The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant ...

14 Pages·2012·0.75 MB·English

Transcriptome-Wide Characterization of miRNA-Directed and
Non-miRNA-Directed Endonucleolytic Cleavage Using
Degradome Analysis Under Low Ambient Temperature in
Phalaenopsis aphrodite subsp. formosana
R
Feng-Ming An1,2 and Ming-Tsair Chan1,2,3,* e
g
1InstituteofBiotechnology,CollegeofBioscienceandBiotechnology,NationalChengKungUniversity,Tainan701,Taiwan u
l
2AcademiaSinica,BiotechnologyCenterinSouthernTaiwan,Tainan741,Taiwan a
3AgriculturalBiotechnologyResearchCenter,AcademiaSinica,Taipei,115,Taiwan r
D
*Correspondingauthor:E-mail,[email protected];Fax:[phone]. Pow
(Received April 10, 2012; Accepted August 7, 2012) apnlo
a
ed
red
Plant microRNAs (miRNAs) regulate gene expression long non-coding RNA; miRNA, microRNA; siRNA, small inter- fro
through post-transcriptional gene silencing. Phalaenopsis fering RNA; sRNA, small RNA; NAT, natural antisense tran- m
aphrodite subsp. formosana is an orchid species native to script; RT–qPCR, quantitative reverse transcription–PCR http
Taiwan,whichhashigheconomicvalueandahighfrequency s
offloralpolymorphism.Todate,fewstudieshavefocusedon The degradome library reported in this paper has been sub- ://ac
a
the regulatory roles of miRNAs and functional small RNAs mittedtoNCBI-GEOunderaccessionnumberGSE35739. d
e
m
(sRNAs)inorchidsalthoughunderstandingtheregulationof ic
flower development and flowering time is potentially .o
u
important. Here, we combined analyses of the transcrip- Introduction p.c
o
tome, sRNAs andthe degradome to identifysRNA-directed m
Phalaenopsisorchidsareoneofthelargestgeneraofflowering /p
transcript cleavages in Phalaenopsis. Degradome analysis c
provided large-scale evidence of conserved and novel plants. They are appreciated for their beauty and represent a p/a
mdainRtNsAR-NdiAregcrtoeudpcsleaanvdagtehoefirtatargrgeetttrtarnanscsrcirpiptst,sawnder4e6idabenutni-- cpoomlymmoerrcpihailsmspsecoicecsuorfwhidigehlyedcuorninogmtichevbalrueee.diMngoropfhhoylobgriicdasl, rticle-a
especially in the floral organs, which exhibit various colors, bs
fied. Low temperature-responsive sRNAs were validated shapes and sizes. In addition to morphology, several studies tra
with normalized reads from an sRNA library and quantita- of Phalaenopsis have investigated the regulation of flower de- ct/5
tive stem–loop reverse transcription–PCR (RT–PCR) ana- velopmentandfloweringtime(Anetal.2011,Hsiaoetal.2011, 3/1
lysis. According to gene ontology (GO) categorization, Suetal.2011).Phalaenopsisaphroditesubsp.formosana,aspe- 0/17
target transcripts of the novel miRNAs and sRNAs are 3
ciesofPhalaenopsisnativetoTaiwan,hasbeenwidelyusedfor 7
functionally involved in metabolic processes or responses /1
breeding hybrids of floral organs. A cool night temperature 82
to stress. One particular homologous gene, Allcontig28452, 6
(24(cid:3)C/18(cid:3)C, day/night) is important for synchronizing spike 7
whichencodesdigalactosyldiacylglycerolsynthase2(DGD2), 66
was found to be targeted by natural antisense transcripts initiationincertainPhalaenopsisspeciesincludingP.aphrodite by
subsp. formosana. Spikes can only be initiated by prolonged g
(NATs)uniquetoPhalaenopsis.Insummary,comprehensive u
exposure to low ambient temperatures (Blanchard and e
analyses of the transcriptome, sRNAs and degradome using Runkle 2006, Chen et al. 2008), suggesting that temperature st o
deep sequencing technology provided a useful platform for plays a key role in flowering time and/or developmental pro- n 0
investigating miRNA-directed and non-miRNA-directed cessesduringphasetransition. 8 A
ePnhdaolaneuncolpesoilsy.tic cleavage in a non-model plant, the orchid RNAmsic(rsoRRNNAAs)st(hmatiRhNavAes)imapreorteanndtorgoelensoiunspnlaonnt-dcoevdeinlogpmsmeanltl pril 201
9
and responses to environmental stimuli. Flowering time and
Keywords: Deep sequencing (cid:2) Degradome (cid:2) Low ambient
floral developmental processes are known to be regulated by
temperature (cid:2) MicroRNA (cid:2) Phalaenopsis (cid:2) Small RNA.
miRNAs in model plants (Nag and Jack 2010). miR156 and
Abbreviations: DGD2, digalactosyldiacylglycerol synthase 2; miR172actsequentially tocontrol thetransitionfromtheju-
GO, gene ontology; GSP, gene-specific primer; HMW, high venile phase to the reproductive phase in Arabidopsis
molecular weight; LMW, low molecular weight; lncRNA, (Aukerman and Sakai 2003, Gandikota et al. 2007) and maize
PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118,availableonlineatwww.pcp.oxfordjournals.org
!TheAuthor2012.PublishedbyOxfordUniversityPressonbehalfofJapaneseSocietyofPlantPhysiologists.
Allrightsreserved.Forpermissions,pleaseemail:[email protected]
PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012. 1737
F.-M.AnandM.-T.Chan
(Lauter et al. 2005). The floral integrator SUPPRESSOR OF transcriptomelibrary.Moreover,wenoticedthatseveralunan-
OVEREXPRESSION OF CONSTANS 1 (SOC1) is regulated by notated sRNAs are abundant, and some of these are also low
miR156-targeted SQUAMOSA PROMOTER BINDING temperatureresponsive(supplementarydatainAnetal.2011).
PROTEIN-LIKE9(SPL9)protein(Wangetal.2009).Otherstu- Toinvestigate thesenovelmiRNAsandsRNAsfurther, weset
dies have indicated that miR319 regulates TCP genes and upanotherdeepsequencing oftheRNAdegradometotryto
subsequently affects the expression of genes for miR164, validatethem.
