SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
NATURE PLANTS | www.nature.com/natureplants 1
1
A trehalose-6-phosphate phosphatase enhances anaerobic germination
tolerance in rice
Tobias Kretzschmar, Margaret Anne F. Pelayo, Kurniawan R. Trijatmiko, Lourd F. Gabunada,
Rejbana Alam, Rosario Jimenez, Merlyn S. Mendioro, Inez H. Slamet-Loedin,
Nese Sreenivasulu, Julia Bailey-Serres, Abdelbagi M. Ismail, David J. Mackill,
Endang M. Septiningsih
Supplementary Information
2 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
2
Supplementary Methods
Near isogenic line (NIL) development to fine map the qAG-9-2
NILs were developed for the quantitative trait locus (QTL), qAG-9-2 by backcrossing selected
BC2F3 progenies to the recurrent parent IR647, and maintaining a small introgression from Khao
Hlan On (KHO) in the QTL region, while selecting against the rest of the genome with
background DNA markers. Seven BC4F3 introgression lines developed from two BC2F3 from the
original mapping populations were used to confirm the presence of the QTL. Selected BC4F4
recombinant families were used to fine map qAG-9-2 (Primers # 1-6, 73-74; SSR markers
RM3769, RM24141, RM24161, RM105), first using 260 lines from 38 different NILs with
overlapping introgressions. Genotyping was performed to identify new recombinants, which
were then phenotyped and further genotyped along with the informative recombinants identified
in the first batch using additional markers (Primers #7-72). Fifty recombinants from nine
different NILs aided further fine mapping (Supplementary Fig.1). NIL-AG1 (IR 93312-30-101-
20-3-66-6) had the smallest introgression at qAG-9-2 and was confirmed to be 99% similar to
IR64 across 4,037 single nucleotide polymorphism (SAG1-) markers using a Rice 6K Illumina
Infinium assay designed by S. McCouch (Cornell University) and run at the Genotyping Services
Laboratory, IRRI (http://gsl.irri.org/). A single SAG1- at 12.326 Mb near qAG-9-2 on
chromosome 9 was polymorphic between IR64 and KHO. NIL66 possesses the KHO SAG1-
(Supplementary Fig. 8).
3
Survival rate experiment
Survival rate experiment under anaerobic germination (AG) in the greenhouse was conducted
following an established protocol6. For the QTL confirmation, 16 trays per replication were used
to accommodate 144 BC4F3 introgression lines and the two parental controls in each tray, thus a
total of 176 entries were used per replication. Seedling survival was scored 21 days after sowing
for two replicates. Randomization for all entries including the parental controls was performed
with Alpha Plus design. Germination was also performed in air on moistened paper towels.
Germination after 7 days was scored. Phenotypic screening for fine mapping was performed with
a similar set up.
De novo-assembly of qAG-9-2 from KHO
A Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with the
Arizona Genomics Institute (AGI). Established methods were used for BAC library
construction36, 41 with high molecular weight (HMW) nuclear DNA cleaved with HindIII (BAC
library OSIKBa available from AGI Resource Center (http://www.genome.arizona.edu/orders/).
BAC clones were spotted onto Hybond Filters using a Genetix Qbot and processed and
hybridized with 32P labeled probes “according to manufacturer’s instructions” (“ATMI”).
TheKHO_360 probe was generated by amplification of KHO genomic DNA in the Os09g20360
region using AG1-360_F and AG1-360_R (#75-76) and cloning of the ~1.4 kb XhoI and SpeI
fragment in pGEM-T Easy (Promega). The KHO_390 probe was obtained via digestion of the
KHO_Os09g20390 clone in pCR4Blunt-TOPO plasmid with PvuI, resulting in a ~1.2 kb
fragment (for cloning of this fragment see below). Probe labeling with 32P was by use of the
DECAprime II kit (Ambion, part no. AM1455) “ATMI”. Hybridization was carried out as
NATURE PLANTS | www.nature.com/natureplants 3
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
2
Supplementary Methods
Near isogenic line (NIL) development to fine map the qAG-9-2
NILs were developed for the quantitative trait locus (QTL), qAG-9-2 by backcrossing selected
BC2F3 progenies to the recurrent parent IR647, and maintaining a small introgression from Khao
Hlan On (KHO) in the QTL region, while selecting against the rest of the genome with
background DNA markers. Seven BC4F3 introgression lines developed from two BC2F3 from the
original mapping populations were used to confirm the presence of the QTL. Selected BC4F4
recombinant families were used to fine map qAG-9-2 (Primers # 1-6, 73-74; SSR markers
RM3769, RM24141, RM24161, RM105), first using 260 lines from 38 different NILs with
overlapping introgressions. Genotyping was performed to identify new recombinants, which
were then phenotyped and further genotyped along with the informative recombinants identified
in the first batch using additional markers (Primers #7-72). Fifty recombinants from nine
different NILs aided further fine mapping (Supplementary Fig.1). NIL-AG1 (IR 93312-30-101-
20-3-66-6) had the smallest introgression at qAG-9-2 and was confirmed to be 99% similar to
IR64 across 4,037 single nucleotide polymorphism (SAG1-) markers using a Rice 6K Illumina
Infinium assay designed by S. McCouch (Cornell University) and run at the Genotyping Services
Laboratory, IRRI (http://gsl.irri.org/). A single SAG1- at 12.326 Mb near qAG-9-2 on
chromosome 9 was polymorphic between IR64 and KHO. NIL66 possesses the KHO SAG1-
(Supplementary Fig. 8).
3
Survival rate experiment
Survival rate experiment under anaerobic germination (AG) in the greenhouse was conducted
following an established protocol6. For the QTL confirmation, 16 trays per replication were used
to accommodate 144 BC4F3 introgression lines and the two parental controls in each tray, thus a
total of 176 entries were used per replication. Seedling survival was scored 21 days after sowing
for two replicates. Randomization for all entries including the parental controls was performed
with Alpha Plus design. Germination was also performed in air on moistened paper towels.
Germination after 7 days was scored. Phenotypic screening for fine mapping was performed with
a similar set up.
De novo-assembly of qAG-9-2 from KHO
A Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with the
Arizona Genomics Institute (AGI). Established methods were used for BAC library
construction36, 41 with high molecular weight (HMW) nuclear DNA cleaved with HindIII (BAC
library OSIKBa available from AGI Resource Center (http://www.genome.arizona.edu/orders/).
BAC clones were spotted onto Hybond Filters using a Genetix Qbot and processed and
hybridized with 32P labeled probes “according to manufacturer’s instructions” (“ATMI”).
TheKHO_360 probe was generated by amplification of KHO genomic DNA in the Os09g20360
region using AG1-360_F and AG1-360_R (#75-76) and cloning of the ~1.4 kb XhoI and SpeI
fragment in pGEM-T Easy (Promega). The KHO_390 probe was obtained via digestion of the
KHO_Os09g20390 clone in pCR4Blunt-TOPO plasmid with PvuI, resulting in a ~1.2 kb
fragment (for cloning of this fragment see below). Probe labeling with 32P was by use of the
DECAprime II kit (Ambion, part no. AM1455) “ATMI”. Hybridization was carried out as
4 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
4
previously described42, followed by visualization with a STORM PhosphorImager “ATMI”.
Positive BAC clones were gathered from the arrayed library and confirmed by secondary
hybridization and PCR. One BAC clone (BAC4), positive for both probes, was sequenced on a
Ion Personal Genome Machine (PGM) (Life Technologies) resulting in a total of 310,000 reads
with an average length of 255 bp. Reads were trimmed, filtered for BAC vector sequence and the
rice genomic sequence was assembled using DNASTAR 11 SeqMan NGen (Lasergene). The
assembly covered chromosome 9 from LOC_Os09g20340 to LOC_Os09g20390. As the region
LOC_Os09g20390 to LOC_Os09g20400, not covered by BAC4, this portion of the qAG-9-2
region was assembled via cloning and Sanger-based sequencing of overlapping amplicons.
Fragments were amplified with primer pairs AMP5.4 to AMP5.8 (#77-86), AG400_B (#87-88),
and AG1-400 (#89-90). Resulting products were cloned into the pGEM-T Easy (Promega) and
sequenced by a service provider (Macrogen) with their in-house M13_F and M13_R primers and
amplicon-specific primers spaced at a distance of ~500 bp along the expected sequence (primers
#91-115) and sequences assembled using DNASTAR 11 SeqMan Pro (Lasergene). The final
KHO qAG-9-2 region was assembled and annotated manually in DNASTAR 11 SeqBuilder
(Lasergene). Alignment of qAG-9-2 from KHO, Nipponbare (MSU7) and the IR64 assembly11
was performed via the MAUVE algorithm in DNASTAR 11 MegAlign Pro (Lasergene).
The 20 kb deletion of IR64 in qAG-9-2 was confirmed via amplification of the deletion flanking
region with primers DFR_F2 and DFR_R2 (#159-160). The resulting ~450 bp fragment was
cloned into pGEM-T Easy and sequenced (Macrogen). DFR_F2 and DFR_R2 in combination
with DFR_LB2 were used as co-dominant markers to track presence/absence of the deletion in
rice germplasm (Supplementary Fig. 2c).
