TENOR:
Database for Comprehensive mRNA-Seq Experiments in RiceO Yoshihiro Kawahara1, Youko Oono1, Hironobu Wakimoto1,2, Jun Ogata1, Hiroyuki Kanamori1,Harumi Sasaki1, Satomi Mori1, Takashi Matsumoto1 and Takeshi Itoh1
1 Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2 BITS. Co., Ltd.

Results of expression proﬁle analyses
low−P, 1dhigh−, low−P, 0hlow−P, 5dlow−P, 10dhigh−P, 10dhigh−P, 5dhigh−P, 1dhigh−salinity, 1hhigh−salinity, 0hvery−low−Cd, 10dvery−low−Cd, 4dvery−low−Cd, 1dJA, 1hflood, 1hosmotic, 1hdrought, 3hflood−cold−osmotic, 0hlow−, very−low−Cd, 0hdrought, 0hcold, 1hlow−Cd, 4dlow−Cd, 1dlow−Cd, 10dJA, 0hdrought, 1hABA, 3hABA, 1hABA, 0hhigh−Cd, 1hhigh−Cd, 0hcold, 12hcold, 6hcoldcold, 3hcold, 1dflood, 12hflood, 6hflood, 3dflood, 1dflood
JA, 1dJA, 12hJAJA, 6hJA, 3hflood, 3hhigh−Cd, 1dhigh−Cd, 12hhigh−Cd, 5ddrought
ABA, 12hABA, 6hosmotic
ABA, 1dosmotic, 6hABAosmotic, 3hosmotic, 12hdrought, 6h high-Cddrought, 1ddrought, 12ha time-course transcriptome analysis of rice reference cultivar (Oryza sativa L.
ssp. japonica cv. Nipponbare) under 10 abiotic stresses (high salinity, high-,low-phosphate, high-, low-, very-low-cadmium, drought, osmotic, cold andflood) and 2 plant hormone treatment conditions (abscisic acids and jasmonicacids) using mRNA-Seq. A large number of abiotic stress responsive genes,

including previously unannotated genes predicted with the RNA-Seq data, weredetected. A hierarchical clustering analysis revealed similarities in responsive

transcriptome among environmental, nutrient, heavy-metal stresses and plant
hormone treatments. Furthermore, tissue-specific and dose-dependent

responsive profiles were also observed. A number of responsive genes encode
transcription factors that likely control responsive transcriptome profiles,

however the timing of induction of the genes were different. In addition, wepromoter regions of responsive genes were shared between different

15001000500conditions. Taken together, these results suggested that rice responsive

transcriptome against various abiotic stresses and plant hormone treatments
was regulated by transcriptional networks and a considerable number ofresources (gene annotation, expression profiles, co-expressed genes,

cis-regulatory elements, etc.) were available in the TENOR (Transcriptome

ENcyclopedia Of Rice) database (http://tenor.dna.affrc.go.jp, Plant Cell Physiol(2015), doi: 10.1093/pcp/pcv179).

Table 1. Summary of samples and stress conditions
Descriptions
150mM NaCl
3 mM KH2PO40 mM NaH2PO450 µM CdSO4
1 µM CdSO4
0.2 µM CdSO4grown without medium
submerged in medium
4ºC
0.6 M Mannitol
100 µM
100 µM
Grown in Yoshida's
mediumTissues
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Root
Shoot, Roota Mizuno et al. (2010) BMC genomics, b Oono et al. (2011) Rice,dcTime points
Read length0h, 1h
360h, 1d, 5d, 10d, 10d+1d rec. e51
0h, 1d, 5d, 10d, 10d+1d rec. e510h, 1h, 12h, 1d, 5d760h, 1d, 4d, 10d760h, 1d, 4d, 10d760h, 1h, 3h, 6h, 12h, 1d760h, 1h, 3h, 6h, 12h, 1d, 3d760h, 1h, 3h, 6h, 12h, 1d760h, 1h, 3h, 6h, 12h
760h, 1h, 3h, 6h, 12h, 1d760h, 1h, 3h, 6h, 12h, 1d760h, 1h, 3h, 6h, 12h, 1d, 3d, 4d, 5d, 10d76

Kawahara et al. (2015) Plant Cell Physiol.
e 10 days after high/low phosphate stress conditions followed by 1 day under normal condition for recovery. These are only for rootsample.