ASYMMETRIC LEAVES1 (AS1), INDOLE-3-ACETIC ACID3/ Degradome analysis is a high-throughput experimental
SHORT HYPOCOTYL2 (IAA3/SHY2) and SMALL AUXIN UP method for identifying the target transcripts of conserved
RNA (SAUR) proteins, which control the size and shape of and novel miRNAs. Based on Parallel Analysis of RNA Ends
the leaf and the flower in Arabidopsis (Koyama et al. 2010, (PARE)analysis,thepartiallydegradedtranscriptswithexposed
Nag and Jack 2010). miR164 also serves to regulate floral 50 phosphates are ligated and amplified by 50-modified
organ formation by suppressing CUP-SHAPED COTYLEDON RLM-RACE (RNA ligase-mediated rapid amplification of D
o
(CUC) genes, members of the NAC family of transcription cDNA ends) following sequencing to generate degradome w
n
factors (Aida et al. 1997, Koyama et al. 2010, Nag and Jack datasets(Germanetal.2009).Thesedatasetsgenerallyshow loa
d
2010). The miR159 family is closely related to the miR319 information about site-specific cleavage and autonomous e
d
family in its mature miRNA sequences of 17–21 nucleotides. cleavageoftheRNAtranscripts.miRNA-directedcleavageisa fro
m
ThetargettranscriptsofmiR159,MYBfamilytranscriptionfac- kindofsite-specificcleavagethatcutsthemiRNA–targetgene h
tors,canalsoberegulatedbymiR319.ThesetwomiRNAssup- duplex normally at nucleotide positions 10–11 relative to the ttp
s
press gibberellin-promoted activation of LEAFY, delaying 50 end of the miRNA sequence (Voinnet 2009). Degradome ://a
flowering(Achardetal.2004,NagandJack2010).Doublemu- analysis has been applied to validate novel miRNA families ca
d
tantsofarf6andarf8,thetargetgenesofmiR167,exhibitser- and their target genes in several plant species such as e
m
ious defects in floral organ formation that result in female Arabidopsis (Addo-Quaye et al. 2008), rice (Li et al. 2010, ic
.o
sterility (Wu et al. 2006, Nag and Jack 2010). Several studies Zhouetal.2010),mosses(Addo-Quayeetal.2009b),soybean up
havealsodemonstratedthatanumberofmiRNAsarecritical (Song et al. 2011), grapevine (Jaillon et al. 2007) and trifoliate .co
m
forresponsestoseveralenvironmentalstimulisuchasdrought orange (Zhang et al. 2011). The same analysis has also been /p
c
(Li et al. 2011), cold (Zhou et al. 2008, Lv et al. 2010) and nu- applied in mammals to describe miRNA-dependent and p
/a
trientdeficiency(Chiouetal.2006,Liangetal.2010).Todate, miRNA-independent endonucleolytic cleavage (Bracken et al. rtic
temperature-responsive miRNAs have mostly been investi- 2011). Some of the miRNAs identified by these analyses were le
-a
gated under freezing or near-freezing conditions, but few stu- found to respond to environmental stimuli in different plant b
s
dies have focused on the miRNAs under low ambient species, such as drought stress in Populus (Li et al. 2011) and tra
c
temperature. One previous study of Arabidopsis found that arbuscular mycorrhizal symbiosis in Medicago truncatula t/5
3
two miRNAs (miR156 and miR169) are up-regulated, and (Devers et al. 2011). These studies confirmed the usefulness /1
0
four (miR163, miR172, miR398 and miR399) are down- ofdegradomesequencingintheinvestigationofnewfunctional /1
7
regulated, under low (16(cid:3)C) ambient temperature (Lee et al. miRNAs and the regulatory roles of their target genes. 37
/1
2010).Thetargetgenefamiliesoftheselowambienttempera- Degradome analysis is also useful for validating target gene 8
2
6
ture-responsivemiRNAswerefoundtobeinvolvedinflowering cleavage mediated by small interfering RNAs (siRNAs) and 7
6
timeregulationandstressresponses(Leeetal.2010). othersmallnon-codingRNAs(sncRNAs). 6 b
Several studies have been conducted with the aim of im- In this study we combined analyses of the transcriptome, y g
u
proving understanding of the orchid transcriptome. Species sRNAsandthedegradometoprovidelarge-scaleexperimental e
s
such as Phalaenopsis (Hsiao et al. 2011, Su et al. 2011) and evidencefortherelationshipbetweenconservedmiRNAsand t o
n
Oncidium (Chang et al. 2011) have been studied using theirtargetgenes.Degradomeanalysisprovideddataabout15 0
8
high-throughputsequencingtechnology.Broadtranscriptome conserved miRNA families with 29 target genes. To discover A
p
informationoforchidspeciesfacilitatesthediscoveryofcandi- more conserved miRNA and novel miRNA precursors in ril 2
date genes, and particularly the study of flower development. Phalaenopsis,additionaldeepsequencingofthetranscriptome 01
9
WepreviouslydemonstratedthatsomeconservedmiRNAsin was conducted and 22 conserved and 471 putative novel
Phalaenopsisareinfluencedbylowambienttemperaturetreat- miRNAprecursorswereidentified.FourteennovelmiRNAcan-
ment(Anetal.2011)andarepotentiallyinvolvedintheregu- didateshadtargettranscripts,ofwhichsevencouldbealigned
lationofflowering.Wealsodemonstratedtheuseofassembled to protein-coding genes. Furthermore, 46 abundant sRNA
contigs from deep sequencing as templates to clarify the groupsthatmediatetheendonucleolyticcleavageof36genes
relationshipbetweenmiRNAsandtheirtargetgeneswhenref- werealsoverifiedbydegradomeanalysis.Targetgenesofabun-
erentialgenomicorexpressedsequencetag(EST)information dantsRNAsarefunctionallyinvolvedinthestressresponseand
islacking(Anetal.2011).Wewereabletoidentifyonlyafew metabolic processes. Expression analysis of raw reads revealed
precursors of miRNAs, so additional deep sequencing was that among the 46 sRNA groups, 25 abundant sRNAs are
included to enhance our data. Twenty-two conserved and up-regulated and 13 are down-regulated by low temperature
471 putative novel miRNA precursors were identified in our treatment. All the abundant sRNAs were confirmed with
