The study of two barley Type I-like MADS-boxgenes as potential targets of epigenetic regulation ...

22 Pages·2012·3.21 MB·English

Kapazoglouetal.BMCPlantBiology2012,12:166
http://www.biomedcentral.com/1471-2229/12/166
RESEARCH ARTICLE Open Access
The study of two barley Type I-like MADS-box
genes as potential targets of epigenetic
regulation during seed development
Aliki Kapazoglou1, Cawas Engineer1, Vicky Drosou1, Chrysanthi Kalloniati3, Eleni Tani1, Aphrodite Tsaballa2,
Evangelia D Kouri3, Ioannis Ganopoulos2, Emmanouil Flemetakis3 and Athanasios S Tsaftaris1,2*
Abstract
Background: MADS-box genes constitute a large family of transcription factors functioning as key regulators of
many processes during plant vegetativeand reproductive development. Type II MADS-box genes have been
intensively investigated and are mostly involved invegetativeand floweringdevelopment. A growing number of
studies ofType I MADS-box genes inArabidopsis, have assigned crucial roles for these genes ingamete and seed
development and have demonstrated that a numberof Type I MADS-box genes are epigenetically regulated by
DNA methylation and histone modifications. However, reports on agronomically important cereals such as barley
and wheat are scarce.
Results: Here wereport the identification and characterization oftwo TypeI-like MADS-box genes, from barley
(Hordeum vulgare),a monocot cereal crop of high agronomic importance. Protein sequence and phylogenetic
analysis showed that theputative proteins are related to Type IMADS-box proteins, and classified them ina distinct
cereal clade.Significant differences in gene expression among seed developmental stages and betweenbarley
cultivars withvarying seed size were revealed for both genes. One of these genes was shown to be induced by the
seed development- and stress-related hormonesABA and JA whereas in situ hybridizations localized theother gene
to specific endosperm sub-compartments.The genomic organization ofthe latter has highconservation withthe
cereal TypeI-like MADS-box homologues and thechromosomalposition ofboth genes is closeto markers
associated with seed quality traits. DNA methylation differences are present in theupstream and downstream
regulatory regions of the barley TypeI-like MADS-box genesin two different developmental stages and inresponse
to ABA treatment which may be associated with gene expression differences.
Conclusions: Two barley MADS-box genes were studied that are related to Type I MADS-box genes. Differential
expression in different seed developmental stages as well as in barley cultivars withdifferent seed sizewas
evidenced for both genes. The two barley TypeI MADS-box genes were found to be induced by ABA and JA. DNA
methylation differences indifferent seed developmental stages and after exogenous applicationof ABA is
suggestive of epigenetic regulation ofgene expression. The study of barley Type I-like MADS-box genes extendsour
investigations of gene regulation during endosperm and seed development ina monocot crop likebarley.
Keywords: MADS-Box, Epigenetic regulation, Chromatin, DNA methylation, Histonemethylation, Seed
development, Endosperm,Retrotransposon, Barley
*Correspondence:[email protected]
1InstituteofAgrobiotechnology(INA),CERTH,Thermi-ThessalonikiGR-57001,
Greece
2DepartmentofGeneticsandPlantBreeding,AristotleUniversityof
Thessaloniki,ThessalonikiGR-54124,Greece
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Kapazoglouetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Kapazoglouetal.BMCPlantBiology2012,12:166 Page2of22
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Background manner where the paternal allele is expressed whereas
In angiosperms, the endosperm of the developing seeds the maternal allele is silenced [19,20]. Both DNA and
is formed as a result of the double fertilization event. histone methylation are responsible for these silencing
Fertilization of the egg cell by a sperm cell from the events [19-21]. The PRC2 Polycomb group complex is
male gametophyte generates the diploid embryo from partially responsible for keeping the maternal allele si-
which the tissues, organs, and shoot meristems of the lent in the female gametophyte and in the seed after
plant will be generated. Fertilization of the adjacent cen- fertilization[19-21].Thisisachievedthroughthehistone
tral cell by a second sperm cell forms a triploid endo- methylation activity of the PRC2 complex, conferred by
sperm which supports embryo growth and development one of its subunits, the histone methyltransferase
by producing storage proteins, lipids and starch [1,2]. MEDEA (MEA) with histone 3 lysine 27 trimethylation
During this process, a large number of genes are acti- activity (H3K27me3). The PRC2 complex also restricts
vated. Epigenetic regulatory considerations become im- the expression of PHE1 to the chalazal domain of the
portant in relation to which parental allele will be endosperm after fertilization. Arabidopsis mea mutants
expressed and in which reproductive tissue, a factor that show upregulated PHE1 expression and form defective
ultimately governs among other things the size of the seed-like structures before fertilization and endosperm
endosperm[3-6]. overproliferation after fertilization [19,20,22]. Further-
Developmental transition of plants from the vegetative more, a distantly located region downstream of paternal
to the reproductivestage and from the floral stage to the PHE1 was found to have a DNA methylation require-
seed stage relies on the activity of MADS-box transcrip- mentfor PHE1expression[22].
