Identification of Differentially-Expressed Genes Associated with Pistil Abortion in Japanese Apricot

13 Pages·2012·0.64 MB·English

Identification of Differentially-Expressed Genes
Associated with Pistil Abortion in Japanese Apricot by
Genome-Wide Transcriptional Analysis
Ting Shi., Zhihong Gao*., Liangju Wang, Zhen Zhang, Weibing Zhuang, Hailong Sun, Wenjun Zhong
CollegeofHorticulture,NanjingAgriculturalUniversity,Nanjing,People’sRepublicChina
Abstract
ThephenomenonofpistilabortionwidelyoccursinJapaneseapricot,andimperfectflowerswithpistilabortionseriously
decreasetheyieldinproduction.Althoughtranscriptomeanalyseshavebeenextensivelystudiedinthepast,asystematic
studyofdifferentialgeneexpressionhasnotbeenperformedinJapaneseapricot.Toinvestigategenesrelatedtothepistil
development of Japanese apricot, high-throughput sequencing technology (Illumina) was employed to survey gene
expressionprofilesfromperfectandimperfectJapaneseapricotflowerbuds.3,476,249and3,580,677tagsweresequenced
from two libraries constructed from perfect and imperfect flower buds of Japanese apricot, respectively. There were 689
significantdifferentially-expressedgenesbetweenthetwolibraries.GOannotationrevealedthathighlyrankedgeneswere
thoseimplicatedinsmallmoleculemetabolism,cellularcomponentorganisationorbiogenesisatthecellularlevelandfatty
acidmetabolism.Accordingtotheresults,weassumedthatlateembryogenesisabundantprotein(LEA),Dicer-like3(DCL3)
Xyloglucanendotransglucosylase/hydrolase2(XTH2),Pectinlyase-likesuperfamilyprotein(PPME1),Lipidtransferprotein3
(LTP3),Fattyacidbiosynthesis1(FAB1)andFattyaciddesaturase5(FAD5)mighthaverelationshipswiththepistilabortion
inJapaneseapricot.Theexpressionpatternsof36differentiallyexpressedgeneswereconfirmedbyreal-time(RT)-PCR.This
is the first report of the Illumina RNA-seq technique being used for the analysis of differentially-expressed gene profiles
related to pistil abortion that both computationally and experimentally provides valuable information for the further
functional characterisation ofgenes associatedwith pistil developmentinwoody plants.
Citation:ShiT,GaoZ,WangL,ZhangZ,ZhuangW,etal.(2012)IdentificationofDifferentially-ExpressedGenesAssociatedwithPistilAbortioninJapanese
ApricotbyGenome-WideTranscriptionalAnalysis.PLoSONE7(10):e47810.doi:10.1371/journal.pone.0047810
Editor:ChristianScho¨nbach,KyushuInstituteofTechnology,Japan
ReceivedJune19,2012;AcceptedSeptember17,2012;PublishedOctober16,2012
Copyright:(cid:2)2012Shietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:SpecializedFundfortheBasicResearchOperatingexpensesProgramofCentralCollege2012,theNationalScienceFoundationofChina(31101526)
andthePriorityAcademicProgramDevelopmentofJiangsuHigherEducationInstitutions(PAPD)forfundingpartofthisstudy.Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
.Theseauthorscontributedequallytothiswork.
Introduction between perfect and imperfect flowers [4]. In addition, morpho-
logicalresearchindicatedthatduringthefirstandsecondtendays
Japanese apricot (Prunus mume Sieb. et Zucc) belongs to the
of December, flower buds of the ‘Daqiandi’ cultivar did not
Rosaceaefamilyoffruitsandisanimportanteconomicfruitcropin
continue to elongate, instead the pistil differentiation stagnated
ChinaandJapan[1].Owingtoitshighnutritionalvalue,thefruit
and gradually disintegrated, which was the key stage of pistil
has not only been used in the preparation of preserved fruit and
abortion of the ‘Daqiandi’ cultivar. The factors leading to the
wine,butcanalsobeusedasadietingredient[1,2].However,the
selectiveabortionofpistilsmayrelatetocatabolismofmacromol-
phenomenonofimperfectflowerswidelyoccursandhasseriously
ecule nutrients in the flower bud [6]. However, the molecular
affected the production yield [3]. The imperfect flowers are
mechanism involved in pistil abortion remains unknown for
characterisedbyeitherpistilsbelowthestamens,witheredpistilsor
Japanese apricot.
an absence of pistils, and such flowers fail to bear fruit [4].