1738 PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012.
DegradomeanalysisofsmallRNAsinorchid
experimental target transcripts using stem–loop quantitative Abundant sRNAs contain information about conserved
reverse transcription–PCR (RT–qPCR). Forty-five of the 46 miRNA families. We identified 15 of these conserved families
abundant sRNAs were detected, and nine of them showed a with 29 target genes using degradome analysis (Table 1). The
>2-fold increase or decrease after 3 months at low tempera- conserved target transcripts were validated using degradome
ture. One particular gene annotated as digalactosyldiacylgly- analysis of SQUAMOSA PROMOTER-BINDING-LIKE (SPL) of
cerol synthase 2 (DGD2) was found to be regulated by a miR156, AUXIN RESPONSIVE FACTOR (ARF) of miR160 and
natural antisense transcript (NAT). High-throughput sequen- miR167,DICERhomolog1-likegenesofmiR162,ARGONAUTE
cing combined with the degradome analysis gave large-scale 1 (AGO1) of miR168, NUCLEAR TRANSCRIPTION FACTOR Y
experimental information to determine the cleavage sites of subunit (NY) of miR169, ethylene-responsive transcription
sRNA–targetpairs,thusmakingitausefulstrategyforstudies factor RAP2-7 and floral homeotic protein APETALA-2 (AP-2)
ofnon-modelorganismsforwhichlesssequenceinformationis of miR172, and transcription factor TCP-like of miR319
available. (Table 1). According to gene ontology (GO) analysis, these D
o
target genes are mainly involved in developmental processes w
n
and the stress response (Supplementary Fig. S2A). Thus the loa
Results d
conservedmiRNAfamiliesplayimportantrolesinmanydevel- e
d
Preparation of Phalaenopsis orchid transcriptome opmentalstages inPhalaenopsis.Besidestheconserved target fro
m
and sRNA library genesthatwereidentified,somenon-conservedtargetgenesof h
knownmiRNAswerealsoidentifiedusingdegradomeanalysis; ttp
The transcriptome library was assembled from fivebatches of s
deepsequencingdatasetstoincreasecoverageofthesequence fboyrmexiaRm15p9le,,ckyisntaesien-einpteroratcetininagseprRoDte1i9naanbdy cmhioRr1is7m2,atriebmosuotmasael ://aca
information.ForsRNA,foursRNAlibrariesweregeneratedfrom d
L19 family protein by miR396 and floral homeotic protein e
m
leaves (with and without low temperature treatment), stalks APETALA 3 of pha-miR5179 (Table 1). These results suggest ic
and flower buds. The low temperature-treated leaf and thatsomenon-conservedtargettranscriptcleavagesmediated .oup
untreated leaf data sets were used for digital gene expression byconservedmiRNAsarespeciesspecific. .co
analysis.Toremoveredundantsequencesandsiftouttheabun- m
dantsRNAsequences,thefoursRNAlibrarieswerepooledinto Identification of novel miRNA-mediated /pc
p
onedataset.Lowabundancesequenceswith<100readswere transcript cleavage /a
removed. Finally, 5,029 abundant sRNA sequences were se- TodiscovermoreconservedmiRNAandnovelmiRNAprecur- rticle
lectedfortargetpredictionanddegradomeanalysis. -a
sors in Phalaenopsis, additional deep sequencing of the tran- bs
scriptome was conducted (see the Materials and Methods). tra
Summary of the degradome library of c
Twenty-twopotentialconservedmiRNAprecursorswereiden- t/5
Phalaenopsis orchid 3
tified(SupplementaryTableS3).Bystructuralpredictionfrom /1
0
RNAextractedfromvarioustissuesandatseveraltimepoints the non-protein-coding transcripts of our transcriptome li- /1
7
from low temperature-treated leaves was mixed to cover all brary, 471 putative novel miRNA sequences were identified 37
possible RNA degradation products. Degradome sequencing (datanotshown).Further,degradomeanalysiswasconducted /18
2
technology was used to discover miRNA-directed and non- tofindthecleavedtranscriptstoconfirmthesenovelmiRNAs 67
6
miRNA-directed endonucleolytic cleavages in Phalaenopsis. (Fig.1A;SupplementaryFig.S4).Only14novelmiRNAcan- 6 b
We obtained 14,356,941 raw reads through this procedure. didates had target transcripts, of which only seven could be y g
After quality filtration, adaptor removal and null insert, aligned to protein-coding genes (Table 2). The homologs of ue
s
13,659,831cleanreads(95.94%)with479,333uniquesequences these target transcripts had putative roles in transcriptional t o
n
were obtained. Among these, (cid:4)99% of sequencing products regulation, RNA processing, photosynthesis, glycolysis, ATP 0
8
were 18–21 nt in length (Supplementary Fig. S1). We catabolic processes and cell communication (Table 2). All of A
p
mappedthe479,333uniquesequencesontoourtranscriptome thesevennovelmiRNAcandidatesthathadtargettranscripts ril 2
libraryandfound443,034(92.43%)sequenceswithatleastone could be detected using stem–loop RT–qPCR. These novel 01
reportedalignment,andonly36,299(7.57%)withoutalignment miRNAs were repressed by low ambient temperature treat- 9
(datanotshown).Theseresultssuggestthatourtranscriptome ment for 3 months (Fig. 1; Supplementary Fig. S4).
library obtained by deep sequencing is (cid:4)90% complete, and Expression of the target transcripts showed that one of the
thus suitable for further studies. The unique sequences that miRNAs, annotated to nin one-binding (NOB) protein, had a
could be mapped to the Phalaenopsis transcriptome library complementary expression pattern to the novel miRNA
werefurtheranalyzed. pha-m1154-5p (Fig. 1B, C). However, the expression patterns
of the other target transcripts of the novel miRNAs were
Experimental target gene validation of conserved
