angiospermsas well as grasses (Poaceae) and
dicotyledonous plants possess such putative orthologous genes, but SLEEPER-family genes were notfound in
gymnosperms, mosses and algae. Most species contain more than one SLEEPER gene. AllSLEEPERscontaina C H
2 2
typeBED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the
BED-zinc finger and dimerization domain, which are hallmarkfeatures inthe SLEEPER family. AlthoughSLEEPER
genes are structurally conserved between species, constructs with SLEEPER genes from grapevine and rice did not
complement the daysleeper phenotype inArabidopsis, when expressed under control of the DAYSLEEPER promoter.
However theseconstructsdid cause a dominant phenotype when expressed inArabidopsis.Rice plant lines withan
insertion in theRICESLEEPER1 or 2locus displayed phenotypic abnormalities, indicating that thesegenes are
functional and important for normal development inrice. We suggest a model inwhich wehypothesize that an
ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution.
Evidence is also presentedfor more recent retroposition events of SLEEPER genes, such as an event in therice
genome, which gave rise to theRICESLEEPER1 and 2 genes.
Conclusions: We propose the ancestral SLEEPER gene was formedafter a process of retro-transpositionduring the
evolution of the first angiosperms. It may have acquired animportant function early on, as mutation of two SLEEPER
genes inrice, likethe daysleeper mutant in A. thaliana gave a developmental phenotype indicative of their
importance for normal plant development.
Background conceivable way: shuffling, addition and deletion of not
The role of transposons in evolution has long been only new coding and regulatory sequences, but of large
greatly underestimated. Viewed as genomic parasites, stretches of chromosomes as well [4,5].
transposons were classified as part of the so-called Although a more detailed classification system is now
“junk-DNA” and largely ignored, even though transpo- being used, two major classes of transposable elements
sons and transposon-remnants make up significant frac- (TE’s) exist: retrotransposons, which transpose by using
tions of eukaryotic genomes [1]. Forty four percent of a RNA intermediate, and DNA transposons, which
the human genome and more than 85% of the maize transpose by cutting their genomic sequence and insert-
genome consists of transposons and their relics [2,3]. ing it elsewhere in the genome. These TE’s are referred
New views have led to the insight that transposons toas“copy-paste”elementsand“cut-paste”elements,re-
have shaped the genomic landscape in almost every spectively [1]. Retrotransposons encode several proteins
that are highly similar to those encoded by retroviruses.
One of these proteins is a reverse-transcriptase that is
*Correspondence:[email protected]
DepartmentofMolecularandDevelopmentalGenetics,InstituteofBiology, able to reverse-transcribe the full-length transposon
LeidenUniversity,Sylviusweg72,2333BE,Leiden,TheNetherlands
©2012Knipetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
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mRNAintoDNA,afterwhichthenewcopyisintegrated transposition of the Ac-element, the first described hAT
in the genome [1]. DNA transposons encode proteins, transposon family member of maize, are not conserved
called transposases, which are able to cut their own cod- in DAYSLEEPER [12]. DAYSLEEPER was found to be
ing sequence from the genomic DNA, by recognizing essential to Arabidopsis thaliana, as displayed by a se-
flanking repeats, and inserting it elsewhere in the gen- vere developmental phenotype in daysleeper mutants.
ome. High transposon activity would be deleterious for The gene most likely codes for a DNA-binding protein,
the host and therefore defense mechanisms have evolved since it was identified through binding to the promoter
to counteract transposase activity. Still, transposons are oftheDNArepairgeneKu70inayeastone-hybridassay
numerous in almost every eukaryotic genome and thus [12]. DAYSLEEPER consists of 696 amino acids, pos-
havesuccessfully managed tosustainthemselves[6]. sesses a DNA binding BED-type zinc finger domain and
Transposons have contributed greatly, not only to ahATdimerizationdomain[12,13].
shaping the genomic landscape, but also to the coding Here we present data on the presence of putative
material of endogenous genes, for instance by giving rise DAYSLEEPER orthologs in angiosperms, including the
to chimeric proteins (reviewed in [5]). Many conserved basal angiosperms. We show that SLEEPER genes are
proteindomainshave now been shownto originate from present in many species, often in multiple copies. Fur-
transposable elements (e.g. BED zinc finger domains) thermore, we postulate a theory on the domestication
[7]. In the process called “domestication” a transposase process oftheancestralSLEEPER gene.