Inmostfloralplants,floralorgansplayasignificantroleinplant
Comparative proteomic analysis has been performed for perfect
sexualreproduction,butonlyafewoftheflowersandovulesthat
and imperfect flowers and the different proteins have been
are initiated actually give rise to mature seeds and fruits [7].
analysed in both perfect and imperfect flowers for the different
Several different mechanisms have been proposed to explain the
stages of young bud, mature bud and blossom flower; moreover,
phenomenon of female sterility, including pistil abortion. Female
glucosemetabolism,starchmetabolismandphotosynthesisrelated
sterility is thought to be triggered by environmental and
to pistil abortion were found [5]. More recently, real-time
nutritional conditions [8,9], low sink strength [10,11], the
quantitative reverse transcription polymerase chain reactions and
influence of pathogens [12], the occurrence of sporophytic or
in situ hybridisation have shown that PmAG mRNA was highly
gametophytic mutations [13], ABCDE model and other related
expressedinthesepals,carpelandstamens,andaweaksignalwas
genes[14–16]andphytohermone[17–20].Morphologicalstudies
detectedintheseedandnutlet.Noexpressionwasdetectedinthe
have shown that pistil development of staminate flowers in the
leavesorpetals,butnosignificantdifferentialexpressionwasfound
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TranscriptionalAnalysisofJapaneseApricot
olive is interrupted after differentiation of the megaspore mother Results
cell. At that stage, no starch was observed in the pistils of the
staminate flowers;theplastids hadfewthylakoid membranesand Statistics of tag sequencing
grana and the staminate flowers appeared very similar to To identify differentially-expressed genes involved in the pistil
proplastids [10]. In Arabidopsis, heat-stress reduced the total development of flowers in Japanese apricot, we used Illumina
number of ovules and increased ovule abortion [21]. Early ovule sequencing on DGE from the perfect (PF) and imperfect (IF)
degeneration was also caused by high temperatures in sweet flower buds. A total of 3,476,249 and 3,580,677 tags were
cherry,andovuledevelopmentwasregulatedbygibberellin(GA) obtained from the PF and IF flower bud libraries, respectively
insweetcherryflowers[9].GAsuppressedthedevelopmentofthe (Table1).Toincreasetherobustnessoftheapproach,single-copy
embryo sacandshortened itslongevityin grapes [20]. tagsinthetwolibraries(141,270inthePFand142,287intheIF
Next-generation high-throughput sequencing technologies pro- library) were excluded from further analysis (Figure 1). After
vide a powerful strategy to identify genome-wide expression. discarding the low quality tags (tags containing ‘N’, adaptor
Genome-wide expression analyses are essential tools for elucidat- sequences and copy number ,2), 3,331,468 and 3,434,800 tags
(clean tags) remained in the PF and IF libraries, from which
ing molecular function. Recent studies have highlighted the
129,933 (PF) and 126,485 (IF) distinct tags were obtained
significance of high-throughput expression data, particularly with
(Figure 1). There were 3,448 more distinct tags in the PF than
the integration of large, diverse data sets, in constructing
in the IF library, possibly representing genes related to pistil
biochemical and regulatory networks in silico [22–25]. Digital
development. The percentage of distinct tags rapidly declined as
gene expression tag profiling (DGE) is a revolutionary approach
copynumberincreased,indicatingthatonlyasmallportionofthe
for expression analysis [26,27]. Driven by Solexa/Illumina
transcripts were expressed at a high level under the conditions
technology, DGE creates genome-wide expression profiles by
tested.
sequencing. This method combines the sequencing serial analysis
The saturation of the library was determined by the identifi-
of gene expression (SAGE) principle with the sequencing
cationofuniquetags.Sequencingwasconsideredtohavereached
technology for generating a digital output proportional to the
saturation when no new unique tags were detected. The results
number of transcripts per mRNA [28]. The benefit of not
shown in Figure 2 indicate that the PF and IF libraries were
requiring pre-synthesised oligonucleotide probes (as in micro-
sequenced to saturation, producing a full representation of the
arrays)allowsthedirectenumerationoftranscripts,whichishighly
transcriptsintheconditionstested.Inbothlibraries,fewerunique
replicable, accurate and comparable across experiments [29,30].
tags were identified as the number of sequencing tags increased,
In addition, the quantification of weakly expressed genes can be
reachingaplateaushortlyafter1Mtagsweresequenced.Nonew
performed,whichcannotbeassessedusingmicroarrays[31].DGE
unique tags were identified as the total tag number approached
also offers researchers a global orthogonal hybridisation array
3 MinthePFlibrary and2.5M intheIFlibrary.
validationmethod,withalmostunlimiteddynamicrange,provid-
The distribution of the clean tag copy number is shown in
ingatuneabledepthofcoverageforraretranscriptdiscoveryand
figure 3. The copy number of clean tags between 2 and 5 was
quantification.Forexample,toexplorethemolecularmechanism
208,111,between6and10was153,194,between11and20was
of plant developmental-stage transition and cell-fate determina-
213,051, between 21 and 50 was 396,739, between 51 and 100
tion, DGE analysis was undertaken of sequential developmental
was 382,603, and .100 was 1,977,770 in the PF library. In
time-points and individual tissue types in the model moss
contrast, the copy number of distinct clean tags was mostly
Physcomitrella patens [24], which was chosen because of the short
distributedbetween2and5,with55.45%ofthetotalcleantagsin
life-cycleandrelativestructuralsimplicityofthisplant.Tovalidate
the PF library (Figure 3). In the IF library, the copy number of
gene expression in the models for trichome development in
clean tags between 2 and 5 was 199,910, between 6 and 10 was
soybean, DGE analysis and RNA-Seq were used to compare the
147,600,between11and20was205,594,between21and50was
transcriptional profiles in wild-type and glabrous soybean lines
395,033, between 51 and 100 was 392,318, and .100 was
[32]. Early developing cotton fibre was analysed by deep
2,094,345. Similar to the PF library, the copy number of distinct
sequencing, and differential expressions of genes in a fuzzless/
cleantagswasmostlydistributedbetween2and5,with54.88%of
lintlessmutantwererevealed[33].DGEsignatureswerealsoused
thetotal clean tagsin theIFlibrary(Figure 3).