unchanged or showed the same trend as the miRNAs under
miRNA families
low temperature treatment (Supplementary Fig. S4). These
Degradomeanalysisgiveslarge-scaleexperimentalevidenceof results suggest that the novel miRNAs were effectors, but
conserved miRNA-mediated cleavage of target transcripts. played only minor roles or fine-tuned the regulation of
PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012. 1739
F.-M.AnandM.-T.Chan
Table 1 Identification of conserved miRNA-directed transcript cleavage using degradome analysis
miR_fam Sequence Target Target description A. thaliana homolog
pha-miR156 TGACAGAAGAGAGTGAGCAC Unigene100268leaf Squamosa promoter-binding-like protein AT3G15270
pha-miR159 TTTGGATTGAAGGGAGCT Allcontig12564 Kinase-interacting KIP1-like protein AT3G22790
TTTGGATTGAAGGGAGCTCTA Allcontig13734 Chorismate mutase AT1G69370
TTTGGATTGAAGGGAGTT Allcontig14627 MYB transcription factor AT3G11440
Allcontig37893 Hydroxyproline-rich glycoprotein familyprotein AT5G21280
pha-miR160 TGCCTGGCTCCCTGTATGCCA Unigene109305leaf Auxinresponse factor16 AT4G30080
pha-miR162 TCGATAAACCTCTGCATCCGG Allcontig57719 Endoribonuclease dicerhomolog 1-like AT1G01040
TCGATAAACCTCTGCATCCGGT
pha-miR164 TGGAGAAGCAGGGCACATGTT Allcontig42093 NAM (no apicalmeristem)-like protein AT5G61430
TGGAGAAGCAGGGCACGTGCA Allcontig6215 NAC domain-containing protein 21/22 AT1G56010
TGGAGAAGCAGGGCACGTGCT D
o
TGGAGAAGCAGGGCACGTGTT w
n
pha-miR166 TCGGACCAGGCTTCATTC Allcontig40248 Homeobox-leucine zipper proteinATHB-14 AT2G34710 lo
a
TCGGACCAGGCTTCATTCC Allcontig57003 Homeobox-leucine zipper proteinATHB-9 AT1G30490 de
d
TCGGACCAGGCTTCATTCCC fro
TCGGACCAGGCTTCATTCCCC m
TCGGACCAGGCTTCATTCCCT h
TCGGACCAGGCTTCATTCCTC ttps
TCGGACCAGGCTTCATTCTCC ://a
c
TCGGACCAGGCTTCATTTCCC a
d
TCGGACCAGGCTTCCTTCCCC em
TCGGACCAGGCTTCGTTCCCC ic
.o
TCGGACCAGGCTTCTTTCCCC u
p
TCGGACCAGGCTTTATTCCCC .c
o
TCGGACCAGGTTTCATTCCCC m
/p
TTCGGACCAGGCTTCATTCCC c
p
pha-miR167 TGAAGCTGCCAGCATGAT Allcontig12108 Auxinresponse factor6 AT1G30330 /a
TGAAGCTGCCAGCATGATC Allcontig12779 Auxinresponse factor8 AT5G37020 rtic
le
TGAAGCTGCCAGCATGATCT -a
TGAAGCTGCCAGCATGATCTA bs
TGAAGCTGCCAGCCTGATCTGA tra
c
pha-miR168 TCGCTTGGAGCAGGTCGGGAC Allcontig26370 Proteinargonaute 1 AT1G48410 t/5
3
TCGCTTGGTGCAGGACGGGAC /1
0
TCGCTTGGTGCAGGCCGGGAC /1
7
TCGCTTGGTGCAGGGCGGGAC 3
7
TCGCTTGGTGCAGGTCGGGA /1
8
TCGCTTGGTGCAGGTCGGGAA 2
6
TCGCTTGGTGCAGGTCGGGAC 76
6
TCGCTTGGTGCAGGTCGGGAT b
TCGCTTGGTGCAGGTCGGGCC y g
TCGCTTGGTGCAGGTCGGTAC ue
s
TCGCTTGGTGCAGGTCTGGAC t o
TCGCTTGGTGCCGGTCGGGAC n 0
TTCGCTTGGTGCAGGTCGGGAC 8 A
TTGCTTGGTGCAGGTCGGGAC p
pha-miR169 CAGCCAAGGATGACTTGCCGA Unigene22902leaf Nuclear transcription factorY subunitA-10 AT5G06510 ril 2
0
CAGCCAAGGATGACTTGCCGG Allcontig17156 Nuclear transcription factorY subunitA-3 AT1G72830 1
9
pha-miR172 AGAATCATGATGATGCTGCAA Allcontig14814 Ethylene-responsive transcription factor RAP2-7 AT2G28550
AGAATCTTGATGATGCTGCAT Allcontig20707 Cysteine proteinaseRD19a AT4G39090
AGAATCTTGATGATGCTGCCT Unigene79533leaf Floral homeotic proteinAPETALA 2 AT4G36920
CGAATCTTGATGATGCTGCAT Allcontig43241 Uncharacterized protein AT2G40430
TGAATCTTGATGATGCTGCAT
TGAATCTTGATGATGCTGCTT
pha-miR319 TTGGACTGAAGGGAGCTCCCT Allcontig18524 Transcription factor TCP-like AT1G53230
Unigene122166leaf Transcription factor TCP-like
pha-miR390 AAGCTCAGGAGGGATAGCGCC Allcontig18726 Leucine-rich repeatprotein kinase-like protein AT1G31420
pha-miR396 TTCCACAGCTTTCTTGAACTG Allcontig58358 Ribosomal protein L19family protein AT5G11750
pha-miR5179 TTTTGCTCAAGACCGCGCAAC Allcontig59930 Floral homeotic proteinAPETALA 3 AT3G54340
pha-miR2950 TTCCATCTCTTGCACACTGGA Allcontig40709 Uncharacterized protein AT1G08760
1740 PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012.
DegradomeanalysisofsmallRNAsinorchid
Fig.1 Cleavagesites,t-plotsignaturesandexpressionanalysisofnovelmiRNAsandtheirtargettranscripts.(A)CleavagefeaturesofmiRNA–
targetpairs.Arrowsindicatethecleavagesitesandrawreadsfromdeepsequencing.Thex-axisrepresentsthenucleotide(nt)positionandthey-
axisrepresentsthenormalizedreadsfromCleaveLandoutputs.(B)ComparisonofrelativeexpressionofnovelmiRNAsunderlowtemperature
withuntreatedconditionsusingstem–loopRT–qPCR.(C)Relativeexpressionofthetargettranscriptsofpha-m1154-5pmiRNA.NL,3-month D
o
untreatedleaf;CL,3-monthlowambienttemperature-treatedleaf. w
n
lo
a
d
e
Table 2 Novel miRNAs and target transcripts identified in this study d
fro
Novel miRNA ID sequence Target Target description Biological process m
h
pha-m0448-3p GATCTTCTATCGGTTGATCT Allcontig28668 Sequence-specific DNA-binding Regulation of transcription, ttp
transcription factor DNA-dependent s
pha-m0568-5p TCCGCATCATCGGCAAGTTC Allcontig12540 Probable splicing factor 3a RNA processing ://ac
a
subunit 1-like d
e
m
pha-m1154-5p TCTTCAATGGCGGCGCTCTT Allcontig51392 Nin one-binding protein - ic
pha-m0582-5p GGTGATCATCGCCGACGGGATC Allcontig30283 Light harvesting chlorophyll Photosynthesis, light harvesting; .ou
p
a b-binding protein protein–chromophore linkage .c
o
pha-m0450-5p TGGGTATTTAGCTGAGATGGT Allcontig60416 Enolase 2-like Glycolysis m
/p
pha-m0684-5p CTGACAGAAGATATAAAGCT Allcontig17124 ATP-dependent transmembrane ATP catabolic process c
p
transporter /a
pha-m1220-5p AAGAATACAAGAAGAGCATAG Unigene105562leaf PREDICTED: uncharacterized Cell communication rticle
protein LOC100826159 -a
b
[Brachypodium distachyon] s
tra
c
t/5
3
gene expression. Other effectors such as transcription factors Via expression analysis of the abundant sRNAs using sequen- /1