losesitsoriginal functionandacquiresnewfunctionality,
creating a novel gene. Various genes in different species
have been found to be domesticated transposases Results
(reviewed in [8]). A recurrent theme in domestication DAYSLEEPERorthologsinthegenomeoforyzasativa
seems to be the conversion of transposases encoded by andvitisvinifera
DNA transposons into important host proteins such as Two genes that are possibly orthologous to DAYSLEE-
chromatin-related proteins and transcription factors. PER, were identified by Benjak et al. (2008) [14] in a
Among these factors are CENP-B, a centromere protein genome-wide analysis of hAT-transposons in the grape-
invertebratesandfungi,theFAR1-FHY3family,involved vine genome and named VINESLEEPER1 and 2. In a
in far-red light signaling in plants and BEAF-32, a study on the transcriptional activity of transposons in
boundary element associated factor in Drosophila mela- rice, several sequences were designated as “DAYSLEE-
nogaster [5,7,9,10]. These elements are derived from, PER-like” [15]. We used the DAYSLEEPER sequence as
pogo, MuDR and hAT super-families of “cut-paste” ele- a query to find the most related sequences in the gen-
ments respectively. This evolutionary trend can be omes of Arabidopsis, grapevine and rice and produced a
explained by the fact that the transposases of these maximum-likelihood phylogenetic tree with bootstrap
elements all contain DNA binding domains and values, depicted in Figure 1, to reveal the relationship
protein-protein interaction domains, since they work in between the highest scoring BLAST hits in the Arabi-
conjunction with host factors to enable the transpos- dopsis, grapevine and rice genomes. This resulted in a
ition process [11]. It seems likely that host partners of clustering of putative SLEEPERs among the hAT-like
these transposases include chromatin remodelers, DNA transposase genes. The four putative orthologs we found
repair genes and/or endonucleases, since one can envis- in the rice genome only partly overlap with the DAY-
age players in these fields to be required for facilitation SLEEPER-like sequences reported by Jiao et al. (2007)
of the “cut-paste” process. Remarkably, very little is [15]. Because of their high identity RICESLEEPER 1 and
known about these potential factors and the steps of 2 are probably the result of a recent duplication event
the transposition process. (Figure 1). One gene in Arabidopsis, At1g15300, was
DAYSLEEPER was first described in 2005 by Bundock found to be related to DAYSLEEPER. A homozygous T-
and Hooykaas [12]. The DAYSLEEPER gene in Arabi- DNA insertion mutant (SALK_020839C) for this gene
dopsis thaliana is an example of molecular domestica- showed normal development. This gene is expressed,
tion of a DNA transposon. DAYSLEEPER shares but may havebecome non-functional by a lack of the N-
extensive homology with members of a large subfamily terminal R/K rich nuclear localization signal which is
of transposable elements, the hAT transposons, which characteristically present in DAYSLEEPER and all RICE-
are widely spread throughout the tree of life and are and VINESLEEPERs or has acquired novel functionality.
found in all eukaryotic branches, except in Trichomonas, In order to determine their cellular localization, YFP-
diatoms, and ciliates [6]. Unlike these elements, fusions were constructed for these SLEEPER genes and
DAYSLEEPER is not able to transpose, since it lacks introduced in Arabidopsis protoplasts. This revealed a
the hallmark repeats essential for this process. Also, a nuclear localization for all SLEEPERs from rice, grape-
number of amino acids shown to be essential for the vine and Arabidopsis, but not for the product of the
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Figure1PhylogenyofDAYSLEEPERhomologsfromArabidopsis,riceandgrapevine.Ricesequenceswereobtained[15]and
supplementedwiththemosthomologoussequencesfromboththeOryzasativa,VitisviniferaandtheArabidopsisthalianagenomes,foundin
genomicdatabasesusingTBLASTNqueries.Geneidenticatorsstartingwith“LOC_Os”“LOC10”or“At”indicatesgenesfromrice,grapevineand
Arabidopsis,respectively.PhylogenywascreatedusingRAxML,withbootstrapvalues[21].