to study the maize RA3 gene [22] and the development of brace
root [34]. In addition, Solexa/Illumina technology was used to
Annotation analysis of the unique tags
analyse gene expression during female flower development in
Thedistincttagswerecomparedagainstthegenomeandgene
cucumber [35]. Overall, the DGE approach has provided more
sequences of peach using BLASTn. Tags with a complete match
valuable tools for qualitative and quantitative gene expression
oronebase-pairmismatchwereconsideredfurther.Theresultsin
analysis than theprevious microarray-based assays [26].
Table1showthataproportionoftags(26.70%inthePFlibrary
Here, we used the DGE method to perform a deep
and27.52%intheIFlibrary)matchedtothepeachgenome,but
transcriptome analysis of pistil abortion in Japanese apricot.
64,599 (49.72% of unique tags) and 58,309 (46.10% of unique
Althoughtranscriptomeanalyseshavebeenextensivelystudiedin
tags) in the PF and IF libraries matched to 17,056 (59.45%) and
the past years, for example, 454-pyrosequencing of the transcrip-
16,386 (57.12%) peach genes. Further analysis revealed that
tome was used in the research of dormant stages in Japanese
57,871 unique tags (44.54%) in the PF library and 52,144
apricot[36],asystematicstudyofdifferentialgeneexpressionhas
(41.23%) inthe IFlibrary matched toonlyonegene sequence in
not been carried out on Japanese apricot. To investigate the
the peach genome (Table 1 and Figure 4), including perfect
differentially-expressed genes related to the pistil development of
matchingtogenesandwitha1bpmismatch.Thesedataindicate
Japaneseapricot,high-throughputsequencingtechnology(Solexa)
that approximately 50% of the transcripts predicted in Japanese
was employedtosurveythegeneexpression profilesfromperfect
apricot are expressed in the perfect or imperfect flowers, with
and imperfectJapanese apricot flowerbuds. moretranscriptspresentintheperfectsample.Figure4showsthat
sense regulation, with clean sequencing tags perfectly mapped to
thesensegenebeing22.84%(antisensewas11.14%)and21.39%
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TranscriptionalAnalysisofJapaneseApricot
Figure1.Distributionoftagexpression.LeftisPFandrightisIF.Totalcleantagsrepresentthesumofallcleantagnumbers;distinctcleantags
representalltypesofcleantags.
doi:10.1371/journal.pone.0047810.g001
(antisense was 9.86%) in PF and IF, respectively, was the main Pathway enrichment analysis for DEGs
regulated model. AQ-valueof#0.05definedthosegenesthatweresignificantly
differentially expressed (enriched). 227 differentially expressed
Gene ontology functional enrichment analysis of genes associated with 12 metabolic and signal transduction
differentially-expressed genes (DEGs) pathways were found (Figure 6). The pathways with the most
Using the P-value #0.05 as the threshold value, 333 differen- unique genes were metabolic pathways (250 genes), cellular
tiallyexpressedgenesthatcouldbecategorisedinto14functional process (66 genes) and genetic information processing (20 genes).
groups were found (Figure 5), which included four cellular Webelievethatthesepathwaysaresignificantinthepistilabortion
components,fourmolecularfunctionsandsixbiologicalprocesses. ofJapaneseapricot,inparticularmetabolicpathwaysandcellular
The four component categories were as follows: anchored to processes. In our study, metabolic pathways (ko01100) are large
membrane(9),ribosome(16),ATPasecomplex(10)andorganelle complexes comprising several metabolic patterns, including: the
part (96). Based on molecular function, the genes were finally biosynthesis of secondary metabolites (ko01110, 66 genes);
classified into four categories: ammonia ligase activity (3), acid- flavonoid biosynthesis (ko00941, 15); oxidative phosphorylation
ammonia (or amide) ligase activity (3), Rho GTPase activator (ko00190, 10 genes); arginine and proline metabolism (ko00330,
activity(2)andtransferaseactivity(37).Additionally,sixbiological eight genes); galactose metabolism (ko00052, seven genes); the
processes were identified: nitrogen cycle metabolism (3), proton biosynthesis of unsaturated fatty acids (ko01040, seven genes);
transport (6), fatty acid metabolism (20), cellular component pyruvatemetabolism(ko00620,sevengenes);nitrogenmetabolism
organisation or biogenesis at the cellular level (44), glutamine (ko00910, six genes) and alanine, aspartate and glutamate
metabolism (3) and small molecule metabolism (81). The genes metabolism (ko00330, six genes). Cellular processes included
involved in small molecule metabolism [GO: 0044281] were the phagosome (ko04145, 14 genes) and endocytosis (ko04144, nine
most significantly enriched in comparison to the other five genes). Genetic information processing only included protein
biologicalprocesses.Forty-fourdifferentiallyexpressedgeneswere processing intheendoplasmic reticulum (ko04141,20genes).