0
possibly coordinate the expression of target transcripts with cingreads,wenormalizedtherawreadsofeachsRNAgroupin /1
7
3
miRNAs. In summary, we conclusively confirmed seven novel millionsofunits(readspermillion).Thefoldchangeofthelow 7
/1
miRNAclassesandtheirtargettranscriptsusingstructuralpre- temperature-treatedgroup(CL)relativetotheuntreatedgroup 8
2
6
diction,expressionanalysisanddegradomeanalysis. (NL) was then calculated. Among the 46 sRNA groups, 25 7
6
Non-miRNA-directed transcript cleavage under showed an increase (CL/NL ratio >1.2) whereas 13 showed a 6 b
long-term low temperature treatment in decrease (CL/NL ratio <0.8) under low ambient temperature y gu
(Table4).Asexpressionanalysis basedonreadsissometimes e
Phalaenopsis orchid inconsistent with other analytic methods (Jayaprakash et al. st o
n
Inadditiontovalidationofnon-conservedtargetgenes,other 2011), we examined the expression of these abundant sRNAs 0
8
non-miRNA-directedtranscriptcleavagescouldalsobeidenti- quantitatively using stem–loop RT–qPCR; 45 of the 46 abun- A
p
fied using degradome analysis. However, several abundant dantsRNAscouldbedetected(Table4).Anotherquantitative ril 2
sRNA sequences could not be aligned to known miRNAs in PCR method (Shi and Chiang 2005) was also used in some 0
1
9
themiRNARegistryDatabase(miRBase,release18).Inaddition, cases and similar results were obtained (Supplementary
they were not aligned to known non-coding RNAs such as Fig.S9).Accordingtostem–loopRT–qPCRanalysis,nineabun-
rRNA, tRNA, small nuclear RNA (snRNA) or small nucleolar dant sRNAs were increased or decreased >2-fold in
(snoRNA).Further,theydidnotconstitutenovelmiRNAclasses Phalaenopsis leaves with low temperature treatment for 3
becausenoputativeprecursorscouldbeidentifiedinourtran- months (Table 4). These low temperature-responsive sRNAs
scriptomelibrary.Degradomesequencingmakesitpossibleto andtheirtargettranscriptswerefurtheranalyzedforexpression
validatecleavageproductsdirectlyfromtheRNAinterference underlowtemperaturetreatmentconditions.Sixofthetarget
genesilencingmechanism.Therefore,weidentifiedpairsof46 transcriptsshowedcomplementaryexpressionpatternstothe
abundantsRNAgroupsand36targettranscripts(Table3).The sRNAs (Fig. 2; Supplementary Fig. S5), whereas two did not
target transcripts are functionally involved in the stress re- (SupplementaryFig.S5).Thelasttranscript,pha-An-04-5,be-
sponse and metabolic processes (Supplementary Fig. S2B). longstothenaturalantisensetranscriptgroupdescribedbelow.
PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012. 1741
F.-M.AnandM.-T.Chan
Table 3 Abundant sRNA-directed transcript cleavage identified using degradome analysis
miR_fam Sequence Target Target description A. thaliana
homolog
pha-An-01 CTTGGATTTATGAAAGACGAA Allcontig15889 Nucleoporin 50 protein AT1G52380
pha-An-02 AAGATGGTGAACTATGCCT Allcontig21308 Protein acclimation of photosynthesis to AT5G38660
environment
pha-An-03 TGGTACTTGGAGAGCAGTTGGA Allcontig25120 Digalactosyldiacylglycerol synthase 1 AT3G11670
TGGTACTTGGAGAGCAGTTGG
pha-An-04-1 TGACAAATCGTTTCGTTGCATC Allcontig28452 Digalactosyldiacylglycerol synthase 2 AT4G00550
TGACAAATCGTTTCGTTGCAT
pha-An-04-2 TTGAGAAGAAATAGACATGAAA
TGAGAAGAAATAGACATGAAA D
o
TGAGAAGAAATAGACATGAAAG w
n
TTGAGAAGAAATAGACATGAA lo
a
d
ATCTTGAGAAGAAATAGACAT e
d
pha-An-04-3 TGAGGCGGAACGAACGATGGCT fro
TGAGGCGGAACGAACGATGGC m
h
TGAGGCAGAACGAACGATGGC ttp
s
TGAGGCGGAACGAACGAT ://a
AGAGGCGGAACGAACGATGGC c
a
d
pha-An-04-4 TTCTTATCAAGCTCAGCAGCCA e
m
TTCTTATCAAGCTCAGCAGCC ic
.o
TCTTATCAAGCTCAGCAGCCA u
p
pha-An-04-5 TAAAGATTTGAGAGGTATGTAA .c
o
m
pha-An-04-6 ATACTGTTGTATAAAATTGCA /p
c
pha-An-04-7 TCTGCCACCAGTGAGGGCTCTT p
/a
TCTGCCACCAGTGAGGGCTCT rtic
pha-An-04-8 CGAACGATGGCTTCAGAAACAT le
-a
pha-An-04-9 CGGAACGAACGATGGCTTCAG b
s
CGGAACGAACGATGGCTTCAGA tra
c
CGGAACGAACGATGGCTTCAT t/5
3
CGGAACGAACGATGGCTTCAGAAA /1
0
pha-An-4-10 TGCTTGAAGAAGTCATTTGAA /1
7
pha-An-05 TAAAGGAATTGACGGAAG Allcontig26230 NA 37
/1
pha-An-06 TCGACGACCATGAGATTGAGCA Allcontig29034 Beta glucosidase 43 AT3G18070 8
2
pha-An-07 TTTCAGGTGGTGGAACAAGAAGAC Allcontig31585 Putative F-box protein AT1G47790 67
6
pha-An-08 ATTCTTGTATATGATCCAGGAC Allcontig35381 Guanylate-binding-like protein AT2G38840 6
b
pha-An-09 GTGAGCGGTAGGTGAGATGTGGTA Allcontig38162 Putative protein AT4G31190 y g
pha-An-10 TCTCCAGGTTGCGGACATCTT Allcontig39608 V-type proton ATPase catalytic subunit A AT1G78900 ue
s
CTCCAGGTTGCGGACATCTTA t o
n
pha-An-11 TAGTTTGTTTGATGGTACGTGC Allcontig39638 Calcium exchanger 7 AT5G17860 0
8
TAGTTTGTTTGATGGTACGTG A
p
pha-An-12 AATTGTAGTCTGGAGAAGCGT Allcontig39904 aarF domain-containing kinase AT1G11390 ril 2
ATTGTAGTCTGGAGAAGCGT 0
1
9
pha-An-13 TCATTAATCAGCCGGAACATCT Allcontig39992 Uncharacterized protein AT4G34320
pha-An-14 CCATTGTCGTCTAGTCCGGTTA Allcontig40220 ZIP metal ion transporter-like protein AT3G08650
pha-An-15 GGGGATGTAGCTCAAATGGGA Allcontig41071 Pentatricopeptide repeat-containing protein AT1G19520
pha-An-16 CGGAAGACATTGTCAGGTG Allcontig41901 Vacuolar-processing enzyme gamma-isozyme AT4G32940
pha-An-17 TGCTTGCAGAACCGAGTCGTC Allcontig42868 Rhomboid family protein AT3G58460
pha-An-18 TTTCAGGTGGTGGAACAAGAAGAC Allcontig45369 Putative endomembrane protein EMP70 precusor AT1G10950
isolog
pha-An-19 AGGACTTAAATACTGCTTTTCGGC Allcontig46951 Pre-mRNA-processing-splicing factor-like protein AT4G38780
AGGACTTAAATACTGTTTTTCGGC
(continued)