Figure 1. RNA-Seq analysis workflow
RNA-Seq reads(FASTQ)

Annotate novel genes(homology & domain search)
Validate correlation of wholeexpression proﬁles between
biological replicate samples(discard erroneous samples: R<0.8)

Preprocessing
by cutadapt
Removing rRNA
derived reads

Novel genestructure (GTF)

Alignmentby TopHat2

Prediction of novel genestructures by Cu inks
and in-house program(PARPNTE)

Ref. genome(FASTA)

known genestructure (GTF)

plastid (GO:0009536)
photosynthesis (GO:0015979)
thylakoid (GO:0009579)
cellular amino acid metabolic process (GO:0006519)
secondary metabolic process (GO:0019748)
generation of precursor metabolites and energy (GO:0006091)
transferase activity (GO:0016740)
catalytic activity (GO:0003824)
metabolic process (GO:0008152)
membrane (GO:0016020)
transporter activity (GO:0005215)
response to biotic stimulus (GO:0009607)
binding (GO:0005488)
extracellular region (GO:0005576)
carbohydrate binding (GO:0030246)
pollen pistil interaction (GO:0009875)
response to stress (GO:0006950)
kinase activity (GO:0016301)
cellular protein modification process (GO:0006464)
nucleotide binding (GO:0000166)
sequence specific DNA binding transcription factor activity (GO:0003700)
DNA binding (GO:0003677)
nucleus (GO:0005634)
response to endogenous stimulus (GO:0009719)
RNA binding (GO:0003723)
nucleobase containing compound metabolic process (GO:0006139)
hydrolase activity (GO:0016787)
lipid metabolic process (GO:0006629)
cytosol (GO:0005829)
mitochondrion (GO:0005739)
cytoplasm (GO:0005737)
translation factor activity, nucleic acid binding (GO:0008135)
protein binding (GO:0005515)
signal transduction (GO:0007165)
cellular component organization (GO:0016043)
intracellular (GO:0005622)
DNA metabolic process (GO:0006259)
cell cycle (GO:0007049)
translation (GO:0006412)
ribosome (GO:0005840)
structural molecule activity (GO:0005198)
biosynthetic process (GO:0009058)
cytoskeleton (GO:0005856)
motor activity (GO:0003774)
carbohydrate metabolic process (GO:0005975)
cellular process (GO:0009987)
transport (GO:0006810)

FDR:Oono et al. (2014) PLoS ONE,Root< 0.0001< 0.001Suppressed genesConditions
High salinity aHigh phosphate b
Low phosphate b
High cadmium c
Low cadmium dVery low cadmium dDrought dFlood dCold dOsmotic d
ABA d
JA d
No treatment d

ShootJAFlood
ABA
Osmotict
Drought
Cold
Very low Cd
Low CdHigh Cd
Low P
High PHigh Salinity
JAFlood
ABA
Osmotic
Drought
Cold
Very low Cd
Low CdHigh Cd
Low P
High PHigh Salinity
Non- responsive

Materials & Methods< 0.01< 0.05Figure 3. GO enrichment analysis of responsive genes
The functional categories of stress- and plant hormone-induced and stress- and plant
hormone-suppressed genes were examined using the Gene Ontology (GO) classification.
The stress- and plant hormone-induced genes tended to have DNA-binding TF activity,
catalytic activity, transporter activity and kinase activity in the category ‘molecularfunction’ . Furthermore, a significantly large number of genes related to ‘transcription’ ,‘response to abiotic stimulus’ , ‘metabolic process’ , ‘transport’ , ‘response to stress’and ‘cellular protein modification process’ were observed. The non-responsive genes
related to ‘cellular component organization’ and ‘signal transduction’ differed from theresponsive genes, and most of them could be housekeeping genes.