5
Cloning, transformation and genotyping of the mutant plants
The full-length coding region of OsTPP7 (LOC_Os09g20390) was amplified from genomic
KHO DNA using TPP_F and TPP_R (#116-117), which introduced AvrII and KpnI restriction
sites. prUbi-F and prUbi-R (#118-119) were used to amplify a 1986 bp fragment of the maize
polyubiquitin promoter from pCAMBIA1300int::pZmUbi::tNOS (provided by Emmanuel
Guiderdoni, CIRAD, France) and introduced with HindIII and AvrII restriction sites.
TPP_P_Hind_F (#120) and TPP_P_AvrII_R (#121) or TPP_P_BamHI_R (#122) were used to
amplify a 1927 bp fragment of the OsTPP7 promoter from genomic KHO DNA. GUS_BamHI_F
and GUS_KpnI_R (#123-124) were used to amplify a 2053 bp fragment of the GUS reporter
gene from pCAMBIA1301 (CAMBIA, Brisbane, Queensland, Australia). The fragments were
introduced into the pCR4Blunt-TOPO vector (Invitrogen) “ATMI”. The overexpression
construct pZmUbi::OsTPP7 (OX) was assembled by ligating the maize polyubiquitin promoter
(HindIII-AvrII fragment) and OsTPP7 (AvrII-KpnI fragment) into the HindIII / KpnI cleaved
pCAMBIA1300int::tNOS. The native promoter construct pOsTPP7::OsTPP7 (AG1-) was
assembled by replacement of the pZmUbi fragment with the pOsTPP7 fragment using HindIII
and AvrII restriction sites. The pOsTPP7::GUS reporter gene construct was assembled by
inserting the TPP_P promoter fragment (1927 bp) and the GUS gene fragment (2053 bp) into the
HindIII / KpnI cleaved pCAMBIA1300int::tNOS.
Agrobacterium-mediated transformation of pZmUbi::OsTPP7 and pOsTPP7::OsTPP7 into IR64
was performed using immature embryos38. pOsTPP7::GUS was transformed into KHO using
calli derived from mature seeds38. The Agrobacterium strain LBA4404 was employed.
Regenerated transgenic plantlets (T0) were transferred to the greenhouse, grown in hydroponic
culture and transplanted into potted soil after three weeks.
NATURE PLANTS | www.nature.com/natureplants 5
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
4
previously described42, followed by visualization with a STORM PhosphorImager “ATMI”.
Positive BAC clones were gathered from the arrayed library and confirmed by secondary
hybridization and PCR. One BAC clone (BAC4), positive for both probes, was sequenced on a
Ion Personal Genome Machine (PGM) (Life Technologies) resulting in a total of 310,000 reads
with an average length of 255 bp. Reads were trimmed, filtered for BAC vector sequence and the
rice genomic sequence was assembled using DNASTAR 11 SeqMan NGen (Lasergene). The
assembly covered chromosome 9 from LOC_Os09g20340 to LOC_Os09g20390. As the region
LOC_Os09g20390 to LOC_Os09g20400, not covered by BAC4, this portion of the qAG-9-2
region was assembled via cloning and Sanger-based sequencing of overlapping amplicons.
Fragments were amplified with primer pairs AMP5.4 to AMP5.8 (#77-86), AG400_B (#87-88),
and AG1-400 (#89-90). Resulting products were cloned into the pGEM-T Easy (Promega) and
sequenced by a service provider (Macrogen) with their in-house M13_F and M13_R primers and
amplicon-specific primers spaced at a distance of ~500 bp along the expected sequence (primers
#91-115) and sequences assembled using DNASTAR 11 SeqMan Pro (Lasergene). The final
KHO qAG-9-2 region was assembled and annotated manually in DNASTAR 11 SeqBuilder
(Lasergene). Alignment of qAG-9-2 from KHO, Nipponbare (MSU7) and the IR64 assembly11
was performed via the MAUVE algorithm in DNASTAR 11 MegAlign Pro (Lasergene).
The 20 kb deletion of IR64 in qAG-9-2 was confirmed via amplification of the deletion flanking
region with primers DFR_F2 and DFR_R2 (#159-160). The resulting ~450 bp fragment was
cloned into pGEM-T Easy and sequenced (Macrogen). DFR_F2 and DFR_R2 in combination
with DFR_LB2 were used as co-dominant markers to track presence/absence of the deletion in
rice germplasm (Supplementary Fig. 2c).
5
Cloning, transformation and genotyping of the mutant plants
The full-length coding region of OsTPP7 (LOC_Os09g20390) was amplified from genomic
KHO DNA using TPP_F and TPP_R (#116-117), which introduced AvrII and KpnI restriction
sites. prUbi-F and prUbi-R (#118-119) were used to amplify a 1986 bp fragment of the maize
polyubiquitin promoter from pCAMBIA1300int::pZmUbi::tNOS (provided by Emmanuel
Guiderdoni, CIRAD, France) and introduced with HindIII and AvrII restriction sites.
TPP_P_Hind_F (#120) and TPP_P_AvrII_R (#121) or TPP_P_BamHI_R (#122) were used to
amplify a 1927 bp fragment of the OsTPP7 promoter from genomic KHO DNA. GUS_BamHI_F
and GUS_KpnI_R (#123-124) were used to amplify a 2053 bp fragment of the GUS reporter
gene from pCAMBIA1301 (CAMBIA, Brisbane, Queensland, Australia). The fragments were
introduced into the pCR4Blunt-TOPO vector (Invitrogen) “ATMI”. The overexpression
construct pZmUbi::OsTPP7 (OX) was assembled by ligating the maize polyubiquitin promoter
(HindIII-AvrII fragment) and OsTPP7 (AvrII-KpnI fragment) into the HindIII / KpnI cleaved
pCAMBIA1300int::tNOS. The native promoter construct pOsTPP7::OsTPP7 (AG1-) was
assembled by replacement of the pZmUbi fragment with the pOsTPP7 fragment using HindIII
and AvrII restriction sites. The pOsTPP7::GUS reporter gene construct was assembled by
inserting the TPP_P promoter fragment (1927 bp) and the GUS gene fragment (2053 bp) into the
HindIII / KpnI cleaved pCAMBIA1300int::tNOS.
Agrobacterium-mediated transformation of pZmUbi::OsTPP7 and pOsTPP7::OsTPP7 into IR64
was performed using immature embryos38. pOsTPP7::GUS was transformed into KHO using
calli derived from mature seeds38. The Agrobacterium strain LBA4404 was employed.
Regenerated transgenic plantlets (T0) were transferred to the greenhouse, grown in hydroponic
culture and transplanted into potted soil after three weeks.
6 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
6
T1 transformants were tested for T-DNA integration via PCR using HPT_F and HPT_R (#125-
126) that amplify a 600 bp fragment of the hygromycin resistance gene of the pCAMBIA1300
series. Homozygous transgenic plants and their wild type siblings were identified by germination
trials on hygromycin (75 µM) containing plates or a PCR scheme that recognized homozygosity.
T2 generation lines were furthermore checked for segregation of the antibiotic resistance trait.
Detailed studies were performed on two independent homozygous transgene insertion lines
(OX1 and OX2) out of 82 independent events generated with seed of the T2 and T3 generation.
Six independent pOsTPP7::OsTPP7 (AG1) lines were produced and TAIL PCRs43 performed on
the T1-derived genomic DNA using primers #127-133 to determine the T-DNA insertion sites
and allow for design of T-DNA flanking primers to determine homozygosity. Detailed studies
were performed on two independent homozygous lines (AG1-1 and AG1-2) of the T2 and T3
generations. The site of T-DNA insertion was outside of a known coding region of a gene in both
lines. Homozygosity for AG1-1 was evaluated by PCR using the insertion flanking AG1-
1_HOMO_F and AG1-1_HOMO_R primers (#134-135) together with the T-DNA left border
primer TDNA-LB4 (#128). Homozygosity for AG1-2 was determined using AG1-2_HOMO_F
and AG1-2_HOMO_R primers (#136-137) together with TDNA-LB4 (#130) (Supplementary
Fig. 4a-d).
For pOsTPP7::GUS plants 196 independent lines were generated, of which more than 50 were
tested positively for GUS staining in germinating tissues of the T1 generation. GUS staining was
performed as previously described44. Five independent lines in the T2 and T3 generation were
used for detailed studies.
A mutant with a T-DNA insertion in the third exon of LOC_Os09g20390 (Supplementary Fig.
4g) in the Dongjin background (CLON PFG_3A-08739.L) was obtained from the Crop
7
Developmental Biology Lab (http://cbi.khu.ac.kr). Seeds (T1) were germinated and the resulting
lines checked for homozygosity with a three primer approach, using the T-DNA insertion
flanking pfg_tpp_F and pfg_tpp_R (#138-139) and pfg_tpp_F in combination with the T_DNA
left border specific primer TLBP2 (#140) (Supplementary Fig. 4g-i). Homozygous mutant lines
and homozygous null segregant siblings were carried into the T2 and T3 generations and used
for phenotypic analysis. OsTPP7 expression was tested via semiquantitative RT-PCR (see
below) using KO_RT_F (#182) and KO_RT_R (#183).