tion factors. With the completion of the Arabidopsis Additional Type I MADS-box genes have been impli-
genome, more than 100 genes encoding for MADS-box cated in gamete and seed development. The AGA-
transcription factors were uncovered which can be MOUS-LIKE23 (AGL23) gene was found to regulate
phylogenetically classified in five clades, termed MIKC, female gametophyte formation and normal embryo de-
Mα, Mβ, Mγ and Mδ (Arabidopsis Genome Initiative, velopment [23]. AGAMOUS-LIKE 80 (AGL80) was
2000) [7,8]. The Mα, Mβ, Mγ subfamilies comprise the shown to be critical for central cell and endosperm de-
Type I lineage, whereas the MIKC and Mδ constitute velopment in Arabidopsis [24]. In agl80 mutants the for-
the Type II lineage. Similarly, phylogenetic analysis mation of the central cell is defective, and endosperm
based on the completed rice genome revealed 44 Type II development fails to initiate after fertilization. Similarly,
MADS-box genes, of the MIKC and Mδ clades, and 35 AGAMOUS-LIKE 61 (AGL61) also termed DIANA, plays
Type I MADS-box genes of the Mα, Mβ, Mγ clades [9]. an important role in central cell and endosperm forma-
The two lineages are proposed to have arisen by a gene tion, in Arabidopsis [16,25]. Like agl80, agl61 mutants
duplication that took place in the common eukaryotic haveaberrantcentralcellmorphology whichdegenerates
ancestor more than a billion years ago [10-13]. MIKC before fertilization occurs, and endosperm development
genes harbor the characteristic MADS-box domain (M), does not take place post fertilization [16,25]. Both genes
the Intervening domain (I), the Keratin-like domain (K) are expressed exclusively in the central cell and early
and the C-terminal domain (C). In contrast, the Type I endosperm and this along with their similar mutant
genes contain only the MADS-box domain (M). Al- phenotype suggests that AGL61 and AGL80 proteins
though theType I genes represent about 60% of the total may function as heterodimers within the central cell. In
MADS genes in Arabidopsis, they have only recently agreement to this, AGL61 and AGL80 proteins were
started to be studied, whereas the Type II genes have found to interact in yeast two-hybrid assays [16,25]. Of
been extensively investigated both structurally and func- equal interestis AGL62with high expression in the early
tionally [8,14-17]. This is due mostly to the fact that nuclear endosperm and sharp decline right before cellu-
Type I MADS genes function during early gametogen- larization [26]. In agl62 mutants, the endosperm cellu-
esis, embryogenesis and seed formation and conse- larizes prematurely indicating that the AGL62 is
quently homozygous mutants are lethal. In addition required for repression of precocious cellularization dur-
their expression is very low during these developmental ing the syncytial phase. AGL62 is under the epigenetic
stages[8,18].AmongthefewTypeIfamilymembersthat control of PRC2 genes, as in prc2 mutants AGL62 fails
have been studied is PHERES 1 (PHE1) [also known as to become silent and endosperm cellularization is
AGAMOUS-LIKE37 (AGL37)], a Mγ-type gene with an arrested [26]. Thus, AGL62 seems to regulate the timing
important role in Arabidopsis gamete and seed develop- of endosperm cellularization, which is triggered epigen-
ment [19]. Epigenetic regulatory mechanisms have been etically by PRC2-mediated AGL62 silencing. Likewise, a
implicated in the transcriptional control of this gene. recent study on AGL36 demonstrated that the expres-
The PHE1 gene is expressed transiently at high levels sion of the AGL36 maternal allele is epigenetically con-
immediately after fertilization in a parentally imprinted trolled in a sequential manner, firstly upregulated by the
Kapazoglouetal.BMCPlantBiology2012,12:166 Page3of22
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function of DEMETER (DME), a DNA glycosylase en- three of the ArabidopsisType I MADS-box genes, PHE1,
zyme responsible for demethylating MEDEA, and then AGL62, and AGL36 are under epigenetic regulation
downregulated during endosperm development by the mediated, in part, by the chromatin repressive enzymatic