involved in the cellular component organisation or biogenesis at
the cellular level [GO: 0071841], which is carried out at the Comparison of gene expression between the two
cellular level and results in the biosynthesis of constituent libraries
macromolecules, assembly, arrangement of constituent parts, or Differences in thetag frequencies that appeared in thePFand
disassembly of a cellular component. Among the significantly IF libraries were used to estimate gene expression levels in
enrichedtranscriptswere20DEGsassociatedwiththeregulation responsetopistilabortion.Thetranscriptsdetectedwithatleasta
of fatty acid metabolism [GO: 0006631], which includes the two-folddifferencebetweenthetwolibrariesareshowninFigure7
chemical reactions and pathways involving fatty acids and (FDR,0.001).Thereddots(468)andgreendots(221)represent
aliphaticmonocarboxylicacidsliberatedfromnaturallyoccurring transcripts with a higher or lower abundance of more than two-
fats andoils byhydrolysis. fold than in the PF library, respectively. The blue dots represent
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TranscriptionalAnalysisofJapaneseApricot
Table1. StatisticsofDGE sequencing.
Summary PF IF
RawData Total 3,476,249 3,580,677
RawData DistinctTag 274,206 271,884
CleanTag Totalnumber 3,331,468 3,434,800
CleanTag DistinctTagnumber 129,933 126,485
AllTagMappingtoGene Totalnumber 1783091 1703889
AllTagMappingtoGene Total%ofcleantag 53.52% 49.61%
AllTagMappingtoGene DistinctTagnumber 64599 58309
AllTagMappingtoGene DistinctTag%ofcleantag 49.72% 46.10%
UnambiguousTagMappingtoGene Totalnumber 1564689 1496735
UnambiguousTagMappingtoGene Total%ofcleantag 46.97% 43.58%
UnambiguousTagMappingtoGene DistinctTagnumber 57871 52144
UnambiguousTagMappingtoGene DistinctTag%ofcleantag 44.54% 41.23%
AllTag-mappedGenes number 17056 16386
AllTag-mappedGenes %ofrefgenes 59.45% 57.12%
UnambiguousTag-mappedGenes number 14106 13486
UnambiguousTag-mappedGenes %ofrefgenes 49.17% 47.01%
MappingtoGenome Totalnumber 776068 849241
MappingtoGenome Total%ofcleantag 23.30% 24.72%
MappingtoGenome DistinctTagnumber 34698 34806
MappingtoGenome DistinctTag%ofcleantag 26.70% 27.52%
UnknownTag Totalnumber 772309 881670
UnknownTag Total%ofcleantag 23.18% 25.67%
UnknownTag DistinctTagnumber 30636 33370
UnknownTag DistinctTag%ofcleantag 23.58% 26.38%
doi:10.1371/journal.pone.0047810.t001
transcripts that differed less than two-fold between the two five-fold in the IF library, while the expression level of 99.3%
libraries, which were arbitrarily designated as ‘‘no difference in unique tags was within the five-fold difference between the two
expression’’. The differentially-expressed genes with five-fold or samples.
greater differences in accumulation are shown in Figure 8 and Of DEGs with differences greater than five-fold (Table 2), 11
Table2.About0.23%totaluniquetagsincreasedbyatleastfive- genes were present at higher levels in the IF library, which were
fold,andabout0.47%totaluniquetagsweredecreasedbyatleast associatedwithdefence(2),metabolism(6),signaltransduction(2)
Figure2.Saturationevaluationofdifferentialexpression.TheleftfigureisPFandtherightfigureisIF.
doi:10.1371/journal.pone.0047810.g002
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TranscriptionalAnalysisofJapaneseApricot
Figure3.Distributionofcleantagcopynumber.LeftisPFandrightisIF.Totalcleantagsrepresentthesumofallcleantagnumbers;distinct
cleantagsrepresentalltypesofcleantags.
doi:10.1371/journal.pone.0047810.g003
Figure4.Alignmentresultsofthecleantags.Thefigureexhibitsthealignmentstatisticsofthetagcopynumberandthetagtype.Note:1PM
(senseorantisense):perfectmatchtogene(senseorantisense);1tag-.1gene:matchtoonegene;1tag-.ngene:matchtomorethanonegene;
1MM (sense or antisense): match to gene (sense or antisense) with 1bp mismatch; PM Genome 1 tag-.1 position: perfect match to genome
sequencewithonebesthit;PMGenome1tag-.nposition:perfectmatchtogenomesequencewithmultiplebesthits;1MMGenome:matchto
genomesequencewith1bpmismatch;UnknownTag:nomatchtogene(senseandanti-sense)andgenomesequence.
doi:10.1371/journal.pone.0047810.g004
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TranscriptionalAnalysisofJapaneseApricot
Figure5.HistogramshowingtheGeneOntologyfunctionalenrichmentofDEGs.
doi:10.1371/journal.pone.0047810.g005
and transport (1). The greatest differences between IF and PF Theactualmeltingcurvesof36genesareshowedintheTableS2.