Plant growth conditions for biochemical and molecular analyses
Seeds were stored at 4 °C after post-harvest processing. Seed dormancy was broken by
incubation at 50 °C For 5 days. Seeds were de-hulled, sterilized in 70% ethanol for 2 min,
washed three times with sterile water and submerged in 8 cm of autoclaved distilled water for 1-
4 days at 30 °C in the dark. AG was performed with at least three biological replicates, each with
≥ 23 individual coleoptiles per analysis. For germination in air, seeds were placed in sterile petri
dishes that contained moistened paper towels and kept for 1-4 days at 30 °C in the dark. For
sucrose treatment, water was substituted with sterile 90 mM sucrose (n=3, ≥ 46 coleoptiles per
analysis). Tissue was rapidly harvested and snapped frozen in liquid nitrogen and stored at -
80°C. For germination assays in the presence of hormones, seeds were germinated in the dark in
air at 23°C and germination was determined every 12 h (0.1-10 µM GA treatment) or 24 h (0.001
– 5 µM ABA treatment). GA treatment was in the presence of 200 µM paclobutrazol. Mock
treatments controlled for the quantity of DMSO used to solubilize hormones.
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
6
T1 transformants were tested for T-DNA integration via PCR using HPT_F and HPT_R (#125-
126) that amplify a 600 bp fragment of the hygromycin resistance gene of the pCAMBIA1300
series. Homozygous transgenic plants and their wild type siblings were identified by germination
trials on hygromycin (75 µM) containing plates or a PCR scheme that recognized homozygosity.
T2 generation lines were furthermore checked for segregation of the antibiotic resistance trait.
Detailed studies were performed on two independent homozygous transgene insertion lines
(OX1 and OX2) out of 82 independent events generated with seed of the T2 and T3 generation.
Six independent pOsTPP7::OsTPP7 (AG1) lines were produced and TAIL PCRs43 performed on
the T1-derived genomic DNA using primers #127-133 to determine the T-DNA insertion sites
and allow for design of T-DNA flanking primers to determine homozygosity. Detailed studies
were performed on two independent homozygous lines (AG1-1 and AG1-2) of the T2 and T3
generations. The site of T-DNA insertion was outside of a known coding region of a gene in both
lines. Homozygosity for AG1-1 was evaluated by PCR using the insertion flanking AG1-
1_HOMO_F and AG1-1_HOMO_R primers (#134-135) together with the T-DNA left border
primer TDNA-LB4 (#128). Homozygosity for AG1-2 was determined using AG1-2_HOMO_F
and AG1-2_HOMO_R primers (#136-137) together with TDNA-LB4 (#130) (Supplementary
Fig. 4a-d).
For pOsTPP7::GUS plants 196 independent lines were generated, of which more than 50 were
tested positively for GUS staining in germinating tissues of the T1 generation. GUS staining was
performed as previously described44. Five independent lines in the T2 and T3 generation were
used for detailed studies.
A mutant with a T-DNA insertion in the third exon of LOC_Os09g20390 (Supplementary Fig.
4g) in the Dongjin background (CLON PFG_3A-08739.L) was obtained from the Crop
7
Developmental Biology Lab (http://cbi.khu.ac.kr). Seeds (T1) were germinated and the resulting
lines checked for homozygosity with a three primer approach, using the T-DNA insertion
flanking pfg_tpp_F and pfg_tpp_R (#138-139) and pfg_tpp_F in combination with the T_DNA
left border specific primer TLBP2 (#140) (Supplementary Fig. 4g-i). Homozygous mutant lines
and homozygous null segregant siblings were carried into the T2 and T3 generations and used
for phenotypic analysis. OsTPP7 expression was tested via semiquantitative RT-PCR (see
below) using KO_RT_F (#182) and KO_RT_R (#183).
Plant growth conditions for biochemical and molecular analyses
Seeds were stored at 4 °C after post-harvest processing. Seed dormancy was broken by
incubation at 50 °C For 5 days. Seeds were de-hulled, sterilized in 70% ethanol for 2 min,
washed three times with sterile water and submerged in 8 cm of autoclaved distilled water for 1-
4 days at 30 °C in the dark. AG was performed with at least three biological replicates, each with
≥ 23 individual coleoptiles per analysis. For germination in air, seeds were placed in sterile petri
dishes that contained moistened paper towels and kept for 1-4 days at 30 °C in the dark. For
sucrose treatment, water was substituted with sterile 90 mM sucrose (n=3, ≥ 46 coleoptiles per
analysis). Tissue was rapidly harvested and snapped frozen in liquid nitrogen and stored at -
80°C. For germination assays in the presence of hormones, seeds were germinated in the dark in
air at 23°C and germination was determined every 12 h (0.1-10 µM GA treatment) or 24 h (0.001
– 5 µM ABA treatment). GA treatment was in the presence of 200 µM paclobutrazol. Mock
treatments controlled for the quantity of DMSO used to solubilize hormones.
8 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
8
RNA extraction, semiquantitative and quantitative RT-PCRs
Embryos and coleoptiles were dissected from seeds and ground in liquid nitrogen. RNA
extraction and clean-up was performed using the RNeasy kit (Qiagen) with an in-column DNAse
digest “ATMI”. Reverse transcription was performed using the GoScript Reverse transcription
system (Promega) “ATMI”. Semi-quantitative RT-PCR was performed for LOC_Os09g20360
(Primers #141-142), LOC_Os09g20370 (Primers #143-144), LOC_Os09g20390 (Primers #145-
146) and LOC_Os09g20400 (Primers #147-148), with α-tubulin (Primers #149-150) as
housekeeping control using 32 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, preceded
by 90 s of initial denaturation at 95 °C and followed by 90 s at 72 °C.
Quantitative RT-PCR was performed using a Lightcycler 480 (Roche) and Roche consumables
(“ATMI”) or MyiQ real-time PCR detection system (Bio-Rad) using iQ SYBR Green supermix
(Bio-Rad)45. Cycling parameters were: 45 cycles of 10 s at 95 °C, 15 s at 60°C and 8 s at 72 °C,
preceded by 5 s of initial denaturation at 95 °C. Primers pairs (#151-158 and 164-181) for genes
of interest and the references Tubulin, Polyubiquitin and Ubiquitin are in Supplementary Table 2.
For Supplementary Fig. 6a-c the REST software (Qiagen)46 set at 3000 iterations was used for
calculation of expression differences and statistical analysis across. For Supplementary Fig. 6d
relative transcript abundance across 4 biological replicates was calculated by the comparative CT
method47 and samples compared by ANOVA using R script. Primer efficiencies were
calculated by REST or manually via a dilution series and included in the analysis48.
9
Whole genome re-sequencing and RNA sequencing analysis
RNA was extracted using the RNeasy-Plant Mini kit (Qiagen) “ATMI”. RNA quality and
integrity was checked on a 2100 Bioanalyzer (Agilent) “ATMI”. Whole genome sequencing and
cDNA-based whole transcriptome sequencing (RNAseq) was performed by Macrogen on an
Illumina Hiseq2000 platform generating 100 bp paired-end reads with an average insert size of
300 bp. The generated Fastq files were processed and analyzed using the software suite CLC-
Genomics-Workbench 7 (Qiagen). All downstream analyses were performed within the CLC-
Genomics-Workbench 7 RNA Seq Analysis suite. EDGE-test was performed to generate fold-
differences and associated false discovery rate (FDR) corrected p-value and p-values.
Differentially expressed genes were compared to those reported in the apical 6mm tip and 5 mm
base of coleoptiles of rice cv. Amaroo seedlings germinated for 72 h in air or under 3% oxygen
(hypoxia)33 by K-means clustering49 and gene ontology50 analyses.
Analysis of T6P, trehalose and sucrose
Quantification of sucrose, trehalose-6-hosphate (T6P) and trehalose was performed by
Metabolomic Discoveries, Germany. Frozen embryo-coleoptile tissue was mechanically
disrupted in a ball mill in liquid nitrogen (Retsch), freeze dried, and 60 mg was mixed with 1 ml
80% (v/v) methanol and incubated for 15 min in a thermoshaker (1000 rpm) at 70 °C. Cellular
debris was removed by centrifugation. The extract supernatant was mixed with nine volumes of
90% (v/v) methanol and incubated for 15 min at 37 °C with vigorous shaking. Precipitated
protein was removed by centrifugation for 15 min at 13500 rpm and the supernatant retained.
Derivatisation and analyses of metabolites by a GC-MS 7890A mass spectrometer (Agilent)
were carried out as described40. Metabolites were identified in comparison to Metabolomic
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8
RNA extraction, semiquantitative and quantitative RT-PCRs
Embryos and coleoptiles were dissected from seeds and ground in liquid nitrogen. RNA
extraction and clean-up was performed using the RNeasy kit (Qiagen) with an in-column DNAse
digest “ATMI”. Reverse transcription was performed using the GoScript Reverse transcription
system (Promega) “ATMI”. Semi-quantitative RT-PCR was performed for LOC_Os09g20360
(Primers #141-142), LOC_Os09g20370 (Primers #143-144), LOC_Os09g20390 (Primers #145-
146) and LOC_Os09g20400 (Primers #147-148), with α-tubulin (Primers #149-150) as
housekeeping control using 32 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, preceded
by 90 s of initial denaturation at 95 °C and followed by 90 s at 72 °C.