PRC2 complex [27]. Understanding the genetic and epi- complex PRC2. Our group has recently characterized
genetic processes controlling the timing of endosperm barley genes encoding a putative PRC2 Polycomb group
cellularization could be of particular interest in agricul- complex [30]. To further focus our research efforts in
ture,asprematureordelayedcellularizationisassociated this area and study potential barley targets of a PRC2
with smallandlargeseed sizeandweight,respectively. complex, we report here the identification and structural
An extensive genome-wide study of 60 Type I MADS- characterization of two barley Type I-like MADS-box
box genes in Arabidopsis uncovered a cell-type-specific genes. Their expression has been studied in different tis-
expression pattern during female gametophyte and early sues and seed developmental stages, in two cultivars
endosperm development [18]. Most genes are expressed with varying seed size, and after exogenous application
in the central cell and antipodal cells in the female gam- of the developmental- and stress-related phytohormone
etophyte and in the chalazal and peripheral endosperm ABA. In addition, their genomic organization was exam-
of 1–2 days after fertilization (DAF) developing seeds. ined and compared to their cereal homologues. Finally,
Thesedata areinagreementwiththeresultsfromearlier the 5’upstream regions were analyzed for conserved cis
functional studies of individual genes like PHE1, AGL23, regulatory elements and the DNA methylation patterns
AGL61, AGL62 and AGL80 and propose a role for Type of upstream and downstream regions were investigated
I MADS-box genes in female gametophyte and endo- in two tissues with differential Type I-like MADS-box
spermdevelopment. gene expression.
Considerably less is known about Type I MADS-box
genes in other plant species, especially monocots. In Methods
wheat, 42 MADS-box genes were identified in silico, of Plantmaterial
which 8 were classified as Type I [28]. In rice, a global- Commercial barley cultivars, Caresse, Byzantio, and
scalemicroarrayexpression analysisidentifiedanexpres- Ippolytos differing in seed size and weight were planted
sion pattern for all Type II and Type I MADS-box genes in the field and were the source oftotal RNA for expres-
during vegetative and reproductive development [9]. sion analysis. For Caresse, the weight of 1000 grains is
Overall Type I MADS-box genes had lower expression 50–55 gr, and 98% of seeds have diameter longer than
levels than Type II MADS-box genes. Certain Type I 2.5 mm, for Byzantio the weight of 1000 grains is 36–42
genes were expressed throughout development, whereas gr and 75% of seeds have diameter longer than 2.5 mm,
others exhibited more specific expression in particular whereas for Ippolytos, seeds weight 25–31 gr per 1000
tissues. For example, four rice Type I genes, of the sub- grains and only 35–45% of seeds have diameter longer
class Mα, were found predominately expressed in seeds than 2.5mm(www.cerealinstitute.gr).
5–20 days after fertilization suggesting a role for these
genes in seed development. Recently, a rice Type I Hormonaltreatment
MADS-box gene, OsMADS87, was reported to be mater- Seven-day-old seedlings (Caresse) grown in a growth
nally expressed in rice endosperm and associated to chamber (16 hours (h) light, 8 h darkness, at 22°C) were
endosperm developmental transitions caused by inter- sprayed with 100 μM ABA, (abscisic acid +/− cis, trans-
specific hybridization[29]. ABA, SIGMA) and 100 μM JA (methyl jasmonate,
Cereal crops account for about 50% of global human ALDRICH). Aerial parts of plants were collected at 6 h
calorific intake with the endosperm of cereal seeds being and 24 h after treatment and immediately stored in li-
one of the most important sources (faostat.fao.org). quid nitrogen. Aerial parts from five plants were pooled
Chief among these monocots are barley, wheat, rice and togetherforRNAextractionforeachtimepoint.Control
maize. Besides human nutrition, cereal crops also repre- plantsweresprayedwithwater plus0.2% Tween.