1742 PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012.
DegradomeanalysisofsmallRNAsinorchid
Table 3 Continued
miR_fam Sequence Target Target description A. thaliana
homolog
pha-An-20 ACATCACTAGCTTACGCT Allcontig56699 Putative quinone-oxidoreductase-like protein AT4G13010
pha-An-21 GTTGGTCGATTAAGACAGCA Allcontig56708 Serine palmitoyltransferase AT5G23670
GTTGGTCGATTAAGACAGC
GTTGGTCGATTAAGACAG
pha-An-22 TACGTGGTGCGAATTGCAGAA Allcontig57541 Putative tyrosine decarboxylase AT2G20340
pha-An-23 TGTAACACTGATGAGGCTGCTG Allcontig57614 Probably inactive receptor-like protein kinase AT2G46850
pha-An-24 GTCTGTAGGTGGCTTTTCA Allcontig57988 Phosphate starvation response 1 protein AT4G28610
pha-An-25 GGTGAAGTGTTCGGATCGCT Allcontig58124 Photosystem I light harvesting complex protein AT3G61470
pha-An-26 CAAACTGAATTGTGGAAGCGAT Allcontig58250 Putative cathepsin B cysteine protease AT4G01610
D
pha-An-27 TGGGCAGAATCCTTTGCAGAC o
w
pha-An-28 GCTCTGAGGGTTGGGCACGG Allcontig61486 Mitochondrial-processing peptidase alpha subunit AT3G16480 n
lo
pha-An-29 ATCTCAGTGGATCGTGGCAGCA Allcontig62311 NA ad
e
pha-An-30 AGGACTTAAATACTGCTTTTCGGC Allcontig6644 Uncharacterized protein AT1G57613 d
pha-An-31 GCAGGGACGTAGTCAACGCGA Allcontig9697 rRNA intron-encoded homing endonuclease – from
GCAGGGACGTAGTCAACGCGAG h
ttp
GCAGGGACGTAGTCAACACGA s
pha-An-32 TGGCGTCCGATGCTTGCAGAA Unigene120313leaf Uncharacterized protein loc100825936 AT2G06040 ://a
c
a
pha-An-33 TCCGGTATCTAATAAGTTC Unigene122289leaf Ethylene-induced calmodulin-binding protein AT1G67310 d
e
pha-An-34 TGAAGATACAGAACATCGGAT Unigene15263leaf Putative protein kinase family protein AT3G57760 m
ic
pha-An-35 TATTGGTAGAAGATGCGGACA Unigene22736leaf Octicosapeptide/Phox/Bem1p domain-containing AT5G57610 .o
u
protein kinase p
.c
pha-An-36 TTTGTTCGATAAGTAGACGAGA Unigene8166leaf Putative F-box/LRR-repeat protein AT5G41840 o
m
pha-An-37 ACAAATTTCAGAGATTGTCGAG Unigene86934leaf Glycerol-3-phosphate acyltransferase 9 AT5G60620 /p
c
p
/a
rtic
le
-a
Thecleavagesitesandsequenceswerevalidatedbydegradome coveredalmostallregionsofthetranscript(Fig.3A).Thecleav- b
s
analysis. The sRNAs pha-An-01, pha-An-11 and pha-An-24 age sites of the 10 abundant sRNAs could be validated using tra
c
wereinducedbylowtemperature(Fig.2B,E;Supplementary degradome analysis (Fig. 3B, C). The overlapping sRNA se- t/5
3
Fig.S5)andrepressedtheexpressionoftheirtargettranscripts quences led us to speculate that a long antisense transcript /1
0
(Fig.2C,F;SupplementaryFig.S5).Thetargettranscriptswere exists and covers a long portion of the coding sequence of /1
7
3
annotatedasnucleoporin50,phosphatestarvationresponse1 the sense transcript (Fig. 3B). Strand-specific RT–PCR con- 7
/1
andcalciumexchanger7(Table3).Pha-An-26,pha-An-30and firmed the existence of a long antisense transcript (Fig. 3D). 8
2
6
pha-An-35 were repressed by low temperature (Fig. 2G, J; cDNA templates of sense and antisense strands were synthe- 7
6
Supplementary Fig. S5). Their target transcripts were anno- sizedbystrand-specificreversetranscriptionprimers(S-RTfor 6 b
tated as cysteine protease, octicosapeptide/Phox/Bemlp sensestrandandAS-RTforantisensestrand)(Fig.3A)follow- y g
u
domain-containingproteinkinaseandanuncharacterizedpro- ingamplificationusinggene-specificprimers(GSPs).Theamp- e
s
tein(Table3,Fig.2I,L;SupplementaryFig.S5).Thefunctions lification products could be clearly seen in the gel image t o
n
ofthese targettranscripts wereunclear. Theabundant sRNAs (Fig.3D),confirmingtheexistenceofthelongantisensetran- 0
8
may be small interfering RNAs (siRNAs) that also directed script.AsthistranscriptcouldgenerateplentyofsRNAsfrom A
p
target transcript cleavage. The results suggest that some of both sense and antisense strands, we speculated that it is a ril 2
the abundant sRNAs were also low temperature responsive NAT. This result also indicated that the 10 abundant sRNAs 01
9
anddirectlyaffectedtheexpressionofthetargettranscripts. couldregulatetheiroriginaltargetsthroughafeedbackmech-
anism (Fig. 3B, C). The target transcript homolog was anno-
Natural antisense transcript of the Phalaenopsis
tatedasdigalactosyldiacylglycerolsynthase2(DGD2)(Table3),
digalactosyldiacylglycerol synthase homolog gene
which islikely toaffect thelipid content ofchloroplast mem-
caused non-miRNA-directed transcript cleavage
branesunderphosphatestarvationconditions(Geetal.2011).