Quantitative RT-PCR was performed using a Lightcycler 480 (Roche) and Roche consumables
(“ATMI”) or MyiQ real-time PCR detection system (Bio-Rad) using iQ SYBR Green supermix
(Bio-Rad)45. Cycling parameters were: 45 cycles of 10 s at 95 °C, 15 s at 60°C and 8 s at 72 °C,
preceded by 5 s of initial denaturation at 95 °C. Primers pairs (#151-158 and 164-181) for genes
of interest and the references Tubulin, Polyubiquitin and Ubiquitin are in Supplementary Table 2.
For Supplementary Fig. 6a-c the REST software (Qiagen)46 set at 3000 iterations was used for
calculation of expression differences and statistical analysis across. For Supplementary Fig. 6d
relative transcript abundance across 4 biological replicates was calculated by the comparative CT
method47 and samples compared by ANOVA using R script. Primer efficiencies were
calculated by REST or manually via a dilution series and included in the analysis48.
9
Whole genome re-sequencing and RNA sequencing analysis
RNA was extracted using the RNeasy-Plant Mini kit (Qiagen) “ATMI”. RNA quality and
integrity was checked on a 2100 Bioanalyzer (Agilent) “ATMI”. Whole genome sequencing and
cDNA-based whole transcriptome sequencing (RNAseq) was performed by Macrogen on an
Illumina Hiseq2000 platform generating 100 bp paired-end reads with an average insert size of
300 bp. The generated Fastq files were processed and analyzed using the software suite CLC-
Genomics-Workbench 7 (Qiagen). All downstream analyses were performed within the CLC-
Genomics-Workbench 7 RNA Seq Analysis suite. EDGE-test was performed to generate fold-
differences and associated false discovery rate (FDR) corrected p-value and p-values.
Differentially expressed genes were compared to those reported in the apical 6mm tip and 5 mm
base of coleoptiles of rice cv. Amaroo seedlings germinated for 72 h in air or under 3% oxygen
(hypoxia)33 by K-means clustering49 and gene ontology50 analyses.
Analysis of T6P, trehalose and sucrose
Quantification of sucrose, trehalose-6-hosphate (T6P) and trehalose was performed by
Metabolomic Discoveries, Germany. Frozen embryo-coleoptile tissue was mechanically
disrupted in a ball mill in liquid nitrogen (Retsch), freeze dried, and 60 mg was mixed with 1 ml
80% (v/v) methanol and incubated for 15 min in a thermoshaker (1000 rpm) at 70 °C. Cellular
debris was removed by centrifugation. The extract supernatant was mixed with nine volumes of
90% (v/v) methanol and incubated for 15 min at 37 °C with vigorous shaking. Precipitated
protein was removed by centrifugation for 15 min at 13500 rpm and the supernatant retained.
Derivatisation and analyses of metabolites by a GC-MS 7890A mass spectrometer (Agilent)
were carried out as described40. Metabolites were identified in comparison to Metabolomic
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10
Discoveries database entries of authentic standards. The LC separation was performed using a
Zorbax SB-Aq column (Agilent), operated by an Agilent 1290 UPLC system (Agilent). The LC
mobile phase was A) 0.1% (v/v) formic acid in water and B) 0.1% (v/v) formic acid in methanol
with a gradient from 0% to 90% B over 5 min, to 95% at 6.5 min and 100% at 8 min, and
subsequently equilibrated. Flow rate was 400 µl/min, and injection volume 1 µl. Mass
spectrometry was performed using a 6540 QTOF/MS Detector (Agilent). Measured metabolite
concentration was normalized to internal standards. Significant concentration changes of
metabolites in different samples were analyzed by Student’s t-test. A p-value of p<0.05 was
considered significant. For absolute quantification, the amount of target metabolite was
compared to known concentrations of reference standards. Five biological replicates were used
for all analyses.
Analysis of monosaccharides, sugar alcohols and amino acids
Homogenized tissue (20 mg) was mixed with 100 µL decanoic acid (1 mg/mL in ethanol) and 1
mL hot ethanol. The mixture was extracted three times by incubation for 5 min in a 75 °C water
bath followed by centrifugation at 12,000 g, t he supernatants combined and dried in 2 mL
microfuge tubes using a SpeedVac concentrator. For derivatization, 135 µL BSTFA with 1%
(w/w) TMCS and 120 µL Pyridine was added, silanized at 75 °C for 1 h in a water bath,
centrifuged at 12,500 g for 30 sec and 1 µL was analyzed using an Agilent 6890 gas
chromatograph coupled to a 5975 MS (Agilent) using a DB-5MS (30 m x 250 µm x 0.25 µm)
column. The inlet was set to 280 °C and the oven at 60 °C for 4 min, ramped to 170 °C at 8
°C/min, then to 300 °C at 4°C/min and then maintained at 300 °C for 30 min. The carrier gas
was helium programmed to flow at 1.2mL/min.
11
The MS was set to scan from 50-650m/z in the electron impact mode at 70eV. The interface,
quadrupole and source were set at 285 °C, 150 °C and 230 °C respectively. Metabolites were
identified by comparison of the experimental mass spectrum to the NIST 11 MS Library
(Gaithersburg, MD, USA). Peak areas were normalized using the internal standard-corrected
method and the values were log transformed. Values were derived from 5 biological and 2
technical replicates. Fold change differences and statistical significance was assessed using the
Welch two-sample t-test with adjusted P-value for multiple corrections using Benjamini &
Hochberg. The relative amounts of the individual metabolites were represented in the form of a
heat map.
TPP enzyme production in E. coli, purification and activity assays
The TPP7 coding sequence (LOC_Os09g20390.1) was amplified from coleoptile cDNA using
the oligonucleotides FLEX_SV1_F and FLEX_SV1_R (#157-158) and cloned into pFN18A
Halo Tag T7 Flexi vector of the pFLEXI bacterial expression system (Promega) “ATMI”. The
construct was subsequently transformed into Single Step (KRX) competent E. coli cells
(Promega) “ATMI”. The recombinant TPP7 with a N-terminal Halo tag was induced in KRX
and purified using the HaloTag Protein Purification system (Promega) “ATMI”. TPP enzyme
activity was monitored spectrophotometrically by colorimetric quantification of released
phosphate from T6P as described previously21, using BIOMOL Green Reagent (Enzo Life
Science) and 0.2 µg purified TPP7 per assay. Substrate concentrations ranged from 0.1 to 1 mM
T6P.
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10
Discoveries database entries of authentic standards. The LC separation was performed using a
Zorbax SB-Aq column (Agilent), operated by an Agilent 1290 UPLC system (Agilent). The LC
mobile phase was A) 0.1% (v/v) formic acid in water and B) 0.1% (v/v) formic acid in methanol
with a gradient from 0% to 90% B over 5 min, to 95% at 6.5 min and 100% at 8 min, and
subsequently equilibrated. Flow rate was 400 µl/min, and injection volume 1 µl. Mass
spectrometry was performed using a 6540 QTOF/MS Detector (Agilent). Measured metabolite
concentration was normalized to internal standards. Significant concentration changes of
metabolites in different samples were analyzed by Student’s t-test. A p-value of p<0.05 was
considered significant. For absolute quantification, the amount of target metabolite was
compared to known concentrations of reference standards. Five biological replicates were used
for all analyses.
Analysis of monosaccharides, sugar alcohols and amino acids
Homogenized tissue (20 mg) was mixed with 100 µL decanoic acid (1 mg/mL in ethanol) and 1
mL hot ethanol. The mixture was extracted three times by incubation for 5 min in a 75 °C water
bath followed by centrifugation at 12,000 g, t he supernatants combined and dried in 2 mL
microfuge tubes using a SpeedVac concentrator. For derivatization, 135 µL BSTFA with 1%
(w/w) TMCS and 120 µL Pyridine was added, silanized at 75 °C for 1 h in a water bath,
centrifuged at 12,500 g for 30 sec and 1 µL was analyzed using an Agilent 6890 gas
chromatograph coupled to a 5975 MS (Agilent) using a DB-5MS (30 m x 250 µm x 0.25 µm)
column. The inlet was set to 280 °C and the oven at 60 °C for 4 min, ramped to 170 °C at 8
°C/min, then to 300 °C at 4°C/min and then maintained at 300 °C for 30 min. The carrier gas
was helium programmed to flow at 1.2mL/min.
11
The MS was set to scan from 50-650m/z in the electron impact mode at 70eV. The interface,
quadrupole and source were set at 285 °C, 150 °C and 230 °C respectively. Metabolites were
identified by comparison of the experimental mass spectrum to the NIST 11 MS Library
(Gaithersburg, MD, USA). Peak areas were normalized using the internal standard-corrected
method and the values were log transformed. Values were derived from 5 biological and 2
technical replicates. Fold change differences and statistical significance was assessed using the
Welch two-sample t-test with adjusted P-value for multiple corrections using Benjamini &
Hochberg. The relative amounts of the individual metabolites were represented in the form of a
heat map.