sent major sources of feedstock, fiber and recently bio-
fuel substrates. Contrary to Arabidopsis and other dicots RNAisolationandfirststrandcDNAsynthesis
where the endosperm is consumed during seed develop- Total RNA wasisolatedfrom roots,shoots,apical meris-
ment, in monocots, such as barley and other cereals, the tems, first leaves of seedlings, flowers before fertilization
endosperm persists and constitutes the nutritional part (immature flowers), seeds 1–3, 3–5, 5–10, 10–15, 15–20
oftheseedcontainingstorageproteinsandstarch. days after fertilization (DAF), and aerial parts after hor-
Considering the implications of Type I MADS-box monal treatments, respectively, using TRI REAGENT
genes on seed development and the agronomic import- (SIGMA) according to the instructions of the manufac-
ance of cereal endosperm, we set out to identify and turer. First strand cDNA synthesis was performed using
characterize Type I MADS-box genes in barley. At least 1.0 μg total RNA, 0.5 μg 3’ RACE Adapter primer,
Kapazoglouetal.BMCPlantBiology2012,12:166 Page4of22
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5’-GGCCACGCGTCGACTAGTAC (T) -3’ (Invitrogen), OPA-2009-3 H [37], the Barley, OPA123-2008, Consen-
17
1 mM dNTPs and 200U of Superscript II (Invitrogen) in sus-Hordeum-OPA123-2008-3 H and the Barley, OWB,
20 μL total volume, according to the specifications of OPA2008-Hordeum-OWB-OPA2008-3 H [38] using the
themanufacturer. HarvEST tool and the comparative map viewer (Cmap)
availableatGramene(http://www.gramene.org/cmap/).
Proteinsequenceanalysis
1895 viridiplantae proteins sequences from UniProt with ExpressionanalysisofbarleyTypeI-likeMADS-boxgenes
a statistically significant hit for the MADS-box domain Qualitative RT-PCR and quantitative real-time RT-PCR
(PF00319: SRF-type transcription factor (DNA-binding wasperformed withcDNA synthesized from1 μg oftotal
and dimerization domain) were collected from the Pfam RNA from roots, stems, meristems, leaves, immature
database [31]. After sequence fragments were filtered flowers, seeds 1–3 DAF, 3–5 DAF, 5–10 DAF, 10–15
out, the resulting set was the subject of an all-against-all DAF and 15–20 DAF and aerial parts of seedlings
similarity detection step using the BLASTalgorithm. We after ABA and JA treatment. For real-time PCR, each
used the bidirectional best hit approach [32] for disco- sample reaction was set up in a PCR reaction mix
vering putative orthologous proteins; we also asked for (20 μl) containing 5 μl of the 1:50 diluted cDNA,
any orthologous sequence to be one of at least twenty 0.25 μM of each primer and 1X Platinum SYBR Green
sequences with the MADS-box domain from the corre- qPCR Supermix-UDG (Invitrogen, Paisley, UK) and
sponding species, so that we reduce the chance of best using the Corbett Rotor Gene 6000. Each reaction was
hits due to lack of sequence context. The multiple align- performed in triplicates. General thermocycler condi-
ment was created with MAFFT [33] and edited with Jal- tions were50°C for 2 min, 95°C for 2 min,then 42 cycles
View version 2.5 [34] after which the phylogenetic tree of 95°C for 15 sec, annealing [53°C for HvOS1 and 57°C
was constructed in the same software with the Neighbor for HvOS2] for 20 sec, extension 72°C for 20 sec, then
Joining method using a BLOSUM62-based distance 72°C for 5 min. To identify the PCR products a melting
measure. The phylogenetic tree was calculated using curve was performed from 65°C to 95°C with observa-
MEGA 3.1 software [35] by the Neighbor-Joining tions every 0.2°C and a 10-s hold between observa-
Method with p-distance correction [36]. Bootstrap tions. Relative quantification was performed using actin
values wereobtainedfrom1000bootstrapreplicates.Ac- as the reference gene and HvActinF / HvActinR as pri-
cession numbers for sequences used for alignments and mers. The barley genes HVA22 and HvADC (arginine de-
phylogenetic analysis areindicated inTable 1. carboxylase 2) which are known to be induced by ABA
and JA, respectively [39,40], were used as positive con-
Genomicorganization trols. All primers used in expression analysis corres-
The genomic sequences of Brachypodium distachyon _ pond to non-conserved regions and are shown in
Bradi2g59120, Brachypodium distachyon_ Bradi2g59190, (Additional file 1: Table S2).