Itisinterestingthatwefound10abundantsRNAsthattargeted AstheNAToftheDGD2homologisonlyfoundinthespecies
a single transcript (Table 3). This is similar to cases that have weused,itmayplayauniqueroleinregulatingthelipidcom-
been reported in model plant species (Lenz et al. 2011). By position of the chloroplast membrane under low ambient
re-analyzingoursRNAlibrary,wefoundmorethanahundred temperature treatment. On the basis of the normalized reads
abundantsRNAsthatcouldbemappedontothistargettran- fromthesRNAlibraries,wenoticedthatsomeofthe10sRNAs
scriptinbothsenseandantisensestrands.ThemappedsRNAs appeared to respond to low ambient temperature (Table 4).
PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012. 1743
F.-M.AnandM.-T.Chan
Table 4 Relative expression level of abundant sRNAs under low WethusinvestigatedtheexpressionofthesesRNAsunderlow
temperature treatment ambienttemperatureandfounddecreasedexpressionineight
miR_fam NL CL Reads qPCR of them after treatment for 3 months (Fig. 3E). Expression of
ratioa ratiob theDGD2transcriptwasslightlydecreasedunderlowambient
pha-An-01 7.4 9.7 1.32 2.03±0.10 temperature treatment (Fig. 3F). That of the antisense tran-
pha-An-02 11.8 12.9 1.09 1.09±0.06 script was slightly increased under low ambient temperature
pha-An-03 126.2 79.3 0.63 0.75±0.02 treatment,buttheexpressionlevelwasmuchlowerthanthat
pha-An-04-1 80.5 45.4 0.56 0.59±0.03 of the DGD2 transcript (Fig. 3F). These results led us to con-
pha-An-04-2 67.2 58.5 0.87 0.57±0.04 clude that these 10 sRNAs were low temperature responsive.
pha-An-04–3 245.5 259.1 1.06 0.66±0.04 However,expressionoftheDGD2homologmightberegulated
coordinatelybytheNAT,sRNAsandotherunknownfactorsin
pha-An-04-4 34.4 16.0 0.46 0.94±0.05
Phalaenopsisorchid. D
pha-An-04-5 5.4 6.0 1.10 0.41±0.02 o
w
pha-An-04-6 6.8 3.5 0.51 0.52±0.03 n
lo
pha-An-04-7 17.8 25.8 1.45 0.42±0.03 Discussion ade
pha-An-04-8 19.3 8.6 0.45 1.06±0.05 d
pha-An-04-9 468.2 451.1 0.96 0.60±0.05 Evolutionary conservation of some miRNA families from
pha-An-04-10 3.7 1.8 0.48 0.79±0.04 and their target transcripts h
ttp
pha-An-05 15.8 38.5 2.43 1.03±0.05 s
pha-An-06 1.7 4.3 2.58 0.89±0.04 Transcriptome-wide information about RNA degradation ://a
products can be collected through deep sequencing (Addo- ca
pha-An-07 13.6 18.9 1.39 0.87±0.03 d
Quaye et al. 2008, Zhou et al. 2010, Bracken et al. 2011, e
m
pha-An-08 8.6 3.7 0.44 0.16±0.01 Devers et al.2011, Li etal. 2011, Song et al. 2011, Zhang etal. ic
pha-An-09 24.9 45.3 1.82 0.64±0.02 2011).Ourprevious studydemonstratedapipelinethatcom- .ou
p
pha-An-10 81.0 77.2 0.95 0.59±0.02 bined analysis of sRNAs and the transcriptome using high- .co
pha-An-11 7.6 13.5 1.76 2.33±0.08 throughput sequencing technology in Phalaenopsis orchid m/p
pha-An-12 26.8 61.1 2.28 0.95±0.04 (An et al. 2011). We found that several conserved miRNA cp
/a
pha-An-13 3.1 4.9 1.59 3.37±0.32 families,miR156,miR162,miR535andmiR528,arelowambient rtic
pha-An-14 5.4 2.6 0.49 0.87±0.03 temperatureresponsive.DifferentialexpressionofsomemiRNA le
-a
pha-An-15 5.6 6.4 1.15 0.61±0.07 familiesalsoimpliedthattheregulationmaybestageortissue b
s
pha-An-16 25.4 44.7 1.76 1.82±0.05 specific(Anetal.2011).Similarly,inArabidopsis,sixconserved tra
c
pha-An-17 41.1 72.2 1.76 0.85±0.10 miRNA families were found to be low ambient temperature t/5
3
pha-An-18 13.6 18.9 1.39 0.78±0.02 responsive(Leeetal.2010).ThetargetsofthemiRNAfamilies /1
0
pha-An-19 97.5 68.3 0.70 0.53±0.02 in Phalaenopsis are similar to thosein Arabidopsis, suggesting /1
7
3
pha-An-20 4.7 15.4 3.26 1.29±0.11 thatthesequencesandfunctionsareevolutionarilyconserved. 7
/1
pha-An-21 134.0 201.9 1.51 1.80±0.05 Thefunctionsofthetargetgenesincludedfloweringtime,floral 82
pha-An-22 7.6 6.7 0.87 1.05±0.03 organ identity and response to stress (Lee et al. 2010). In the 67
6
pha-An-23 3.2 5.0 1.57 0.64±0.07 current study, the target genes of the conserved low ambient 6 b
pha-An-24 2.9 14.9 5.10 5.56±0.19 temperature-responsive miRNA families were validated using y g
u
pha-An-25 25.3 45.3 1.79 ND degradome analysis, and found to be functionally involved in es
pha-An-26 5.7 2.2 0.39 0.31±0.07 various developmental stages and stress responses t on
(Supplementary Fig. S2A). Two of the putative target genes 0
pha-An-27 4.3 5.8 1.35 1.63±0.05 8
of the low temperature-responsive miRNAs identified in our A
pha-An-28 6.0 14.3 2.40 1.67±0.10 p
pha-An-29 2.5 4.9 1.94 1.10±0.03 previous study (table S2 in An et al. 2011), SQUAMOSA ril 2
PROMOTER-BINDING-LIKE (SPL) and DICER homolog 1-like, 0
pha-An-30 67.2 38.7 0.58 0.38±0.03 19
were validated using degradome analysis in this study
pha-An-31 13.5 17.8 1.32 0.66±0.04
(Table 1). miR162 mediates the degradation of DICER tran-
pha-An-32 1.5 3.1 2.00 0.67±0.05
scripts,whichisrequiredforthesRNA-mediatedgenesilencing
pha-An-33 20.4 32.5 1.59 1.67±0.17
mechanism as the self-feedback control of the entire system.