At1g15300 gene, which is present in the cytosol and three strongly conserved motifs, that we designated
which wethereforenamed CYTOSLEEPER (Figure 2). SLEEPERmotifs1to3(Figure3).SLEEPERmotif1encom-
passes part of the BED-zinc finger, raising the possibility
SLEEPERstructureandconserveddomains that SLEEPERs might bind conserved sites in different
We found that genes coding for SLEEPERs are con- species.SLEEPERmotif2doesnotoverlapwithanyofthe
served between different species. SLEEPERs contain conserved hAT blocks and is, in contrast to SLEEPER
hAT motifs that are widely conserved in hAT transpo- motif1 and 3, not exclusive to SLEEPERs, since BLAST
sases. Six hAT motifs are generally found in hAT searches using this motif also yield hAT transposases in
transposases from various species, which are named various species that lack SLEEPERmotif1 and 3. SLEE
motif A to F [16]. SLEEPERs contain a K/R rich nuclear PERmotif3 overlaps largely with hAT block E. This hAT
localization domain (NLS) adjacent to a BED-type zinc block is part of the hAT dimerization domain, in con-
finger at their N-terminal region and have a hAT junction with hAT block D and F, suggesting that other
transposase-like dimerization domain at the C-terminus SLEEPERs can dimerize like DAYSLEEPER and other
(Figure 3) [12,16]. Like hAT transposases, SLEEPERs are hAT transposases [13]. Localization of SLEEPERs is nu-
generally present in the nucleus (Figure 2B,C,D). In clear in Arabidopsis protoplasts, but CYTOSLEEPER,
DAYSLEEPER, the C-terminal dimerization domain is which lacks the K/R rich conserved array adjacent to
functional as well (M. Knip, unpublished results), allow- the BED-zinc finger, is present in the cytosol, suggest-
ing DAYSLEEPER to homodimerize. Like DAYSLEEPER, ing that this domain is indeed necessary for nuclear
RICE- and VINESLEEPERs lack the amino acids neces- localization of SLEEPERs (Figure 2). The divergent se-
sary for transposition and the genes are not flanked by quence of CYTOSLEEPER, represented by the long
hATrepeat sequences(data not shown). branch-length in Figure 3, and the lack of an apparent
The SLEEPERs form a separate group when compared phenotype in mutant plants indicate that this gene
to other hAT-transposases (Figure 1). SLEEPERs contain might be pseudogenized or has obtained a different
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A
B
C
D
Figure2(Seelegendonnextpage.)
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(Seefigureonpreviouspage.)
Figure2SubcellularlocalizationofSLEEPERproteinsfromdifferentspeciesinArabidopsisthalianacell-suspensionprotoplasts.A.
CYTOSLEEPER:YFP.B.VINESLEEPER2:YFP.C.RICESLEEPER3:YFP.D.DAYSLEEPER:YFP.Imagesintheleftcolumnarebrightfieldimages,themiddle
columndepictsthefluorescentimageandtherightcolumnmergedimagesoftheothertwocolumns.Thescalebarrepresents20μm.