Oryza sativa_OsMADS65_Q9XJ61 (Os01g0922800),
and Zeamays_ZmMADS1_GRMZM2G171650_B4FML Insituhybridization
1, were downloaded from the Phytozome database In situ hybridization experiments were performed as
(http://www.phytozome.net/). Thesequencesofthetwo it has been described previously [41]. Briefly, seeds
full length barley Type-like MADS-box ESTs were used were fixed in 4% (w/v) paraformaldehyde supplemen-
to interrogate the barley database http://webblast.ipk- ted with 0.25% (v/v) glutaraldehyde in 10 mM so-
gatersleben.de/barley/index.php for detection of gen- dium phosphate buffer (pH 7.4) for 4 h in a vacuum
omic sequences. Genomic organization of exons and aspirator. Fixed tissues were block-stained in 0.5% (w/v)
introns was obtained using the mRNA-to-genomic safranin, dehydrated through ethanol series, embed-
alignment Spidey tool, in NCBI (http://www.ncbi. ded in paraffin and cut into 8 mm-thin sections.
nlm.nih.gov/spidey). Detection of retroelements was Antisense RNA probes labelled with digoxigenin-11-
performed with the MASiVE and LTRharvester tools rUTP (Boehringer Mannheim, Mannheim, Germany)
(http://tools.bat.ina.certh.gr/masive/) (http://tools.bat. were originated from PCR-generated templates incorpo-
ina.certh.gr/ltrharvester/) and homology was visua- ratingT3 polymerase sites. The probe was designed close
lized with Circoletto (http://tools.bat.ina.certh.gr/cir- tothe3’-UTRofthegeneanditslengthwas202bp.Sec-
coletto/), all three tools developed in-house at INA, tions were prepared for hybridization as described before
by the Bioinformatics Analysis Team (BAT). [42]andhybridizedovernightat42°Cin50%(v/v)forma-
mide, 300 mM NaCl, 10 mM Tris–HCl pH 7.5, 1 mM
Mappinginsilico EDTA, 0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrroli-
An in silico visual comparative analysis was performed done,0.025%(w/v)bovineserumalbumin(BSA),10%(v/v)
against the Barley, OPA 2009, Consensus-Hordeum- dextran sulfate and 60 mM DTT. After hybridization, the
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Table1TypeIMADS-boxsequencesusedforalignmentsandphylogenetictreeconstruction
Organism Genename Accessionnumber TypeI
Cerealtype-I-like
Hordeumvulgare HvOS2 HM130526.1TC178280
Triticumaestivum TaAGL33 ABF57950.1Q1G159
Brachypodiumdistachyon Bradi2g59190 Bradi2g59190
Hordeumvulgare HvOS1 HM130525.1BG365393
Triticumaestivum TaAGL42 ABF57942.1Q1G167
Brachypodiumdistachyon Bradi2g59120 Bradi2g59120
Zeamays ZmB4FML1 NP_001140218.1
Orysasativa OsMADS65 Os01g0922800Q9XJ61
Mα
Arabidopsisthaliana AtAGL62 NP_200852.1Q9FKK2
Arabidopsisthaliana AtAGL61 NP_850058.1A5AZX6
Arabidopsisthaliana AtAGL23 NP_176715.1
Arabidopsisthaliana AtAGL28 NP_171660.1
Poplartrichocarpa AGL62-like XP_002313197.1B9HN34
Vitisvinifera AGL62-like A5AZX6
Ricinuscommunis AGL62-like B9S7W9
Orysasativa OsMADS71 Os06g22760.1
Orysasativa OsMADS78 Os09g02830.1
Orysasativa OsMADS79 Os01g74440.1
Mβ
Arabidopsisthaliana AtAGL47 NP_200380.1
Arabidopsisthaliana AtAGL82 NP_200697.1Q9FIM0
Poplartrichocarpa PtMADS-I-like XP_002301840
Orysasativa OsMADS90 Os07g04170
Orysasativa OsMADS91 Os01g11510.1
Orysasativa OsMADS96 OsO1g67890
Mγ
Arabidopsisthaliana PHE1AGL37 NP_176712.1O80805
Arabidopsisthaliana PHE2AGL38 NP_176709.2Q7XJK8
Arabidopsisthaliana AGL80 NP_199678.1Q9FJK3
Orysasativaindica OsAGL80-like EAY73584.1Q75IC5
Orysasativajaponica OsjAGL35-related BAD81343.1
Orysasativa OsMADS82 Os04g24800
Orysasativa OsMADS83 Os04g24810
Orysasativa OsMADS85 Os04g25920
Poplartrichocarpa PtAGL80-like XP_002329991.1B9I7J4
Petuniahybrid PhAGL80-like B6DT62
Ricinuscommunis RcAGL80-like B9S273
Vitisvinifera VvAGL80-like A5BJU9
sections were treated with a solution containing visualized with anti-digoxigenin antibodies conjugated
500 mM NaCl, 1 mM EDTA, 10 mM Tris–HCl and with alkaline phosphatase. Images were processed using
50μg/mlRNaseA.Finally,sectionswerewashedseveral Photoshop 7 software (Adobe Systems Inc., San Jose,
times in a 2xSSC solution. Hybridization signals were CA, USA).