TPP enzyme production in E. coli, purification and activity assays
The TPP7 coding sequence (LOC_Os09g20390.1) was amplified from coleoptile cDNA using
the oligonucleotides FLEX_SV1_F and FLEX_SV1_R (#157-158) and cloned into pFN18A
Halo Tag T7 Flexi vector of the pFLEXI bacterial expression system (Promega) “ATMI”. The
construct was subsequently transformed into Single Step (KRX) competent E. coli cells
(Promega) “ATMI”. The recombinant TPP7 with a N-terminal Halo tag was induced in KRX
and purified using the HaloTag Protein Purification system (Promega) “ATMI”. TPP enzyme
activity was monitored spectrophotometrically by colorimetric quantification of released
phosphate from T6P as described previously21, using BIOMOL Green Reagent (Enzo Life
Science) and 0.2 µg purified TPP7 per assay. Substrate concentrations ranged from 0.1 to 1 mM
T6P.
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12
Statistical analysis
Normality of the data sets analyzed by t-tests was confirmed graphically by histograms.
For coleoptile (Fig. 1b-c and Supplementary Fig. 3c), shoot and root length measurements
(Supplementary Fig. 3e) “n” equals the total number of individual seedlings sampled per
experiment, pooled from three independent biological replications (individual beaker or petri
dish set-up). Seeds that had not germinated at all at the time of sampling were not included in the
analysis and hence the “n” differed between genotypes. The “n” displayed in the figure legends
therefore represents the minimum “n”. Mean and standard error of means (s.e.m.) are displayed,
with a two-tailed t-test analysis.
For amylase assays (Fig. 1d and Supplementary Fig. 3d) “n” equals the number of independent
biological replications (individual beaker set-up). Though normality of the data set could not be
confirmed due to small sample; size standard deviation (s.d.) for error display and two-tailed t-
test for statistical analysis was chosen.
For T6P-related metabolite assays (Fig. 2a-c) “n” equals the number of independent biological
replications (individual beaker set-up). Though skewness was present, two tailed t-tests were
chosen for statistical analysis.
For OsTPP7-catalyzed de-phosphorylation assays (Supplementary Fig. 5c) “n” equals the
number of independent biological replications. For error display the standard error of means
(s.e.m.) was chosen. For statistical analysis a two-tailed t-test was chosen.
For glucose sensitivity assays (Supplementary Fig. 5f) “n” equals the total number of individual
seedlings sampled for the experiment, which were pooled from three independent biological
13
replications (individual beaker or petri dish set-up). For error display the standard deviation (s.d.)
was chosen. For statistical analysis a two-tailed t-test was chosen.
For metabolomics (Fig. 2) and transcriptomics analysis (Fig. 3h) detailed description of the
statistical methodology are given in the respective sections above.
NATURE PLANTS | www.nature.com/natureplants 13
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12
Statistical analysis
Normality of the data sets analyzed by t-tests was confirmed graphically by histograms.
For coleoptile (Fig. 1b-c and Supplementary Fig. 3c), shoot and root length measurements
(Supplementary Fig. 3e) “n” equals the total number of individual seedlings sampled per
experiment, pooled from three independent biological replications (individual beaker or petri
dish set-up). Seeds that had not germinated at all at the time of sampling were not included in the
analysis and hence the “n” differed between genotypes. The “n” displayed in the figure legends
therefore represents the minimum “n”. Mean and standard error of means (s.e.m.) are displayed,
with a two-tailed t-test analysis.
For amylase assays (Fig. 1d and Supplementary Fig. 3d) “n” equals the number of independent
biological replications (individual beaker set-up). Though normality of the data set could not be
confirmed due to small sample; size standard deviation (s.d.) for error display and two-tailed t-
test for statistical analysis was chosen.
For T6P-related metabolite assays (Fig. 2a-c) “n” equals the number of independent biological
replications (individual beaker set-up). Though skewness was present, two tailed t-tests were
chosen for statistical analysis.
For OsTPP7-catalyzed de-phosphorylation assays (Supplementary Fig. 5c) “n” equals the
number of independent biological replications. For error display the standard error of means
(s.e.m.) was chosen. For statistical analysis a two-tailed t-test was chosen.
For glucose sensitivity assays (Supplementary Fig. 5f) “n” equals the total number of individual
seedlings sampled for the experiment, which were pooled from three independent biological
13
replications (individual beaker or petri dish set-up). For error display the standard deviation (s.d.)
was chosen. For statistical analysis a two-tailed t-test was chosen.
For metabolomics (Fig. 2) and transcriptomics analysis (Fig. 3h) detailed description of the
statistical methodology are given in the respective sections above.
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14
Supplementary Figures
a
b
RM5535RM8303 RM24433RM219 RM7175RM23911
RM3769 RM105RM5526
RM3769 RM105
RM3769 RM105RM24141 GST2bpGST6bp
Ann11bp RM24161PDC3u1
L LLLLHHHHHMLL
Survival
100.5 kb
804.8 kb
Ann11bp RM24161Drebdws4bp TP80-90_6UE400_G3
AG400_GC3
L
HHHL L
EP40_G1TPP_E3
TP80-90_1 Sdhups5bp
L
L
~50 kb
Survival
A B C DEF GHI J KL M
ND, E, O
J P
K-1 K-2 QA, IR64
61251411011321
#Family
#Family
310
11
16
12
~ 20 kb deleted in IR64
15
Supplementary Figure 1 | Fine mapping of qAG-9-2
qAG-9-2 was delimited to a region of ~50 kb using 260 BC4F4 lines from 38 recombinant
progenies with different sizes of introgression in the region of the QTL (A-M) a, Solid boxes
represent homozygous KHO introgression regions. Seedling survival of AG is represented by L
(Low: 1-19%), M (Medium: 28%) and H (high: 35-50%). b, A second round of fine mapping
using selected families (A, D, E, O, J, and K, which splits to K-1 and K-2) and additional 50
BC4F4 lines from 9 NIL-AG1s (N-Q) narrowed down qAG-9-2 to a region of 46.5 kb. Successive
dominant markers predicted a 20.9 kb deleted region in IR64.
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14
Supplementary Figures
a
b
RM5535RM8303 RM24433RM219 RM7175RM23911
RM3769 RM105RM5526
RM3769 RM105
RM3769 RM105RM24141 GST2bpGST6bp
Ann11bp RM24161PDC3u1
L LLLLHHHHHMLL
Survival
100.5 kb
804.8 kb
Ann11bp RM24161Drebdws4bp TP80-90_6UE400_G3
AG400_GC3
L
HHHL L
EP40_G1TPP_E3
TP80-90_1 Sdhups5bp
L
L
~50 kb
Survival
A B C DEF GHI J KL M
ND, E, O
J P
K-1 K-2 QA, IR64
61251411011321
#Family
#Family
310
11
16
12
~ 20 kb deleted in IR64
15
Supplementary Figure 1 | Fine mapping of qAG-9-2
qAG-9-2 was delimited to a region of ~50 kb using 260 BC4F4 lines from 38 recombinant
progenies with different sizes of introgression in the region of the QTL (A-M) a, Solid boxes
represent homozygous KHO introgression regions. Seedling survival of AG is represented by L
(Low: 1-19%), M (Medium: 28%) and H (high: 35-50%). b, A second round of fine mapping
using selected families (A, D, E, O, J, and K, which splits to K-1 and K-2) and additional 50
BC4F4 lines from 9 NIL-AG1s (N-Q) narrowed down qAG-9-2 to a region of 46.5 kb. Successive
dominant markers predicted a 20.9 kb deleted region in IR64.
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16
a
b
KHOqAG9-2region
10 kb
Os09g20360 Os09g20370 Os09g20380 Os09g20390 Os09g20400
BAC-based de novo assembly Sequenced amplicons
Drebdws4bp AG400_GC3OsTPP7
MSU7
KHO
IR64
A B C D E
A B C D E
bp
A=Os09g20360Expressed protein
B=Os09g20370Expressed protein
C=Os09g20380Transposon protein
D=Os09g20390 E=Os09g20400Expressed proteinOsTPP7
d
IR 6
IR26
IR 42
IR 64
IR74
PSB Rc 82
NSIC Rc 222
NSIC Rc 238
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INDEL22.2 Mb 22.8 Mb
c
Os09g20380 Os09g20400Os09g20390
DELETION
OsTPP7
17
Supplementary Figure 2 | Differences of qAG-9-2 region between KHO, IR64 and MSU7
and distribution of the OsTPP7 deletion across IRRI germplasm
a, Schematic of the qAG-9-2 region, assembled via a hybrid approach of Bacterial Artificial
Chromosome (BAC) deep sequencing and sequencing of overlapping amplicon clones. White
boxes on left and right indicate fine mapping flanking markers. Black boxes indicate gene
models (MSU ID) and grey arrows indicate direction of gene transcription. b, Mauve alignment
of the qAG-9-2 region from the Nipponbare reference assembly (MSU7), KHO and IR6411. First
row indicates size and relative position. Numbered rectangles indicate gene models (A-E =
LOC_Os09g20360 to LOC_Os09g20400 indicated in panel a). Green areas indicate well aligned
portions and grey areas indicate deletions. LOC_Os09g20390 encodes OsTPP7.