DEGsweretheDCL3(dicer-like3)geneandthevacuolarATPase The expression level of each gene in the perfect and imperfect
subunit F family protein gene, both of which were present 8.95- flower buds was compared with its abundance from the
fold higherin theIFlibrarythan inthePFlibrary. sequencing data of DGE sequencing (Figure 9). The apparent
Forty-nine DEGs were less abundant in the IF library. Those discrepancies with respect to ratio can be attributed to the
presentfive-foldormoreinthePFlibraryarealsolistedinTable2, essentiallydifferentalgorithmsdeterminedbythetwotechniques.
inwhich43geneswere classified asdefence (5),development (2), In the analysis of gene profiles, the DGE method generates
metabolism (27), signal transduction (3), transcription (2) and absolute ratherthan relative expression measurements. However,
transport (4). The highest DEG was the FAD5 (fatty acid theresultsshowedthatexpressionofthese36geneswasconsistent
desaturase 5) gene, which was present at levels 14.82-fold higher between the qRT-PCR and the DGE analyses. Transcripts from
than PFlevels. highly abundant Illumina tags appeared at the expected high
expression level in the quantitative PCR analyses. Additionally,
Real-time RT-PCR analysis high-foldchangeswereobservedforgenesthatshowedlowcopy-
ToconfirmthereliabilityofIlluminasequencingtechnology,36 numbersinthePFlibrarybuthighabundancesintheIFlibrary.
genes were randomly selected for quantitative RT-PCR assays. For example, CPK13 (calcium-dependent protein kinase 13;
Figure6.Histogramillustratingpathwayenrichmentanalyses.
doi:10.1371/journal.pone.0047810.g006
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TranscriptionalAnalysisofJapaneseApricot
Figure8.Differentially-expressedtagsintheIFtissuelibrary.
The x-axis represents fold-change of differentially expressed unique
tags in the imperfect library. The y-axis represents the number of
Figure 7. Comparison of gene expression levels between the uniquetags(log10).Differentiallyaccumulatinguniquetagswithafive-
twolibraries.Forcomparinggeneexpressionlevelsbetweenthetwo folddifferencebetweenlibrariesareshownintheredregion(99.30%).
libraries, each library was normalised to 1 million tags. Red dots Theblue(0.23%)andgreen(0.47%)regionsrepresentuniquetagsthat
representtranscriptsmoreprevalentintheinfectedleaflibrary,green are up- and down-regulated more than five-fold in the IF library,
dotsshowthosepresentatalowerfrequencyintheinfectedtissueand respectively.
blue dots indicate transcripts that did not change significantly. The doi:10.1371/journal.pone.0047810.g008
parametersFDR,0.001andlog2ratio$1wereusedasthethreshold
tojudgethesignificanceofgeneexpressiondifference. thosedetectedbytheSolexa-basedsequencingmethod,thetrends
doi:10.1371/journal.pone.0047810.g007
of up- and down-regulation were similar. The lower expression
level detected by RT-PCR could be due to the difference in
ppa004141m) showedno expression in the PFlibrary, whereas it sensitivity between the two technologies [37]. Solexa sequencing
was detected at levels of 8.86-fold in the IF library. It was has been documented to be more sensitive for the estimation of
significantly up-regulated by more than 15-fold in the RT-PCR gene expression, especially for low-abundance transcripts, com-
analysis. Similarly, DCL3 (ppa021659m) was induced by more pared tomicroarrays and real-timeRT-PCR [38].