function as DAYSLEEPER. SLEEPER genes from Arabi- for SLEEPER-like sequences in the monocotyledonous
dopsis, grapevine and rice, do not have introns in their (Poaceae) and dicotyledonous species searched. In
coding sequences. Most other SLEEPER genes contain databases of species beyond the plant realm, namely Sac-
one intron between the 5’ UTR and their start-codons. charamyces cerevisiae and Drosophila melanogaster, no
DAYSLEEPER shares ~50% identity (61-69% similarity) similar sequences were found (standard settings: Max
with the VINESLEEPERs and between 36 and 43% target sequences = 100, expect threshold = 10, word
identity with the RICESLEEPERs (51-58% similarity) at size = 3, NCBI BLAST [17]). Also, the EST library for
the amino acid level (Table 1). RICE- and VINESLEE- gymnosperm species Ginkgo biloba (data not shown)
PERs vary in length between 684 and 753 amino acids and a mixed Pinus-species library (TIGR plant tran-
(Figure 3, Table 1). The increased length of RICESLEE- script assemblies [18]) did not yield any significant hits
PER4 is mainly caused by the acquisition of extra coding (Additional file 1: Table S1), neither did queries in data-
sequence at its N-terminus. The relatively large size of basesofthelycophyteSelaginellamoellendorffii(Additional
CYTOSLEEPER is predominantly due to an additional file 1: Table S1) and the moss Physcomitrella patens
stretch of amino acids between the second and third of (Phytozome [19]) (data not shown). However, lower angio-
three conserved motifs, which is not found in other sperm EST databases (Ancestral Angiosperm Genome
SLEEPERs. Project; http://ancangio.uga.edu/content/est-assemblies)
yieldedhitsin several species of different orders, namely
SLEEPERsareonlypresentinhigherplants Perseaamericana(order:Laurales),Liriodendrontulipifera
An important question is where and when the SLEEPERs (order:Magnoliales),Nupharadvena(order:Nymphaeales)
have emerged in evolution. To answer this question and Amborella trichopoda (order: Amborellales) (Table 2).
SLEEPERmotif1 and 3 consensus sequences and DAY- These data indicate that SLEEPER genes belong to an
SLEEPER were used in TBLASTN searches in genomic angiosperm specific gene family and that formation of
and EST databases from several organisms. Queries with the first SLEEPER gene coincided with the evolution of
the SLEEPERmotifs yielded exclusively high-scoring hits angiosperms.
Figure3SchematicoverviewofthestructureofhATtransposasegenes.hATtransposasegenespossessanN-terminalNLS,followedbya
BED-typezincfingerdomainandconservedhAT-blocksAuntilF.ThelastthreehATblocks(D-F)makeupthehATdimerizationdomain.Three
highlyconservedmotifs(1–3)wereidentifiedbyaligningallSLEEPERsfromArabidopsisthaliana,VitisviniferaandOryzasativa.SLEEPERmotifsare
depictedusingWeblogo3.0[40].
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Table1HomologyoftheVINESLEEPERsandRICESLEEPERs Although high similarity exists between RICESLEEPERs,
toDAYSLEEPER wechosetodesignatetheRICESLEEPERswithindividual
Comparedto Codingsequence numbers, namely 1 to 4. The coding sequence of RICE
DAYSLEEPER(696AA’s) length(AA’s) SLEEPER1 and 2 are almost identical (97% sequence
Identity Consensus identity), as are RICESLEEPER3 and 4, OLIMSLEEPER2a
positions(%) positions(%)
and2bandPOPSLEEPER2band2c.Thesemaytherefore
CYTOSLEEPER 30.1 42.1 799 be relatively recent duplications, which had been shown
VINESLEEPER1 48.4 60.7 689 previously for the genes in Olimarabidopsis pumila by
VINESLEEPER2 55.9 68.5 675 Halletal.[22].Indicotyledonousplants,allrecentdupli-
RICESLEEPER1 43.0 58.1 722 cations seem to have occurred in the DAYSLEEPER-
RICESLEEPER2 43.3 58.0 722 branch of the phylogeny shown in Figure 4. When look-
ing closer at the rice genome, there is no evidence for a
RICESLEEPER3 35.7 51.4 684
segmental duplication of the genomic location of the
RICESLEEPER4 37.4 53.8 752
RICESLEEPER1 and 2 genes, since there is no apparent
Similarityandidentityvalues,aswellasthelengthoftheSLEEPER,are
depicted.ThenumberswereobtainedusingAlignXintheInvitrogenVector sequencehomologyorsyntenyoftheregionsurrounding
NTIsuite(Invitrogen®). these genes. The close relatives of Arabidopsis thaliana,
namely Olimarabidopsis pumila, Arabidopsis arenosa
SLEEPERsarefrequentlycopiedinseveralspecies and Capsella rubella, have homologs of the CYTOSLEE
TBLASTN searches using the amino acid sequence PER gene, but these genes are not depicted in the phyl-
of DAYSLEEPER in genomic databases of several ogeny,sincethecompletegenomesequenceofthesespe-
sequenced angiosperm species (Figure 4), revealed that cies was not available at the time of the analysis
SLEEPER genes are present in all these queried genomes (Figure4).