TENORURL

1h 3h 6h 12h 1d 3d 4d 5d 10d

ABA

OsmoticDrought

Cold

Very low Cd
Low CdHigh Cd

Low phosphate

High phosphate

High Salinity

5 10 15 20
TF enrichment score

Figure 4. TF enrichment analysis of responsive genes
Significant enrichment of transcription factors in abiotic stress- and plant
hormone-responsive genes was tested by Fisher’ s exact test using enriched
TFs in non-responsive genes as a background level. Enrichment scores(-log10(FDR)) were calculated based on corrected p-values by FDR. We
found that the peaks of TF enrichment scores and peak time points weredifferent among the different conditions. For drought, osmotic, flood, ABA
and JA treatment conditions, the fraction of TFs was highest at relatively
early time points, such as 1 or 3 hours after the treatments were applied.
Even among the same cadmium stress conditions, the timing of TF induction
varied depending on the concentration of cadmium. The result indicates thatinduction of TFs is a typical occurrence when environmental changes occur,
but the timing of TF induction differs among different conditions.

ShootEVENINGATREBETALGLHCB21TATCCAOSAMYSREATMSD
AMYBOX2TATCCAYMOTIFOSRAMY3DTATCCACHVAL21IBOX
IBOXCORENTNODCON1GM
OSE1ROOTNODULE
BOXIINTPATPB−10PEHVPSBDCARGCW8GATGCBP2ZMGAPC4PYRIMIDINEBOXHVEPB1ERELEE4TBOXATGAPBMARABOX1GT1CONSENSUSPYRIMIDINEBOXOSRAMY1ASORLREP3ATTATABOX4TATAPVTRNALEU
MARTBOXSEF1MOTIFTATABOXOSPALPOLASIG2TATABOX3HDZIP2ATATHB2POLASIG3AACACOREOSGLUB1ANAERO1CONSENSUSCPBCSPORSEF4MOTIFGM7SPOLASIG1TATABOX5SP8BFIBSP8BIB
LECPLEACS2NTBBF1ARROLBELRECOREPCRP1WBBOXPCWRKY1ACGTABOXP1BSACGTOSGLUB1DPBFCOREDCDC3GT1GMSCAM4TATABOX1TATABOX2ABRERATCALRHERPATEXPA7GADOWNATMYBPLANTQARBNEXTAT.GBOXATPIN2REALPHALGLHCB21ACGTTBOXABREMOTIFAOSOSEMACGTABREMOTIFAOSOSEM
BP5OSWXZDNAFORMINGATCAB1X2SSEEDPROTBANAPACANBNNAPA
ABREATRD22PALINDROMICCBOXGMABADESI2ABADESI1ACGTSEED3ABREOSRAB21CGCGBOXATE2FBNTRNRVOZATVPPACGTABREMOTIFA2OSEMEMBP1TAEMGBOXLERBCSCACGTGMOTIFIRO2OS
CMSRE1IBSPOA
PALBOXAPCUPRMOTIFIIATCPRFPCCHSRAV1BATABREBNNAPA
ABRETAEM
SGBFGMGMAUX28
ACGTROOT1ABREATCONSENSUS
BOXIIPCCHSRYREPEATVFLEB4RYREPEATBNNAPA
RYREPEATGMGY2RYREPEATLEGUMINBOXPALBOXLPCUP2ATMSD
SITEIIATCYTC
UP1ATMSD
DRE2COREZMRAB17CBFHVDRECRTCOREATANAERO2CONSENSUS
WBOXNTCHN48
BOXLCOREDCPALMYBPZM
CDA1ATCAB2E2FCONSENSUS
LTRE1HVBLT49