than 5-fold. In addition, FAD5 (ppa027208m) showed no 60 DEGs were found to have differences greater than five-fold
expression in the IF library, while it was detected 14.26-fold in (Table 2), 54 of which were associated with metabolism (33),
the IF library, and was significantly down-regulated in the RT- defence (7), signal transduction (5), transport (5), development (2)
PCR analysis. These results confirmed the reliability of the and transcription (2). These transcripts possibly encode genes
transcriptome analysis. responding for pistil abortion or programmed cell death (PCD),
whichwerebroadlygroupedintothefollowingcategoriesbasedon
Discussion theirknown rolesinother plant systems:
Illuminasequencingtechnology,ahigh-throughputsequencing
Defence response genes
approach, was utilised to estimate gene expression in libraries
There were two up-regulated genes and five down-regulated
prepared from perfect and imperfect flower bud tissues. The
genesinthedefenceresponsegroup.Amongthedefenceresponse
results (Figure 2) provide estimates of gene expression as
genes, CPK13 [39], CPR30 F-box and associated interaction
determined by the frequency that any given tag (representing a
domains-containing protein [40], ENODL5 (early nodulin-like
transcript) is sequenced. The data indicate that there is sufficient
protein 5) [41], FRO2 (ferric reduction oxidase 2) [42,43], and
coverage depth to reach saturation, i.e. there is a complete
MAPKKK15 (mitogen-activated protein kinase kinase kinase 15)
assessment of all transcripts present in the libraries. This study
[44] have been widely studied in plant pathogen resistance. The
demonstrated differential transcript abundance and regulation in
lateembryogenesisabundantprotein(LEA)familyproteinshowed
the development of pistils in Japanese apricot between perfect
lessexpressionintheimperfectflowerbuds,whichwasconsistent
flower buds and imperfect flower buds. The phenomenon of
with previous research on the comparative proteomic analysis of
imperfect flowers widely occurs and seriously affects the produc-
pistil abortion in Japanese apricot and belonged to the group of
tionyield;thereforeitisnecessarytoidentifythegenesrelatedto
down-regulated genes [45]. This was similar to many other cold
the molecular mechanism of pistil abortion in the Japanese
resistant proteins [46], a number of which enhance freezing
apricot.Theseresultsrepresentthefirstlarge-scaleinvestigationof tolerancebyputativelystabilisingmembranes,orincreasinglevels
geneexpressioninthepistilabortionanalysisofJapaneseapricot. ofprotectiveosmolytes[8].Astudyonchickpeasshowedreduced
17,056 putative genes were identified among the Illumina ovule size, reduced ovule viability, missing embryo sacs, and
sequencing tags for the PF library and 16,386 were identified for impairedpistil functionintemperature-sensitive cultivars forcold
theIFlibrary.Thesteady-statetranscriptlevelforasetofselected stress [47]. In addition, LEA proteins contain hydrophilic side-
genes was confirmed by real-time (RT)-PCR. Although the chains thought to interact with and protect hydrophilic proteins
differences in gene expression did not match the magnitude of duringdesiccationbycausingproteinaggregation[48,49].Water
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TranscriptionalAnalysisofJapaneseApricot
Table2. Listofdifferentially expressed genes changed byfive-foldormore in theIFlibrary.
Gene log2Ratio(IF/PF) Description
Up-regulatedgenes
Defence
ppa004141m 8.86 Calcium-dependentproteinkinase13(CPK13)
ppa018301m 8.86 CPR30F-boxandassociatedinteractiondomains-containingprotein
Metabolism
ppa000615m 8.86 EMB3011RNAhelicasefamilyprotein
ppa010131m 8.67 Peptidyl-tRNAhydrolasefamilyprotein
ppa006743m 8.67 AGD2Pyridoxalphosphate(PLP)-dependenttransferasessuperfamilyprotein
ppa006182m 8.86 Proteinkinasesuperfamilyprotein
ppa021659m 8.95 Dicer-like3(DCL3)
ppa005799m 8.77 Pre-mRNA-splicingfactorCWC26
Signaltransduction
ppa012743m 8.77 SLY2F-boxfamilyprotein
ppa003105m 8.67 Leucine-richrepeatproteinkinasefamilyprotein
Transport
ppa013294m 8.95 VacuolarATPasesubunitFfamilyprotein
Down-regulatedgenes
Defence
ppa002249m 28.61 Early-responsivetodehydrationstressprotein(ERD4)
ppa016718m 28.99 ENODL5earlynodulin-likeprotein5
ppa002203m 29.37 FRO2ferricreductionoxidase2
ppa006485m 29.61 Mitogen-activatedproteinkinasekinasekinase15(MAPKKK15)
ppa021261m 210.04 Lateembryogenesisabundantprotein(LEA)familyprotein
Development
ppa001970m 28.71 Glutamine-richprotein23(GRP23)
ppa016920m 28.99 BTB/POZdomain-containingprotein
Metabolism
ppa023612m 25.96 Lipidtransferprotein3(LTP3)
ppa011478m 27.11 Plantinvertase/pectinmethylesteraseinhibitorsuperfamilyprotein
ppa023515m 27.18 Cysteineproteinasessuperfamilyprotein
ppa009726m 28.61 ABI-1-like1(ABIL1)
ppa025960m 28.61 BEN1NAD(P)-bindingRossmann-foldsuperfamilyprotein
ppa015204m 28.61 SYDP-loopcontainingnucleosidetriphosphatehydrolasessuperfamilyprotein
ppa002676m 28.61 Pentatricopeptiderepeat(PPR)superfamilyprotein
ppa005209m 28.81 XF1FAD/NAD(P)-bindingoxidoreductasefamilyprotein
ppa026851m 28.81 Subtilisin-likeserineendopeptidasefamilyprotein
ppa007640m 28.91 IRX9Nucleotide-diphospho-sugartransferasessuperfamilyprotein
ppa003553m 28.91 P-loopcontainingnucleosidetriphosphatehydrolasessuperfamilyprotein
ppa022113m 28.91 3-ketoacyl-CoAsynthase7(KCS7)
ppb019226m 29.08 Plantinvertase/pectinmethylesteraseinhibitorsuperfamilyprotein
ppa007503m 29.15 PLC-likephosphodiesterasessuperfamilyprotein
ppa017270m 29.3 Alpha/beta-Hydrolasessuperfamilyprotein
ppa005976m 29.55 Pectinlyase-likesuperfamilyprotein(PPME1)
ppa018639m 29.61 CytochromeP450,family735,subfamilyA,polypeptide1(CYP735A1)
ppa013439m 29.76 RING/U-boxsuperfamilyprotein
ppa004479m 29.95 Fattyacidbiosynthesis1(FAB1)
ppa020149m 210.16 Alphadioxygenase
ppa005749m 210.55 Purpleacidphosphatase22(PAP22)
ppa004364m 210.55 Non-specificphospholipaseC3(NPC3)
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TranscriptionalAnalysisofJapaneseApricot
Table2. Cont.