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Genomewalkingand5’upstreamsequenceanalysis MADS-box domain and the absence of a K-domain,
Genome walking experiments were conducted on leaf characteristic of Type I MADS-box proteins. These can-
genomic DNA obtained from the barley Caresse cultivar. didates were: BG365393 and TC178280 (TIGR database,
The Clontech genome walking kit was used and proce- www.tigr.org) (see Materials and Methods). During the
dureswereconductedaccordingtomanufacturer’sspecifi- course of our investigations another study characterizing
cations. Sequence information was obtained by doing these sequences with respect to their role in the
threeprogressiveroundsofgenomewalkingusingESTse- vernalization process was reported [43]. These authors
quencedataasstartingpoints.Statisticallysignificantpre- designated these sequences as ODDSOC1(HvOS1) and
diction for CpG islands was performed using the online ODDSOC2(HvOS2), respectively. In our recent study on
predictor,whichispartofthesequencemanipulationsuite the identification and characterization of two SOC1-like
at http://www.bioinformatics.org/SMS/index.html. The gene homologues from barley [44] it was shown that
prediction of the putative cis acting elements was accom- two HvSOC1-like proteins containing the K-box domain
plished using theTSSP /Prediction of PLANT Promoters are closely related to the Type II MADS-box proteins
algorithm (Using RegSite Plant DB, Softberry Inc.) in the whereas they are more distantly related to ODDSOC1
SoftBerry database (http://linux1.softberry.com/cgi-bin/ and ODDSOC2. However, since Greenup et al. (2010)
programs/promoter/tssp.pl) and PlantCARE (http://bio [43] were the first to report these genes we will utilize
informatics.psb.ugent.be/webtools/plantcare/html/). The the nomenclature introduced by these authors and here-
positionoftheputative5’upstreamregulatoryelementsis after will be referring to the sequences BG365393 and
indicatedaccordingtothestartcodon(ATG). TC178280 as HvOS1 and HvOS2, respectively.
HvOS1 is composed of 834 nt encoding a putative pro-
DNAmethylationassays tein of 167 aa, and harboring a 5’UTR of 76 nt and a
Genomic DNA was prepared from immature flowers 3’UTRof253nt.HvOS2iscomposedof1319ntencoding
andfrom1–3DAFseeds(Caresse)withQiagencolumns a putative protein of 159 aa, and containing a 5’UTR of
following the protocol of the manufacturer (Qiagen 382 nt, and a 3’UTR of 460 nt. An alignment of MADS-
Plant genomic DNA kit). Cytosine DNA methylation box proteins (Type I and Type II) from different plants is
was analyzed by restricting 1 μg of genomic DNA from shown in Figure 1. The two barley putative protein
each sample with the methylation-dependent enzyme sequencesHvOS1 andHvOS2showhighsimilarity tothe
McrBC (NEB Biolabs), according to the manufacturer’s wheat sequences that have been previously classified as
instructions, and PCR-amplifying equal quantities of Type I MADS-box sequences [28]. HvOS1 has 96% iden-
McrBC-treated and untreated samples. Primers used are tity with the wheat TaAGL-42 sequence and HvOS2
shownin(Additionalfile1:TableS2). shares 92% identity with the wheat TaAGL-33 sequence.