and often in multiple copies (Plant Genome Database Unlike CYTOSLEEPER, genes clustering with VINE-
[20]). Figure 4 depicts a maximum likelihood-tree SLEEPER1 do code for a K/R-rich putative nuclear
with bootstrap values, generated with the RAxML algo- localization domain. Most dicotyledonous species ana-
rithm [21]. Many genomes appear to have several DAY- lyzed also have a homolog in both the CYTOSLEEPER,
SLEEPER homologs. SLEEPER genes possess the three as well as the DAYSLEEPER cluster (Figure 4). Excep-
SLEEPERmotifs and were distinguished from hAT tions are poplar, which has three POPSLEEPERS cluster-
transposase sequences by a BLAST score of over 400, ing with DAYSLEEPER, Lotus japonicus, which has
whereas hAT-like sequences typically did not score LOTUSSLEEPER2 clustering with DAYSLEEPER and
higher than 200. LOTUSSLEEPER1, which has diverged from other SLEE-
It is clear to see a clustering of SLEEPER genes from PERs and Carica papaya, which apparently has only one
Poaceae, separated from those of dicotyledonous plants, SLEEPER. This might suggest that SLEEPERs clustering
which form two groups, grouping with either CYTO- with DAYSLEEPER are functionally more conserved
SLEEPER or DAYSLEEPER (Figure 4). LOTUSSLEEPER1 than CYTOSLEEPER-clustering SLEEPERs. It has to be
is exceptional in that it has diverged rather far from the notedthattwoauxiliarySLEEPER-likegeneswereidenti-
other SLEEPERs in dicotyledonous plants. Since VINE- fied in Carica papaya. These genes showed BLAST
SLEEPER1 and 2 were described by Benjak et al. [14] (TBLASTN) values of justbelow 400 in relation to DAY-
andtheseproteinsclusterinseparategroups,wedecided SLEEPER and did not possess a conserved SLEEPERmo-
to use a similar naming scheme for all SLEEPERs. We tif1. These genes were therefore not included in
found synteny between the genomic regions in which Figure 4. If they were included in the alignment, these
theVINESLEEPER2andDAYSLEEPERgenesreside,sug- sequences cluster with LOTUSSLEEPER1, albeit with
gesting they are homologs (Additional file 2: Figure S1). verylong branch-length (datanot shown).
Table2EvidenceofSLEEPERgeneexpressioninlowerAngiosperms
Species Sleeperdomain1. Sleeperdomain3. FulllengthDAYSLEEPER
Perseaamericana b4_ep_c61270,b4_c39392 b4_c14697,b4_c9266,b4_ep_c32228 b4_c2641,b4_c7656
Nupharadvena b3_c39269 b3_c17103,b3_c9604 b3_c707,b3_c1078
Liriodendrontulipifera b3_c3339,b3_c108364 b3_c2953,b3_c39743 b3_c2953,b3_c21053
Amborellatrichopoda b4_c220959,b4_c97395 b4_c12734 ESThitstooshort
TBLASTNsearcheswereperformedontheESTdatabasesoftheAAGP(AncestralAngiospermGenomeProject;http://ancangio.uga.edu/).OnlyuniqueESTsare
shown.Thecut-offscoreforESTsfoundwiththefull-lengthDAYSLEEPERsequenceTBLASTNqueryis400.QuerieswereperformedwithSLEEPERmotif1and3and
thefull-lengthaminoacidsequenceofDAYSLEEPER.
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Figure4PhylogenetictreedepictingSLEEPERgenesfromvariousspecies.Darkgrey=CYTOSLEEPERcluster.Grey=DAYSLEEPERcluster.
Lightgrey=Poaceaecluster.Sequencesthatwereusedforcomplementationstudieshaveablacklogo.*Contains1DAYSLEEPERgene(D),and
CYTOSLEEPER(C).XThesespeciescontainoneDAYSLEEPERortholog(shown)andaCYTOSLEEPERortholog(notdepicted).Thenumberinsidethe
symbolisthenumberassignedtoeachgene.ThetreeiscreatedfromproteinsequencesalignedwithClustalW[33],processedbytheRaxML
algorythm,withbootstrapvaluesenabled[21].Clustershavebeengivenacolor.