LOC_Os09g20380 encodes an unclassified transposable element that is partially deleted in IR64
and KHO. c, Schematic of the ~21 kb deletion region containing OsTPP7 as found in IR64.
Arrows indicate primer positions for co-dominant marker. Orange area indicates position within
deletion. d, Presence (white boxes) and absence (black boxes) of SNPseek37 core SNPs across
the larger qAG-9-2 region for 90 IRRI-derived varieties and breeding lines that are represented in
the 3000 genomes project12 (NSIC Rc222 and NSIC Rc238 are not present in SNPseek and were
added from IRRI-internal data). Each row represents a different genotype/breeding line (pink
lines indicating the subset named on the left) and each column a core SNP. The data shows
distribution of the OsTPP7-containing deletion across a representative range of IRRI-derived
cultivars including very early cultivars (IR6) and recently released varieties (NSIC Rc238).
NATURE PLANTS | www.nature.com/natureplants 17
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16
a
b
KHOqAG9-2region
10 kb
Os09g20360 Os09g20370 Os09g20380 Os09g20390 Os09g20400
BAC-based de novo assembly Sequenced amplicons
Drebdws4bp AG400_GC3OsTPP7
MSU7
KHO
IR64
A B C D E
A B C D E
bp
A=Os09g20360Expressed protein
B=Os09g20370Expressed protein
C=Os09g20380Transposon protein
D=Os09g20390 E=Os09g20400Expressed proteinOsTPP7
d
IR 6
IR26
IR 42
IR 64
IR74
PSB Rc 82
NSIC Rc 222
NSIC Rc 238
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xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx
xxxxxx
xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx
xxx
xxx xxx
xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx
xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx
xxx
xxx xxx xxx xxxxxx
xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx
xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx
xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx
xxx xxx xxx xxx xxx xxx xxx xxx
xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx
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xxxxxx xxx xxx xxx xxx
xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx
xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx
xxx xxx xxx xxxxxx
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xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx
xxx xxx
xxxxxx
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xxx xxx xxx
xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx
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xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx
xxx xxx
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xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx
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INDEL22.2 Mb 22.8 Mb
c
Os09g20380 Os09g20400Os09g20390
DELETION
OsTPP7
17
Supplementary Figure 2 | Differences of qAG-9-2 region between KHO, IR64 and MSU7
and distribution of the OsTPP7 deletion across IRRI germplasm
a, Schematic of the qAG-9-2 region, assembled via a hybrid approach of Bacterial Artificial
Chromosome (BAC) deep sequencing and sequencing of overlapping amplicon clones. White
boxes on left and right indicate fine mapping flanking markers. Black boxes indicate gene
models (MSU ID) and grey arrows indicate direction of gene transcription. b, Mauve alignment
of the qAG-9-2 region from the Nipponbare reference assembly (MSU7), KHO and IR6411. First
row indicates size and relative position. Numbered rectangles indicate gene models (A-E =
LOC_Os09g20360 to LOC_Os09g20400 indicated in panel a). Green areas indicate well aligned
portions and grey areas indicate deletions. LOC_Os09g20390 encodes OsTPP7.
LOC_Os09g20380 encodes an unclassified transposable element that is partially deleted in IR64
and KHO. c, Schematic of the ~21 kb deletion region containing OsTPP7 as found in IR64.
Arrows indicate primer positions for co-dominant marker. Orange area indicates position within
deletion. d, Presence (white boxes) and absence (black boxes) of SNPseek37 core SNPs across
the larger qAG-9-2 region for 90 IRRI-derived varieties and breeding lines that are represented in
the 3000 genomes project12 (NSIC Rc222 and NSIC Rc238 are not present in SNPseek and were
added from IRRI-internal data). Each row represents a different genotype/breeding line (pink
lines indicating the subset named on the left) and each column a core SNP. The data shows
distribution of the OsTPP7-containing deletion across a representative range of IRRI-derived
cultivars including very early cultivars (IR6) and recently released varieties (NSIC Rc238).
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18
a b
0
20
40
60
80
surv
ival
(%)
KHO IR64 NIL-AG1
a
b
c
0
40
60
Shoot Root
Leng
th (m
m)
IR64NIL-AG1KHO
***
***
******
***
***
20
c
Os09g20400 (E)Expressed protein
Os09g20360 (A)Expressed protein
Os09g20390 (D)OsTPP7
TUBULIN
Os09g20370 (B)Expressed protein
IR64 NIL-AG1 GC
AIR H2O AIR H2O
Days after seeding
0
10
20
30
40
0 2 3 4
IR64
NIL-AG1
KHO
Col
eopt
ilele
ngth
(mm
)
d
e
0
10
20
30
0 1 2 3 4
Days after seeding
IR64
NIL-AG1
KHO
α-am
ylas
e ac
tivity
(mm
olm
in-1
g-1pr
otei
n)
1
3
5
7
Moc
k
0.01 0.
1 1 2 3 4 5
T50
(day
)
ABA (µM)
IR64NIL-AG1
f
g h
- GA + GA - GA + GA
40
60
80
100
Mock PAC+0 PAC+0.1 PAC+10
T50
(hou
r)
GA (µM)
IR64NIL-AG1
IR64 NIL-AG10
10
20
30
40
Col
eopt
ilele
ngth
(mm
)
a b
c
d
19
Supplementary Figure 3 | qAG-9-2 and candidate gene analysis and qAG-9-2-dependent
AG-survival and seedling vigor phenotypes
a, Semi-quantitative RT-PCR for all genes in the qAG-9-2 candidate region performed with
mRNA obtained from IR64 and NIL-AG1 after four days of growth in the dark under air (AIR)
or submergence (H2O). α-tubulin (TUBULIN) served as a housekeeping control and genomic
DNA (GC) as a PCR control. b, Survival rates of the tolerant parent KHO, the susceptible parent
IR64 and NIL-AG1 after 21 days of growth under submergence (n = 24 ± s.e.m.), different
letters denote p<0.001. c, Means of coleoptile lengths after 2-4 days of growth in the dark under
submergence (DGDS) (n = 69, ± s.e.m., p<0.001 for all timepoints relative to IR64). d, Means
of α-amylase activity after 1-4 DGDS (n = 4, ± s.d., p<0.05 for all timepoints relative to IR64). e,
Root and shoot total length of IR64 (white bars), NIL-AG1 (light grey bars) and KHO (dark grey
bars) seedlings after four days of growth in the dark under aerobic conditions (n = 209, ± s.e.m.),
*** = p<0.001. f-g, Germination time (T50, time for half of all seeds to germinate) in the dark
under aerobic condition in the presence of GA plus 200 µM paclobutazol (PAC) (f) or ABA (g)
(n = 20). Mock=H20. h, Means of coleoptile lengths after 4 DGDS in the absence (- GA) and
presence (+ GA) of 10 µM GA3 (n = 132, ± s.e.m.) Different letters indicate differences with
p<0.001.
NATURE PLANTS | www.nature.com/natureplants 19
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
18
a b
0
20
40
60
80
surv
ival
(%)
KHO IR64 NIL-AG1
a
b
c
0
40
60
Shoot Root
Leng
th (m
m)
IR64NIL-AG1KHO
***
***
******
***
***
20
c
Os09g20400 (E)Expressed protein
Os09g20360 (A)Expressed protein
Os09g20390 (D)OsTPP7
TUBULIN
Os09g20370 (B)Expressed protein
IR64 NIL-AG1 GC
AIR H2O AIR H2O
Days after seeding
0
10
20
30
40
0 2 3 4
IR64
NIL-AG1
KHO
Col
eopt
ilele
ngth
(mm
)
d
e
0
10
20
30
0 1 2 3 4
Days after seeding
IR64
NIL-AG1
KHO
α-am
ylas
e ac
tivity
(mm
olm
in-1
g-1pr
otei
n)
1
3
5
7
Moc
k
0.01 0.