pha-An-34 1.9 3.2 1.64 0.62±0.08
TheputativetargetgeneofmiR156,SPL,isknowntoregulate
pha-An-35 2.1 5.4 2.60 0.45±0.04
thetransitionfromjuvenilephasetoadultphase.TheSPLgenes
pha-An-36 15.4 7.6 0.50 0.61±0.04
subsequently promotethe expression ofmiR172, which nega-
pha-An-37 7.4 5.6 0.75 0.55±0.08 tivelyregulatestheAP-2-liketranscriptionalfactors.Weidenti-
aRatio=readspermillionoflowtemperature-treatedleaf(CL)/readspermillion fied two target transcripts that encode the AP-2 domain and
ofcontrolgroup (NL).
havebeendescribedasethylene-responsivetranscriptionfactor
bRelativeexpressionoflowtemperature-treatedleaf(CL)tocontrolgroup(NL);
18S rRNAwasused asendogenous control. RAP2-7 and floral homeotic protein APETALA-2, which are
1744 PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012.
DegradomeanalysisofsmallRNAsinorchid
D
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d
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h
ttp
s
://a
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Fig.2 Cleavagesites,t-plotsignaturesandexpressionanalysisoflowtemperature-responsivesRNAsandtheirtargettranscripts.(A),(D),(G),(J) /18
2
Cleavage features of sRNA–target pairs. Arrows indicate the cleavage sites and raw reads from deep sequencing. The x-axis represents the 6
7
nucleotide(nt)positionandthey-axisrepresentsthenormalizedreadsfromCleaveLandoutputs.(B),(E),(H),(K)Relativeexpressionoflow 66
temperature-responsive sRNAs using stem–loop RT–qPCR. (C), (F), (I), (L) Relative expression of the target transcripts of each sRNA. NL, by
3-monthuntreatedleaf;CL,3-monthlowambienttemperature-treatedleaf. gu
e
s
t o
n
functionally involved in flowering time regulation and floral 0
Limitation of assembled contigs for identification 8
organidentity(Table1).However,onlyoneTOE-likehomolog A
of novel miRNAs p
could be identified in the transcriptome library. The homolo- ril 2
giesoftheAP2-likegenesarealsoambiguous,makingitimpos- We used structural prediction to find putative precursors in 01
9
sible to exclude functionally redundant genes. The putative the transcriptome library to identify novel miRNA classes.
target genes of miR528 and miR535 were not validated using Although many precursors could be identified using bioinfor-
degradome analysis. As degrading mRNAs are short lived, maticsanalysis,evidencetoprovetheywerenovelmiRNAswas
increasing the amount of sample or scale of deep sequencing weak. By performing degradome analysis, which gives direct
mayincreasetheprobabilityofidentifyingcleavedtranscriptsin evidence of site-specific cleavage of miRNA–target pairs, we
thefuture.Theabundanceofthereadsoncleavagesitesshows found 14 putative novel miRNAs, but even though we per-
that sRNA-directed endonucleolytic cleavage predominates formeddeepsequencingforthetranscriptomeofPhalaenopsis
overothernon-specific degradation processes. Additional val- orchidfivetimes,annotationscouldbefoundforonlysevenof
idationisrequiredforthosesRNA–targetpairswithlowreads their target transcripts. These results reveal the weakness of
onthecleavagesitetodistinguishsRNA-specificcleavagefrom deep sequencing for identifying novel miRNA precursors. The
non-specificcleavages. assembled contigs could still not cover the entire
PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012. 1745
F.-M.AnandM.-T.Chan
D
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://a
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b
Fig.3 Validationofnaturalantisensetranscriptcomplementarytodigalactosyldiacylglycerolsynthase2(DGD2)homolog.(A)Over100abun- stra
dant sRNAs from the sRNA library mapped on to the sense and antisense strands of the DGD2 transcript. (B) Multiple cleavage sites were c
identified using degradome analysis. Arrows indicate the cleavage sites and raw reads from deep sequencing. (C) Cleavage signatures are t/53
represented by t-plot. The x-axis and y-axis represent normalized reads and nucleotide position, respectively. (D) Detection of DGD2 sense /10
and antisense transcripts using strand-specific RT–PCR. Gene-specific primers (GSPs) are marked in (A). S-RT, sense strand-specific reverse /17
3
transcription primer; AS-RT, antisense strand-specific reverse transcription primer. (E) Relative expression of 10 sRNAs targeting the DGD2 7
/1
transcriptusingstem–loopRT–qPCR(3months).(F)RelativeexpressionofDGD2andantisensetranscripts(3months).NL,untreatedleaf;CL, 8
2
lowambienttemperature-treatedleaf. 6
7
6
6
b
transcriptome.AstheprecursorsofmiRNAsaredestinedtobe species (Table 1). These conserved miRNAs are expressed at y g
u
processed into mature form, they have less chance of being higherlevelsthannon-conservedmiRNAs,whichprovidesfur- e
s
identifiedthanothermRNAtranscripts.Theincompletetran- therevidenceoftheconservationofmiRNA-mediatedregula- t o
n
scriptomealsoresultsinbiasesinjudgmentofthestructureand tionamongplantspecies.UnlikeabundantmiRNAs,thetarget 0
8
annotations.Ifgenomicsequencesarenotavailable,additional transcriptsofotherabundantsRNAsinPhalaenopsisorchidare A
p
experimental evidence such as expression analysis of miRNAs involved in metabolic processes, not in any developmental ril 2
andprecursorsanddegradomeanalysisfortargetidentification stage,butsomeofthemarestressresponsive(Supplementary 01
9
is needed to validate the putative precursors from bioinfor- Fig. S2B). Ion homeostasis is a dynamic process under cold
maticdata. stress conditions. Elevation of the cytosolic calcium (Ca2+)
levelisaprimarystepinresponsetolowtemperature.Calcium
Several functional sRNAs identified by degradome
alsoinducestheexpressionofseveralcold-relatedgenesinclud-
analysis were species specific and low temperature
ing COR6 and KIN1 (Monroy and Dhindsa 1995, Knight et al.
responsive
1996). Our results suggest that sRNA-directed gene silencing
A previous report found that only a very small proportion of mayalsoregulateionhomeostasisunderlowambienttempera-
miRNA families are present in more than one organism, ture treatment in Phalaenopsis. As the 46 abundant sRNAs
whereas most are species specific (Lenz et al. 2011). Several identified here may also be conserved among plant species
examples in our transcriptome and sRNA library also showed but have yet to be annotated, we used the abundant sRNAs
conservation of miRNA-mediated regulation among plant toperformaBLASTNsearchagainsttheunannotatedsRNAsin
1746 PlantCellPhysiol.53(10):1737–1750 (2012) doi:10.1093/pcp/pcs118 !TheAuthor2012.