Root

Stress/plant hormone treatments and time−points
FDR:< 0.0001< 0.001< 0.01< 0.05

Mean CDS Mean Mean exp.
length (bp) # exons level (RPK)# Loci

http://tenor.dna.affrc.go.jp

w/ mRNA support

35,4691,580.0

912.0

4.3

327.9

ab initio prediction w/ EST support

2,400

1,067.0

1,067.0

3.0

59.2

RAP-DB predicted genes

8,121

541.3

541.3

1.6

4.7

742

2,069.1

552.8

2.3

40.1

PARPNTE assembled non-coding genes

2,274

1,281.2

-

2.0

48.5

Cufflinks assembled genes

1,263943.4

-

1.8

22.5

RAP-DB representative genes

Expression profile viewer (EPV)

Unannotated genes

Acknowledgements:
This work was supported by a grant from the Ministry of Agriculture,Forestry and Fisheries of Japan (Genomics for Agricultural Innovation,RTR-0001).
Publication:
Plant Cell Physiol. 2015 Nov 16. doi: 10.1093/pcp/pcv179.
Database URL:
http://tenor.dna.affrc.go.jp
Contact:y.kawahara@affrc.go.jp>= 0.05

Figure 5. Enrichment of cis-regulatory elements in promoter regions ofinduced genes
Significantly enriched cis-regulatory elements in each condition wererepresented by colored boxes based on the significant levels. The TATA box
element was significantly enriched in the promoter regions of almost all
responsive genes, suggesting that transcription of abiotic stress- and plant
hormone-responsive genes is strictly regulated by canonical TFs.

Responsive gene search

Detection of di erentiallyexpressed genes by
Fishers’s exact test

Mean transcript
length (bp)

PARPNTE assembled protein-coding genes

1h 3h 6h 12h 1d 3d 4d 5d 10dFlood

Keyword search

Calculation of RPK

RootJA>= 0.05

Table 2. Statistics of annotated (RAP-DB) and newly identified “RNA-Seq” genes
Category1500

Induced genes

components are shared between different stress signaling pathways. All

1000

Euclidean distanceendoplasmic reticulum (GO:0005783)
nucleolus (GO:0005730)
mitochondrion (GO:0005739)
nucleobase containing compound metabolic process (GO:0006139)
cytoplasm (GO:0005737)
ribosome (GO:0005840)
translation (GO:0006412)
cytosol (GO:0005829)
RNA binding (GO:0003723)
structural molecule activity (GO:0005198)
translation factor activity, nucleic acid binding (GO:0008135)
oxygen binding (GO:0019825)
photosynthesis (GO:0015979)
thylakoid (GO:0009579)
biosynthetic process (GO:0009058)
motor activity (GO:0003774)
cellular component organization (GO:0016043)
nucleus (GO:0005634)
intracellular (GO:0005622)
lipid binding (GO:0008289)
signal transduction (GO:0007165)
catabolic process (GO:0009056)
cellular process (GO:0009987)
cellular protein modification process (GO:0006464)
kinase activity (GO:0016301)
nucleotide binding (GO:0000166)
response to stress (GO:0006950)
carbohydrate binding (GO:0030246)
pollen pistil interaction (GO:0009875)
cellular amino acid metabolic process (GO:0006519)
response to biotic stimulus (GO:0009607)
extracellular region (GO:0005576)
transferase activity (GO:0016740)
transporter activity (GO:0005215)
membrane (GO:0016020)
transport (GO:0006810)
hydrolase activity (GO:0016787)
lipid metabolic process (GO:0006629)
catalytic activity (GO:0003824)
metabolic process (GO:0008152)
binding (GO:0005488)
carbohydrate metabolic process (GO:0005975)
response to abiotic stimulus (GO:0009628)
sequence specific DNA binding transcription factor activity (GO:0003700)
transcription, DNA dependent (GO:0006350)
DNA binding (GO:0003677)
chromatin binding (GO:0003682)

Shoot

Timecourse after the treatments

500

Euclidean distance

Figure 2. Hierarchical clustering of whole transcriptome profiles
Hierarchical clustering of whole transcriptome profiles for shoot and root
under the abiotic stress and plant hormone treatment conditions based on
euclidean distance and Ward’ s clustering method. Representative clusters
were shown in dashed rectangles. Several abiotic stresses and plant
hormone treatments had a similar effect on the overall transcriptome profile.
The result suggests that some of the responsive signaling cascades areshared among those stresses.