1 1 2 3 4 5
T50
(day
)
ABA (µM)
IR64NIL-AG1
f
g h
- GA + GA - GA + GA
40
60
80
100
Mock PAC+0 PAC+0.1 PAC+10
T50
(hou
r)
GA (µM)
IR64NIL-AG1
IR64 NIL-AG10
10
20
30
40
Col
eopt
ilele
ngth
(mm
)
a b
c
d
19
Supplementary Figure 3 | qAG-9-2 and candidate gene analysis and qAG-9-2-dependent
AG-survival and seedling vigor phenotypes
a, Semi-quantitative RT-PCR for all genes in the qAG-9-2 candidate region performed with
mRNA obtained from IR64 and NIL-AG1 after four days of growth in the dark under air (AIR)
or submergence (H2O). α-tubulin (TUBULIN) served as a housekeeping control and genomic
DNA (GC) as a PCR control. b, Survival rates of the tolerant parent KHO, the susceptible parent
IR64 and NIL-AG1 after 21 days of growth under submergence (n = 24 ± s.e.m.), different
letters denote p<0.001. c, Means of coleoptile lengths after 2-4 days of growth in the dark under
submergence (DGDS) (n = 69, ± s.e.m., p<0.001 for all timepoints relative to IR64). d, Means
of α-amylase activity after 1-4 DGDS (n = 4, ± s.d., p<0.05 for all timepoints relative to IR64). e,
Root and shoot total length of IR64 (white bars), NIL-AG1 (light grey bars) and KHO (dark grey
bars) seedlings after four days of growth in the dark under aerobic conditions (n = 209, ± s.e.m.),
*** = p<0.001. f-g, Germination time (T50, time for half of all seeds to germinate) in the dark
under aerobic condition in the presence of GA plus 200 µM paclobutazol (PAC) (f) or ABA (g)
(n = 20). Mock=H20. h, Means of coleoptile lengths after 4 DGDS in the absence (- GA) and
presence (+ GA) of 10 µM GA3 (n = 132, ± s.e.m.) Different letters indicate differences with
p<0.001.
20 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
20
Supplementary Figure 4 | Analysis of native promoter OsTPP7, OsTPP7 overexpression and
knock-out T-DNA insertion OsTPP7 transgenic lines
BLAST of TAIL-PCR amplicons for native promoter OsTPP7 lines AG1-1 (a) and AG1-2 (b),
showing sites of T-DNA insertions in the SALK genome browser (http://signal.salk.edu/cgi-
bin/RiceGE). Three primer homozygosity PCRs for AG1-1 (c) and AG1-2 (d) with two T-DNA
insertion flanking primers and one T-DNA left border primer as depicted (e). Lower bands
correspond to the mutant allele and upper bands correspond to the WT allele. NTC = no
b
c
Gene track
BAC track
cDNA track
TAIL track
d e
T-DN
A
f
a
OX
1 H
MO
X1
HW
OX
2 H
MO
X2
HW
AG
1-1
HM
AG
1-1
HW
AG
1-2
HM
AG
1-2
HW
AG
1-3
HM
AG
1-3
HW
NIL
-AG
1N
TC
Ladd
er
OsTPP7
TUBULIN
AG
1-1
HW
AG
1-1
HM
IR64
NTC
Ladd
er
AG
1-2
HW
AG
1-2
HM
IR64
NTC
Ladd
er
T-DNA
1 2
3
1 = PFG_TPP_F
2 = PFG_TPP_R
3 = TDNA-LB2
1-1
HWKOC
1-3
HMKO
1-2
HE
Ladd
er
DJCont
WTD
ongj
in
KO
KO
C
DN
A C
NTC
Ladd
er
OsTPP7
TUBULIN
g h iPFG_3A08739.L
Gene track
BAC track
cDNA track
TAIL track
21
template control. f, Semi-quantitative RT-PCR detection of OsTPP7 and α-tubulin (TUBULIN)
from mRNA obtained from embryos with coleoptiles after four days of growth in dark and under
submergence for 2 independent homozygous constitutive maize POLYUBIQUITIN promoter
(pZmUbi::OsTPP7; OX HM) lines and their respective null segregant (OX HW) lines, and 3
independent homozygous native promoter (pOsTPP7::OsTPP7; AG1 HM) lines and their
respective null segregants (AG1 HW) lines. Red box depicts lines used for further studies.
Under the observed conditions there were no obvious OsTPP7 transcript differences between
OX1, OX2, AG1-1 and AG1-2. The three bands for OsTPP7 correspond to three splice variants,
with splice variant 1 (LOC_Os09g20390.1) being predominant. g, Site of T-DNA insertion in
OsTPP7 exon 3 (CLON PFG_3A-08739.L) and primer positions for determination of
homozygosity. h, Demonstration of homozygosity by genomic PCR for T2 individuals of CLON
PFG_3A-08739.L in the Dongjin background. Bands in the upper panel correspond to the WT
allele amplified with the T-DNA flanking primers; bands in the lower panel correspond to the
mutant allele, amplified with one T-DNA flanking primer and one T-DNA left border primer.
KO=knockout T-DNA insertion line; DJ Cont=Dongjin control; HW=homozygous wild type
allele; HE=hemizygous allele; HM=homozygous mutant allele. i, Semi-quantitative RT-PCR for
OsTPP7 transcript in the Dongjin wildtype, the OsTPP7 T-DNA insertion homozygous mutant
(KO), the OsTPP7 T-DNA insertion null segregant (KOC) a Dongjin genomic control (DNA C)
and a no-template control (NTC). α-tubulin (TUBULIN) served as a housekeeping control.
NATURE PLANTS | www.nature.com/natureplants 21
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
20
Supplementary Figure 4 | Analysis of native promoter OsTPP7, OsTPP7 overexpression and
knock-out T-DNA insertion OsTPP7 transgenic lines
BLAST of TAIL-PCR amplicons for native promoter OsTPP7 lines AG1-1 (a) and AG1-2 (b),
showing sites of T-DNA insertions in the SALK genome browser (http://signal.salk.edu/cgi-
bin/RiceGE). Three primer homozygosity PCRs for AG1-1 (c) and AG1-2 (d) with two T-DNA
insertion flanking primers and one T-DNA left border primer as depicted (e). Lower bands
correspond to the mutant allele and upper bands correspond to the WT allele. NTC = no
b
c
Gene track
BAC track
cDNA track
TAIL track
d e
T-DN
A
f
a
OX
1 H
MO
X1
HW
OX
2 H
MO
X2
HW
AG
1-1
HM
AG
1-1
HW
AG
1-2
HM
AG
1-2
HW
AG
1-3
HM
AG
1-3
HW
NIL
-AG
1N
TC
Ladd
er
OsTPP7
TUBULIN
AG
1-1
HW
AG
1-1
HM
IR64
NTC
Ladd
er
AG
1-2
HW
AG
1-2
HM
IR64
NTC
Ladd
er
T-DNA
1 2
3
1 = PFG_TPP_F
2 = PFG_TPP_R
3 = TDNA-LB2
1-1
HWKOC
1-3
HMKO
1-2
HE
Ladd
er
DJCont
WT
Don
gjin
KO
KO
C
DN
A C
NTC
Ladd
er
OsTPP7
TUBULIN
g h iPFG_3A08739.L
Gene track
BAC track
cDNA track
TAIL track
21
template control. f, Semi-quantitative RT-PCR detection of OsTPP7 and α-tubulin (TUBULIN)
from mRNA obtained from embryos with coleoptiles after four days of growth in dark and under
submergence for 2 independent homozygous constitutive maize POLYUBIQUITIN promoter
(pZmUbi::OsTPP7; OX HM) lines and their respective null segregant (OX HW) lines, and 3
independent homozygous native promoter (pOsTPP7::OsTPP7; AG1 HM) lines and their
respective null segregants (AG1 HW) lines. Red box depicts lines used for further studies.
Under the observed conditions there were no obvious OsTPP7 transcript differences between
OX1, OX2, AG1-1 and AG1-2. The three bands for OsTPP7 correspond to three splice variants,
with splice variant 1 (LOC_Os09g20390.1) being predominant. g, Site of T-DNA insertion in
OsTPP7 exon 3 (CLON PFG_3A-08739.L) and primer positions for determination of
homozygosity. h, Demonstration of homozygosity by genomic PCR for T2 individuals of CLON
PFG_3A-08739.L in the Dongjin background. Bands in the upper panel correspond to the WT
allele amplified with the T-DNA flanking primers; bands in the lower panel correspond to the
mutant allele, amplified with one T-DNA flanking primer and one T-DNA left border primer.
KO=knockout T-DNA insertion line; DJ Cont=Dongjin control; HW=homozygous wild type
allele; HE=hemizygous allele; HM=homozygous mutant allele. i, Semi-quantitative RT-PCR for
OsTPP7 transcript in the Dongjin wildtype, the OsTPP7 T-DNA insertion homozygous mutant
(KO), the OsTPP7 T-DNA insertion null segregant (KOC) a Dongjin genomic control (DNA C)
and a no-template control (NTC). α-tubulin (TUBULIN) served as a housekeeping control.