RICE-andVINESLEEPERcauseadominantphenotype (Figure 5A,B). Furthermore, these plants formed small
whenexpressedinArabidopsis siliques, suggesting issues with seed development
To assess functionality of the SLEEPER genes found in (Figure 5D-G). Interestingly, we did not observe differ-
other species, we attempted to complement the dayslee- ences between plants containing the various constructs.
per phenotype with coding sequences from rice and However, we did observe differences in phenotype sever-
grapevine under control of the 3.6 kb upstream region ity among plants that were direct descendants of a pri-
of DAYSLEEPER, including the 5’UTR. We found that mary transformant (data not shown). This suggests that
the daysleeper phenotype cannot be complemented by the observed phenotype is associated to SLEEPER abun-
these constructs, although we were able to restore the dance, influenced by DAYSLEEPER hetero- or homozy-
wild-type phenotype with GFP:DAYSLEEPER constructs. gosity or the number of T-DNA inserts. DAYSLEEPER
We found seedlings with the daysleeper phenotype des- overexpression under control of the strong 35S pro-
pite the presence of either one of the RICESLEEPERS moter results in a similar phenotype as described above
(Figure 5C)orVINESLEEPERS(not shown). [12], also we observed similar phenotypic traits in some
Interestingly, the complementation constructs did in- plants when trying to complement daysleeper mutant
voke a dominant phenotype in Arabidopsis plants with plants with a GFP:DAYSLEEPER construct (data not
the DAYSLEEPER-gene still present. Such plants made shown). Complementation of daysleeper was not found
an excess of rosette leaves, often curled, and were with the coding sequence of At1g15300 (CYTOSLEE-
delayed in formation of inflorescences and in flowering PER) under control of the DAYSLEEPER promoter
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A B
C
D E F G
Figure5PhenotypeofArabidopsisplantsexpressingVINE-orRICESLEEPERs.A.DAYSLEEPER+/−plantexpressingpDAYSLEEPER::RICESLEEPER4.
B.DAYSLEEPER+/−plantexpressingpDAYSLEEPER::RICESLEEPER3.C.daysleepermutantharboringpDAYSLEEPER::VINESLEEPER1:HA.D.Siliquesfrom
Col-OplantsE.SiliquesfromDAYSLEEPER+/−plantexpressingpDAYSLEEPER::VINESLEEPER2.F.SiliquesfromDAYSLEEPER+/−plantexpressing
pDAYSLEEPER::RICESLEEPER3.G.SiliquesfromDAYSLEEPER+/−plantexpressingpDAYSLEEPER::RICESLEEPER4.PlantsdepictedinAandBare8weeks
old.Thescalebarsrepresent1cm.
region. Multiple plants of four individual T -DNA ~1500bp upstream of the start codon (Figure 6). For the
insertion lines were extensively analyzed, but none RICESLEEPER1 gene, no UTR’s other than the predicted
of these revealed a rescue of the daysleeper pheno- intronless 574 bases directly adjacent to the start codon
type, or resulted in DAYSLEEPER overexpression-like couldbeamplified.
phenotypes. To study whether RICESLEEPER mutation would re-
sult in similar developmental defects as seen in the A.
RICESLEEPER1andRICESLEEPER2 thaliana daysleeper mutant, two rice T-DNA insertion
RICESLEEPER 1 and 2 have nearly identical coding lines were obtained (Postech, Functional Genomics La-
sequences and probably both have arisen from relatively boratory) [24,25]. RICESLEEPER1 is disrupted by a T-
recent duplication events. A comparison between the DNA insertion in the coding sequence at approximately
RICESLEEPER1 and 2 loci can be seen in Figure 6. 1700 bp from the start codon (line: PFG_1D-01516).