Stress/plant hormone treatments

genes and its regulatory networks responsible for the adaptation, we performed

found that a number of cis-regulatory elements significantly enriched in theRoot

flood−cold−osmotic, 0hlow−Cd, 10ddrought, 3hABA, 0hdrought, 0hJA, 0hdrought, 1hvery−low−Cd, 10dvery−low−Cd, 4dvery−low−Cd, 1dhigh−salinity, 1hhigh−salinity, 0hhigh−Cd, 1hhigh−Cd, 0hflood, 6hflood, 3h1hflood flood,flood, 3dflood, 1dflood, 12hlow−P, 10d+1d recov.
low−P, 10dlow−P, 1dhigh−P, 1dlow−P, 5dhigh−, low−P, 0hhigh−P, 10d+1d recov.
high−P, 5dhigh−P, 10ddrought, 12hdrought, 6hdrought, 1dosmotic, 1hdrought osmotic,12hosmotic, 6hosmotic, 3hosmotic
ABA, 12hABA, 6hABAABA, 1dABA, 3hABA, 1hcold, 12hcold, 6hcold, 3hcold
cold, 1hcold, 1dhigh−Cd, 1d12hhigh-Cd high−Cd,high−Cd, 5dJA, 12hJA, 6hJA, 1dJAJA, 3hJA, 1hlow−Cd, 4dlow−Cd, 1dlow-Cdlow−, very−low−Cd, 0hHigh Salinity, 1hHigh phosphate, 1dHigh phosphate, 5dHigh phosphate, 10dLow phosphate, 1dLow phosphate, 5dLow phosphate, 10dHigh cadmium, 1hHigh cadmium, 12h
High cadmium, 1dHigh cadmium, 5d
Low cadmium, 1d
Low cadmium, 4d
Low cadmium, 10d
Very−low cadmium, 1d
Very−low cadmium, 4d
Very−low cadmium, 10dCold, 1hCold, 3hCold, 6hCold, 12hCold, 1dDrought, 1hDrought, 3hDrought, 6hDrought, 12hDrought, 1dOsmotic, 1hOsmotic, 3hOsmotic, 6hOsmotic, 12hABA, 1hABA, 3hABA, 6hABA, 12hABA, 1dFlood, 1hFlood, 3hFlood, 6hFlood, 12hFlood, 1dFlood, 3dJA, 1hJA, 3hJA, 6hJA, 12hJA, 1dHigh Salinity, 1hHigh phosphate, 1dHigh phosphate, 5dHigh phosphate, 10dLow phosphate, 1dLow phosphate, 5dLow phosphate, 10dHigh cadmium, 1hHigh cadmium, 12hHigh cadmium, 1dHigh cadmium, 5d
Low cadmium, 1dLow cadmium, 4dLow cadmium, 10dVery−low cadmium, 1d
Very−low cadmium, 4d
Very−low cadmium, 10dCold, 1hCold, 3hCold, 6hCold, 12hCold, 1dDrought, 1hDrought, 3hDrought, 6hDrought, 12hDrought, 1dOsmotic, 1hOsmotic, 3hOsmotic, 6hOsmotic, 12hABA, 1hABA, 3hABA, 6hABA, 12hABA, 1dFlood, 1hFlood, 3hFlood, 6hFlood, 12hFlood, 1dFlood, 3dJA, 1hJA, 3hJA, 6hJA, 12hJA, 1dShootEnriched PLACE cis−element

Plant have the ability to adapt various growing conditions. To understand theearly time points and smaller
impact stresses on transcriptome

Abstract

Link to:GBrowseEPV
RAP-DB
RiceXPro
CoExp

Coexpressed genes (CoExp)

Genome Browser (GBrowse)

Zoom incis-elements within 1kb promoter regions