Gene log2Ratio(IF/PF) Description
ppa019741m 210.97 Xyloglucanendotransglucosylase/hydrolase2(XTH2)
ppa025833m 211.32 Alpha/beta-Hydrolasessuperfamilyprotein
ppa020405m 211.33 GDSL-likeLipase/Acylhydrolasesuperfamilyprotein
ppa027208m 214.26 Fattyaciddesaturase5(FAD5)
ppa016543m 214.82 Fattyaciddesaturase5(FAD5)
Signaltransduction
ppa015093m 28.91 CyclinD6
ppa016219m 28.91 Stigma-specificStig1familyprotein
ppa012463m 29.61 PollenOlee1allergenandextensinfamilyprotein
Transcription
ppa011751m 28.61 Mybdomainprotein24(MYB24)
ppa008450m 29.23 Mybdomainprotein73(MYB73)
Transport
ppa004487m 28.61 MATEeffluxfamilyprotein
ppa010364m 28.61 TIP1;3tonoplastintrinsicprotein1;3
ppa000945m 28.81 HA9H(+)2ATPase9
ppa006913m 29.61 AAC2ADP/ATPcarrier2
Variousfunctions
ppa014641m 28.71 Plantproteinofunknownfunction(DUF868)
ppa020792m 28.71 ARMrepeatsuperfamilyprotein
ppa022037m 28.81 Uncharacterizedproteinfamily(UPF0016)
ppa014616m 28.81 Proteinofunknownfunction(DUF1278)
ppa021534m 210.16 Proteinofunknownfunction(DUF679)
ppa008861m 210.33 Familyofunknownfunction(DUF716)
doi:10.1371/journal.pone.0047810.t002
Figure 9. Digital gene expression tag profiling and quantitative real-time PCR analysis of the expression of randomly selected
genes.Allreal-timePCRreactionswererepeatedthreetimesandthedataarepresentedasthemean6SD.Thex-axisindicatesthedifferentgenes.
They-axisshowstheexpressionlevels:theleftshowstherelativeexpressionlevelbyqRT-PCRandtherightshowstagnumberpermilliontagsby
DGE.
doi:10.1371/journal.pone.0047810.g009
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TranscriptionalAnalysisofJapaneseApricot
stressimposedduringthedevelopmentofflowersisamajorfactor istypicallydepositedintotheapoplasminahighlymethylesterified
increasing the rate of abortion, and long-term or frequent water condition. Pectin methylesterase in the apoplasm catalyses the
deficits during this period might decrease yield [50,51]. In our demethylesterificationoftheseHGAs,exposingcarboxylresidues,
study, LEA protein expression in perfect flower buds was higher which can be cross-linked by calcium [70]. These changes
than that in imperfect flower buds. Therefore, we speculate the significantly affect the rheological properties of the cell wall as
reduction of this temperature-resistant protein might increase the wellasitsporosityandionicstatus.Itfollowsthatthetightcontrol
ratio of pistil abortion. There were both up-regulated and down- ofPMEactivity,bothspatiallyandtemporally,occupiesapivotal
regulated genes in this category. Temperature and water stress position in the control of cell-wall growth and development [71].
response-related proteins were associated with hot or cold An important post-transcriptional mechanism of regulation of
temperature stresses and may also play important roles in pistil theseenzymesisrepresentedbyproteinaceousinhibitors[72].Our
abortion inJapanese apricot. previousresearchfoundthatthepistilofimperfectflowersstopped
differentiation in early December and finally disintegrated, while
Genes involved in RNA metabolism the pistil of perfect flowers continued to differentiate and
In our study, there were six up-regulated genes and down- developed perfectly (Shi et al., 2011). This phenomenon might
regulatedgenesinvolvedindifferentkindsofmetabolism,suchas result fromthestrain formation ofcellwalls.