HvOS1 and HvOS2 putative protein sequences share
Results 88.9% identity with each other. In Type II MADS-box
IdentificationofTypeI-likeMADS-boxgenesinbarley proteins the characteristic three subdomains of the
andproteinsequenceanalyses K-region,K1,K2andK3areevidentasshowninFigure1.
Initial efforts to identify orthologues of Type I MADS- The barley HvOS1 and HvOS2, wheat TaAGL-33 and
box genes in barley EST databases via BLASTand other TaAGL-42, as well as their closest relatives rice
sequence similarity-based approaches failed to result in OsMADS65 and maize ZmB4FML1 harbor no regions
significant hits. Additional efforts using degenerate pri- with similarity to K regions, as expected for Type I
mers designed using Arabidopsis sequence data and bar- MADS-box proteins. A separate alignment of the barley
ley cDNA from multiple cultivars did not result in target HvOS1 and HvOS2 and different Type I MADS-box
gene isolation either (data not shown). Hence a more sequences was constructed for more clarity (Figure 2).
rigorous bioinformatics approach was employed and a Most of the similarity between the barley HvOS1 and
monocot-specific C-terminus probe was generated using HvOS2 and the other Type I MADS-box sequences is
existing sequence data for MADS-box putative proteins. concentrated in the MADS-box domain (first 80 aa),
Thesequenceofthisprobe wasthefollowing: whereas the rest of the sequences show a large degree of
GAGXXVNGXOXXXNXDXXXXOOQXXLKEIAXWX divergence. A phylogenetic tree was constructed from a
XQNNAOXXDANOLEKLEOLLTOALRNTKXKKMLXO largenumberofMADS-boxproteinsfromdifferentplants
ONXG, where X is any amino acid and O is a possible (Figure 3). Three of the subclasses of Type I, Mα, Mβ
gap. This probe was used to isolate several cereal Type and Mγ, are shown grouping out into separate clades.
I-like MADS-box sequences from available EST data- The barley HvOS1 and HvOS2 putative proteins, and
bases and the GenBank non-redundant CDS translated their putative cereal orthologues, namely, wheat TaAGL-
database. Of particular interest were two hits from the 33 and TaAGL-42, brachypodium Bradi2g59190 and
barley EST database, chosen based on the presence of a Bradi2g59120, rice OsMADS65, and maize ZmB4FML1,
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MADS-box domain
I
II
I
II
K1 domain K2domain K3domain
I
II
Figure1AminoacidsequencecomparisonsbetweenTypeIMADS-boxproteinsandTypeIIMADS-boxproteinsfromdifferentplants.
ThenameofeachsequenceconsistsofitsUniprotIDorGenBankaccessionnumber,followedbythespeciesabbreviationaccordingtoUniprot.
TheK-boxdomains,K1,K2andK3oftheTypeIIMADS-boxproteinsareshowninboxesandindicatedwithredbars.TheMADS-boxdomainis
alsoindicatedwitharedbar.NoK-boxispresentintheType-Iproteins.Identicalaminoacidsareshownindarkblueandsimilaraminoacidsin
lightblue.
cluster together forming a distinct subclass (Figure 3). Ippolytos examined. Conversely, HvOS2 transcripts
As mentioned above due to the absence of a typical are present in roots, shoots, apical meristem, leaves,
K-domain which is the hallmark of Type-I proteins, we and immature flowers in both Caresse and Ippolytos
named these barley sequences as Type I-like HvMADS- (Figure 4A).
box sequences. Real time PCR analysis was employed to examine and
compare the expression of barley Type I-like HvMADS-
ExpressionanalysisoftheTypeI-likeHvMADS-boxgenes box genes at different seed developmental stages and in
indifferenttissuesandduringseeddevelopment different cultivars, Caresse (a large-seed cultivar) and
End point PCR analysis showed that HvOS1 tran- Ippolytos(a small-seedcultivar) (Figure 4B).
scripts were detected only in immature flowers in For HvOS1, an induction of approximately 8 fold in
Caresse, whereas they were present in all tissues of Caresse 1–3 DAF and 3–5 DAF seed, and a decrease
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MADS-box domain
Figure2AminoacidsequencealignmentsbetweenbarleyHvOS1,HvOS2,andTypeIproteinsfromdifferentplants.Thenameofeach
sequenceconsistsofitsGenBankaccessionnumber,alsoshowninTable1.Identicalaminoacidsareshownindarkblueandsimilaraminoacids
inlightblue.TheMADS-boxdomainisindicatedwitharedbar.