RICESLEEPER2 is predicted to have an intron in its 5’ The T-DNA insertion in the RICESLEEPER 2 locus is
UTR, whereas RICESLEEPER1 is predicted to be intron- located in the 3’UTR of the gene (line: PFG_1B-21919).
less. To verify these predictions, we designed primers Presence of the T-DNA was verified by PCR (data not
based on available mRNA and ESTsequences and tried shown,Additionalfile3:TableS2).
to amplify the 5’ UTR from rice cDNA (PlantGDB [20] Hygromycin resistant heterozygous seeds were
and GenBank) (Additional file 1: Table S1). The PCR- obtained and grown and progeny of these plants ana-
fragments we obtained were isolated and sequenced. We lyzed.Forbothinsertionlinesonlywild-typeandhetero-
found two different transcripts for RICESLEEPER2, zygous plants were identified, indicating that plants
which we named “A” and “B” (Figure 6). Gene model A containing an insert in both copies of either RICESLEE-
corresponds with the predicted transcript (Rice Genome PER1 or RICESLEEPER2 might be lethal at a very early
Browser [23]), whereas the transcript depicted in gene stage. Hygromycin-resistant progeny of the RICESLEE-
model B contains an unspliced UTR that stretches to PER2 insertion mutants reached about half the height of
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Figure6ComparisonoftheRICESLEEPER1and2loci.Codingregionsandpartofthe5’and3’UTR’sare~97%identical.RICESLEEPER1and2
eachhaveobtainedanew5’UTRsequence,whichisnothomologoustothatoftheotherlocus.AandBdisplaytwodifferenttranscription
modelsfoundbyPCRforRICESLEEPER2.TranscriptionmodelBhasmostlikelyacquiredsequencematerialfromaretrotransposoninsertioninan
intronontheoppositestrand.Shortduplicationswerefoundflankingthezonesofhomologyinbothgenes,whichareshowninarrowedboxes.
ThesmallarrowsrepresentPCRprimers,whichweredesignedonavailablericegeneexpressiondata,andwereusedtoobtainRICESLEEPER1and
2transcriptsfromacDNAlibrary.PrimerdescriptionscanbefoundinAdditionalfile3:TableS2.
wild-type plants (Figure 7A,B). RICESLEEPER1 insertion phylogenetically clusters with the SLEEPERs (Figure 1),
mutants also remained somewhat smaller than wild-type but its amino acid sequence is only 30.1% identical to
plants (approximately two thirds of wild-type height), DAYSLEEPER (Table 1). A homozygous insertion mutant
but not as small as RICESLEEPER2 mutants. RICESLEE- (SALK_020839C)displaysnophenotypeanditscodingse-
PER2 mutants produced a normal amount of seeds, but quence cannot complement the daysleeper phenotype.
RICESLEEPER1 mutant plants produced mostly empty However,itseemslikelythatCYTOSLEEPERhasacquired
panicles, yielding only very few seeds (Figure 7C,D), in- novelfunctionality,sinceitseemsthataselectivepressure
dicating a lethal embryo defect. Organs of both insertion existstomaintainCYTOSLEEPER.Wecalculatedtheratio
mutants developed normally. However, yellow discolora- of the number of non-synonymous substitutions per non-
tions were observed in RICESLEEPER1 mutant plant synonymous site (Ka) to the number of synonymous sub-
leaves (Figure 7C insert), which are not present in wild- stitutions per synonymous site (Ks), to determine if selec-
typeplants(Figure7Dinsert),orRICESLEEPER2mutant tion pressure exists to maintain CYTOSLEEPER. Ka/Ks
plants(not shown). ratio(0,29)issimilartothatofDAYSLEEPER(0,28),when
comparing these genes in Arabidopsis thaliana and Cap-
Discussion sellarubella(Additionalfile4:FigureS2).