RNA, protein,pectinandfatty acidmetabolism. FAB1[73]andFAD5[74]areinvolvedinunsaturatedfattyacid
The EMB3011 RNA helicase family protein [52], peptidyl- biosynthesis.Unsaturatedfattyacidplaysaveryimportantrolein
tRNA hydrolase family protein [53], DCL3 [54,55] and pre- lipidbiosynthesisandcellwallorganisation.Thesetwogeneswere
mRNA-splicing factor CWC26 [56,57] are involved in RNA down-regulatedinpistilabortion,andtheirexpressionwashigher
processing,includingsmallRNAmetabolism.DCL3produces24- in the perfect flower buds than in the imperfect flower buds.
nt small interfering RNAs (siRNAs) at heterochromatic loci [58]. Therefore,wehypothesisethattheformingofanimperfectflower
PlantscanutilisesiRNAstoguidedenovoDNAmethyltransferases has a crucialrelationship withthecell-wall organisation.
fortheestablishmentofsequence-specificDNAmethylation[59],
which has several functions in angiosperms, such as transposon Materials and Methods
silencing and transcriptional gene silencing (TGS) [60]. In
Arabidopsis thaliana, double mutants between dicer-like 1 and dicer- Plant materials
like 3 exhibit a delay in flowering that is caused by increased The ratio of imperfect flowers of Japanese apricot cultivars
expression of the floral repressor FLOWERING LOCUS C [61]. Daqiandi is about 76%. While the pistil of a perfect flower
This delayed-flowering phenotype is similar to that of autono- continues to differentiate and develop perfectly, the pistil of an
mous-pathwaymutants,andthefloweringdelaycanbeovercome imperfect flower stops differentiation in early December and
byvernalisation[61,62].Infact,smallRNAsplayimportantroles finally disintegrates. We chose two types of flower buds of this
in the termination of floral stem cells, especially miR172 and periodfromDaqianditreesgrownintheNationalFieldGenBank
miR165/166[63]. We have found that miR319, miR319a, forJapaneseApricot,Nanjing,JiangsuProvince,China.Allofthe
miR319e, miR160, miR393, miR394 are significantly differen- sampleswerecollectedandimmediatelyfrozeninliquidnitrogen
tially-expressed between perfect and imperfect flower buds, and andstored at 280uC.
that most of their targets were transcription factors or F-box
proteins[64].Inourstudy,thegenesinvolvedinRNAmetabolism DGE library construction and Illumina sequencing
were up-regulated in pistil abortion, and their expression was To construct DGE libraries, total RNA was extracted from
higherintheimperfectflowerbudsthanthatintheperfectflower perfect andimperfect flowerbuds using themethod accordingto
buds. These data provide clues to the molecular mechanism of Meisel et al. [75]. Library construction was carried out at the
pistil abortion regulated by small RNA metabolism in Japanese Beijing Genomics Institute (BGI, Shenzhen, Guangdong, China),
apricot. usingIllumina’sDGEtagprofilingtechnology.Themainreagents
andsuppliesweretheIlluminaGeneExpressionSamplePrepKit
Cell-wall organisation: pectin and fatty acid metabolism and the Solexa Sequencing Chip (flowcell), and the main
In this study, we have found many genes related to cell-wall instruments were the Illumina Cluster Station and the Illumina
organisation.Peptidyl-tRNAhydrolasehasconnectionstocell-wall HiSeqTM2000System.6mgoftotalRNAwasextracted,oligo(dT)
carbohydrates and morphogenesis and belongs to a highly magnetic bead adsorption was used to purify mRNA, and then
interconnected core network of G-protein signalling [65]. LTP3 oligo(dT)swereusedasprimerstosynthesisethefirstandsecond-
is involved in cutin synthesis during the fibre primary cell wall strandcDNA.The59endsoftagscouldbegeneratedbytwotypes
synthesisstage [66]. of endonuclease: NlaIII or DpnII. The bead-bound cDNA was
XTHs are cell-wall enzymes that catalyse the cleavage and subsequently digested by the restriction enzyme NlaIII, which
molecular grafting of xyloglucan chain functions in the loosening recognisedandcuttheCATGsites.Thefragments(exceptforthe
andrearrangementofthecellwall[67,68].Inourpreviousstudy, 39 cDNA fragments) connected to oligo(dT) beads were washed
wefoundthat XTH2 expression was higherintheperfectflower awayandtheIlluminaadaptor1wasligatedtothesticky59endof
buds than that in the imperfect flower buds [45]. As they are the digested bead-bound cDNA fragments. The junction of
involved in the modification of the load-bearing cell-wall Illumina adaptor 1 and the CATG site was the recognition site
components, they are believed to be very important in the of MmeI, which is a type of endonuclease with separated
regulation of growth and development [69]. Hyodo et al. (2003) recognitionanddigestionsites.Thisenzymecut17bpdownstream
showed that XTH9 tends to be expressed strongly in rapidly of the CATG site, producing tags with adaptor 1 ends. After
dividing andexpanding tissues in Arabidopsis [68]. removing 39 fragments with magnetic bead precipitation, the
Plant invertase/pectin methylesterase inhibitor superfamily Illuminaadaptor2wasligatedtothe39endsoftags,acquiringtags
protein and PPME1 pectin lyase-like superfamily protein, two withdifferentadaptorsatbothendstoformataglibrary.After15
down-regulated genes, are involved in pectin metabolism. The cyclesoflinearPCRamplification,105bpfragmentswerepurified
homogalacturonan(HGA)fraction,amajorcomponentofpectins, by 6% TBE PAGE gel electrophoresis. After denaturation, the
PLOSONE | www.plosone.org 10 October2012 | Volume 7 | Issue 10 | e47810