thereafter, was observed. In Ippolytos, HvOS1 was DAF seeds whereas they increased slightly in 10–15
induced in 1–3 and 3–5 DAF seeds by approximately 2 DAF as compared to immature flowers. In Caresse 15–
fold. Transcript levels dropped by about 2 fold in 5–10 20 DAF seeds transcript levels increased by about 2 fold,
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SEPALATA-AGAMOUS-SOC1
(Type II)
99 HvOS2
96 TaAGL33
41 Bradi2g59190 Cereal
68 OsjMADS65 Type I-like
Bradi2g59120 MADS-box
100 73 HvOS1
99 TaAGL42 MADS box
ZmMADS-I-like
94 100 OsMADS78
51 OsMADS79
OsMADS71
74 100 AtAGL28 Ma
AtAGL23
100 AtAGL61
AtAGL62
94
98 PtAGL62-like
OsMADS90
100 OsMADS91
100 AtAGL82 Type I
49 AtAGL47 MADS-box
PtMADS-like
OsMADS96
96 AtAGL80
100
PtAGL80-related
100
31 AtPHE1
100 AtPHE2
99 100 OsiAGL80-related
OsjAGL35-related
100 OsMADS83
OsMADS82
100
100 OsMADS85
0.1
Figure3PhylogenetictreeofMADS-boxproteinsfromdifferentplants.PhylogenetictreeshowingthedifferentcladesofMADS-boxTypeI
familyproteins.TheTypeIIMADS-boxSEPALATA,AGAMOUSandSOC1familiesareshownasacondensedbranch.Thesequencesusedandtheir
accessionnumbersareshowninTable1.BarleyHvOS1andHvOS2membersareinbold.Numbersindicatebootstrapvalues(1000=100%).
whereas in Ippolytos 15–20 DAF they decreased by 15–20 DAFseeds (Figure 4B). Expression of HvOS2 was
about 5fold (Figure 4B). examined in another large-seed cultivar, Byzantio.
A marked increase of approximately 8 fold in HvOS2 Similar to Caresse, HvOS2 transcript levels were signifi-
transcript accumulation was observed in Caresse seeds cantly lower in 5–10, 10–15 DAF seeds as compared to
1–3 DAF and 3–5 DAF, as compared to immature Ippolytos(AdditionalFile2).
unfertilized flowers. Expression levels dropped thereafter More detailed expression of the HvOS2 gene within
in 5–10, 10–15 and 15–20 DAF seeds to levels compar- the seed was examined by in situ localizations using
able to those of unfertilized flowers. In the small-seed crosssectionsofseedsandDIG-labelledantisenseprobe.
cultivar, Ippolytos, HvOS2 exhibited substantial tran- Strong hybridization signal was observed in the aleurone
script accumulation in seeds 1–3 DAF and 3–5 DAF. In layer and the first layer of endosperm cells adjacent to
5–10 DAF and 10–15 DAF seeds HvOS2 had a pro- thealeuronelayerthatwasabsentfromthesense-control
nounced increase of approximately 10–15 fold, in con- (Figure 4C), suggesting cell-specific expression in the
trast to Caresse. In Caresse seeds 15–20 DAF there was endosperm. In addition, strong hybridization signal was
a slight increase of HvOS2 transcript. Conversely, a sub- observed in embryo cells between sections hybridized
stantial decrease of about 10 fold was seen in Ippolytos with antisense as well as sense probes suggesting no
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A) R AM YS L IF
HvOS1
HvOS2 Caresse
HvACTIN
HvOS1
HvOS2
Ippolytos
HvACTIN
B)
HvOS1
o
ati 12 *
r 10 *
n
o
i 8
s
s
e 6 * *
r
p 4 *
x
e *
2
e
v
i 0
t
a
l C5 C6 C7 C8 C9 C10 IP5 IP6 IP7 IP8 IP9 IP10
e
r
seed developmental stage
o
i HvOS2
t
a
r 30
r
n *
o 25
si
s 20
pre 15 * *
ex 10 * * *
e
v 5
ti
a 0
el
r C5 C6 C7 C8 C9 C10 IP5 IP6 IP7 IP8 IP9 IP10
seed developmental stage
C)
antisense sense
e
e
a
a
p
p
Figure4(Seelegendonnextpage.)