DAYSLEEPERconservation The highly conserved DNA-binding domain, which
All SLEEPERs have highly conserved features in spans the location of the second α-helix of the BED-zinc
the form of their N-terminally located BED-zinc finger finger [7], might hint to a conserved recognition se-
DNA binding domain, flanked by a nuclear localization quence for all SLEEPERs. SLEEPERmotif 3 is located in
domain and the C-terminal dimerization domain. These the dimerization domain of the SLEEPER coding se-
partly overlap with SLEEPERmotif1 and 3 respectively, quence. The dimerization domain is essential for DAY-
whereas SLEEPERmotif2 is localized adjacent to the SLEEPERfunction, sinceaC-terminaltruncationlacking
dimerization domain, but has no overlap or homology this domain is not able to rescue the daysleeper pheno-
to any known functional domain or motif. The type (M. Knip; unpublished results). The high conserva-
CYTOSLEEPER gene seems to be a divergent homolog tion of the dimerization domain in SLEEPER genes also
of DAYSLEEPER. CYTOSLEEPER possesses the SLEE- offers the theoretical possibility of heterodimerization
PERmotifs, but has lost its nuclear localization signal, between SLEEPERs, for instance in the case of DAY-
which is highly conserved in other SLEEPERs. This se- SLEEPER and CYTOSLEEPER. Heterodimerization can
quencedivergenceandthelackofthenuclearlocalization theoretically take place, since expression patterns of
motif might indicate pseudogenization. CYTOSLEEPER these genes overlap in several tissues (Arabidopsis eFP-
has relatively well conserved SLEEPERmotifs and browser [26], data not shown). The possibility of
Knipetal.BMCPlantBiology2012,12:192 Page10of15
http://www.biomedcentral.com/1471-2229/12/192
A B C D
Figure7RICESLEEPER1andRICESLEEPER2T-DNAinsertionmutants.A,D.Wild-typeplantsofcultivarsDongjinandDaesan,respectively.B.
RICESLEEPER2insertionmutantplants(PFG_1B-21919,Dongjincultivar).C:RICESLEEPER1insertionmutantplants(PFG_1D-01516,Daesancultivar).
TheinsertsinC,DshowleafsectionsofrespectivelyaRICESLEEPER1insertionmutantplantandawild-typecv.Daesanplant.Plantswere
photographed80daysaftergermination.Scalebarsrepresent10cm.
heterodimerization is even likely in the case of RICE- were also observed in some daysleeper mutant plants
SLEEPER1 and 2, since their coding sequences are al- complementedwithaGFP:DAYSLEEPERconstruct(data
most identical and their expression patterns partly not shown). The fact that SLEEPERs cause this pheno-
overlap [23] . We have found that nuclear heterodi- type suggests that they are at least partially functionally
merization is possible in vivo for DAYSLEEPER and similar to DAYSLEEPER. Interestingly, the clustering of
RICESLEEPER4 (Figure 2) in a Bi-molecular fluores- CYTOSLEEPER with other SLEEPERs, such as VINE-
cence complementation (BiFC) assay in Arabidopsis SLEEPER1, suggests that other species possess func-
protoplasts, using DAYSLEEPER:YC and YN:RICE- tional SLEEPERs that are derived from the same
SLEEPER4 fusion proteins (data not shown). The abil- duplication as the CYTOSLEEPER gene. In poplar, none
ity to heterodimerize may offer an interesting layer of of the SLEEPER genes found cluster with CYTOSLEE-
complexity to the function of SLEEPER proteins in PER, suggesting that a SLEEPER derived from the dupli-
several species. cation eventmentioned above, waslostinthisspecies.
SLEEPERcomplementation RICESLEEPER1and2
Although complementation of DAYSLEEPER is not RICESLEEPER1 and 2 are highly similar and have arisen
found with constructs containing other SLEEPERs, these from a duplication event (Figure 6). We suggest that
constructs cause a dominant phenotype in Arabidopsis these RICESLEEPER genes are relatively recently dupli-
(Figure 5). The transformed plants display developmen- cated retrogenes. In the rice genome many retrocopies
tal issues: delayed formation of the inflorescence and ir- and retrogenes can be found, which could be explained
regular and increased formation of leaves, fasciation and by the overall high activity of LTR retrotransposons in
dwarfismhavebeenobservedinalllines.Thisphenotype this species [27]. Retrocopied genes are devoid of
resembles the overexpression phenotype of plants bear- introns, since they are derived from mRNA sequences
ing a 35S:DAYSLEEPER construct [12] and it is probable and are flanked by short non-transposon-derived dupli-
that this effect is caused by increased expression of cations. Both RICESLEEPER1 and 2 meet these criteria
SLEEPER genes in these plants. This is further substan- (Figure 6). Recent retrocopies often possess a relic poly-
tiated by the fact that mild overexpression phenotypes A tail, derived from the mRNA they originated from