22 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
22
0
30
60
90
120
150
0 0.2 0.4 0.6 0.8 1
Pho
spha
te p
rodu
ctio
n(n
mol
min
-1m
g-1pr
otei
n)
T6P (mM)
0
0.3
0.6
0.9
1.2
1.5
-0.2 0 0.2 0.4 0.6 0.8 1
[T6P
] V-1
T6P (mM)
OsTPP7
TEVprotease
TEVELU Lad
75 kD
50 kD
37 kD
25 kD
20 kD
Lad ELU CE+ CE-
a b
IR64 OX1 OX2e
Grown on 0.4 M glucose
Fres
h w
eigh
t (m
g)
0
40
80
120
160
IR64 OX1 OX2
0.4 M glucose
0.4 M sorbitol
***
f
dc
23
Supplementary Figure 5 | In vitro catalytic activity of E. coli expressed recombinant
OsTPP7 and high glucose sensitivity phenotype of OsTPP7 overexpression seedlings
a,b Coomassie blue stained SDS-PAGs with the final elution of Halo-Tag purified OsTPP7
(ELU), the TEV-protease used to cleave OsTPP7 from the resin-bound Halo-Tag (TEV), crude
protein extract from rhamnose-induced E. coli (CE+, arrow indicates induced OsTPP7), crude
protein extract from glucose inhibited culture (CE-) and the Precision Plus protein standard
(Lad). Apparent mass in kD of selected protein bands is indicated. Expected size for OsTPP7 is
41 kD (arrow). Expected size for TEV is 50 kD. c, Michaelis Menten kinetics for recombinant
OsTPP7, as monitored by phosphate release in relation to T6P concentration (n = 6, ± s.e.m.). d,
Hanes-Woolf plot of the same data set, suggesting an apparent Km = 0.2 mM by trend-line
regression. The calculated turnover rate (Kcat) was ~ 0.1/s. e, Phenotypes of IR64 and two
transgenic lines constitutively expressing OsTPP7 (OX) after 2 weeks of growth on plates
containing 0.4 M glucose. f, Total fresh weight of IR64 and two OX lines after 2 weeks of
growth on plates containing either 0.4 M glucose or 0.4 M sorbitol (n = 12 ± s.d., *** =
p<0.001).
NATURE PLANTS | www.nature.com/natureplants 23
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
22
0
30
60
90
120
150
0 0.2 0.4 0.6 0.8 1
Pho
spha
te p
rodu
ctio
n(n
mol
min
-1m
g-1pr
otei
n)
T6P (mM)
0
0.3
0.6
0.9
1.2
1.5
-0.2 0 0.2 0.4 0.6 0.8 1
[T6P
] V-1
T6P (mM)
OsTPP7
TEVprotease
TEVELU Lad
75 kD
50 kD
37 kD
25 kD
20 kD
Lad ELU CE+ CE-
a b
IR64 OX1 OX2e
Grown on 0.4 M glucose
Fres
h w
eigh
t (m
g)
0
40
80
120
160
IR64 OX1 OX2
0.4 M glucose
0.4 M sorbitol
***
f
dc
23
Supplementary Figure 5 | In vitro catalytic activity of E. coli expressed recombinant
OsTPP7 and high glucose sensitivity phenotype of OsTPP7 overexpression seedlings
a,b Coomassie blue stained SDS-PAGs with the final elution of Halo-Tag purified OsTPP7
(ELU), the TEV-protease used to cleave OsTPP7 from the resin-bound Halo-Tag (TEV), crude
protein extract from rhamnose-induced E. coli (CE+, arrow indicates induced OsTPP7), crude
protein extract from glucose inhibited culture (CE-) and the Precision Plus protein standard
(Lad). Apparent mass in kD of selected protein bands is indicated. Expected size for OsTPP7 is
41 kD (arrow). Expected size for TEV is 50 kD. c, Michaelis Menten kinetics for recombinant
OsTPP7, as monitored by phosphate release in relation to T6P concentration (n = 6, ± s.e.m.). d,
Hanes-Woolf plot of the same data set, suggesting an apparent Km = 0.2 mM by trend-line
regression. The calculated turnover rate (Kcat) was ~ 0.1/s. e, Phenotypes of IR64 and two
transgenic lines constitutively expressing OsTPP7 (OX) after 2 weeks of growth on plates
containing 0.4 M glucose. f, Total fresh weight of IR64 and two OX lines after 2 weeks of
growth on plates containing either 0.4 M glucose or 0.4 M sorbitol (n = 12 ± s.d., *** =
p<0.001).
24 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
24
OX1 OX2 KHO NIL-AG1 IR64
TUBULIN
OsTPP7
0
2
4
6
8
10
2 days
OsT
PP
7 ex
pres
sion
3 days 4 days
**
*a
0
2
4
6
8
10
AIR H2O
OsT
PP
7 ex
pres
sion
*b
c
0
0.5
1
1.5
2
2.5
3CIPK15
IR64
NIL66
0
1
2
3
4
Rel
ativ
e m
RN
A le
vel
OsTPP7
0
0.5
1
1.5
2
2.5
3
3.5SnRK1A
0
2
4
6
8
10
Rel
ativ
e m
RN
A le
vel
MYBS1
0
0.5
1
1.5
2
2.5EXP11
b
a a
a
a
b
a
a
bb
b
a
aa a
b
ab
ab
0
2
4
6
8ADH1
a a
b b
AIR H2O
AIR H2O AIR H2OAIR H2O
AIR H2O AIR H2O
d
0
1
2
3EXP3
abb
b
a
AIR H2O
0
400
800
1200
1600
AIR H2O
RAMY3D
a a
b b
AIR H2O0
0.5
1
1.5
2
2.5
Rel
ativ
e m
RN
A le
vels
GH17
ab
ab
b
a
AIR H2O
25
Supplementary Figure 6 | OsTPP7 transcript abundance between treatments and
quantitative PCR validation of RNAseq data on selected genes that are implicated in AG
OsTPP7 transcript levels determined by use of quantitative RT-PCR on RNA isolated from NIL-
AG1 embryos (dark grey bar) and coleoptiles (light grey bar) grown for 2-4 days in dark and
under submergence (H2O) or aerobic conditions (AIR) (a-c). a, OsTPP7 transcript abundance
(Expression) after 2-4 days under submergence is shown relative to the 2 day coleoptile-embryo
samples (white bar). b, Expression of OsTPP7 under submerged conditions relative to aerobic
conditions (white bars) in coleoptile-embryo samples after 2 days. POLYUBIQUTIN, UBIQITIN
and ACTIN served as references transcripts. Average fold changes ± standard errors as calculated
by REST software after 3000 iterations (n = 3), * = p<0.05. c, Semi-quantitative RT-PCR of
OsTPP7 transgene and α-TUBULIN (TUB) mRNA of 2 week old seedling leaves of two
independent transgenic IR64 lines constitutively expressing pZmUbi::OsTPP7 (OX1 and OX2),
KHO, NIL-AG1 and IR64. The transcript is absent in KHO and NIL-AG1, which contain native
OsTPP7 alleles, but present in OX lines which are constitutively expressing OsTPP7.
d, Determination of selected transcript levels by quantitative RT-PCR on RNA isolated from
IR64 (white bars) and NIL-AG1 (grey bars) embryo-coleoptile tissue of seedlings grown for 4
days in the dark and under submergence (H2O) or aerobic conditions (AIR). POLYUBIQUTIN
served as the reference transcript as levels of UBIQITIN and TUBULIN mRNAs were less
consistent. Data represent mean ± s.e.m. of four independent experiments (n=4). Significant
differences between genotypes and treatments were determined by ANOVA and are denoted by
different letters, p≤0.05.
NATURE PLANTS | www.nature.com/natureplants 25
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
24
OX1 OX2 KHO NIL-AG1 IR64
TUBULIN
OsTPP7
0
2
4
6
8
10
2 days
OsT
PP
7 ex
pres
sion
3 days 4 days
**
*a
0
2
4
6
8
10
AIR H2O
OsT
PP
7 ex
pres
sion
*b
c
0
0.5
1
1.5
2
2.5
3CIPK15
IR64
NIL66
0
1
2
3
4
Rel
ativ
e m
RN
A le
vel
OsTPP7
0
0.5
1
1.5
2
2.5
3
3.5SnRK1A
0
2
4
6
8
10
Rel
ativ
e m
RN
A le
vel
MYBS1
0
0.5
1
1.5
2
2.5EXP11
b
a a
a
a
b
a
a
bb
b
a
aa a
b
ab
ab
0
2
4
6
8ADH1
a a
b b
AIR H2O
AIR H2O AIR H2OAIR H2O
AIR H2O AIR H2O
d
0
1
2
3EXP3
abb
b
a
AIR H2O
0
400
800
1200
1600
AIR H2O
RAMY3D
a a
b b
AIR H2O0
0.5
1
1.5
2
2.5
Rel
ativ
e m
RN
A le
vels
GH17
ab
ab
b
a
AIR H2O
25
Supplementary Figure 6 | OsTPP7 transcript abundance between treatments and
quantitative PCR validation of RNAseq data on selected genes that are implicated in AG
OsTPP7 transcript levels determined by use of quantitative RT-PCR on RNA isolated from NIL-
AG1 embryos (dark grey bar) and coleoptiles (light grey bar) grown for 2-4 days in dark and
under submergence (H2O) or aerobic conditions (AIR) (a-c). a, OsTPP7 transcript abundance
(Expression) after 2-4 days under submergence is shown relative to the 2 day coleoptile-embryo
samples (white bar). b, Expression of OsTPP7 under submerged conditions relative to aerobic
conditions (white bars) in coleoptile-embryo samples after 2 days. POLYUBIQUTIN, UBIQITIN
and ACTIN served as references transcripts. Average fold changes ± standard errors as calculated
by REST software after 3000 iterations (n = 3), * = p<0.05. c, Semi-quantitative RT-PCR of
OsTPP7 transgene and α-TUBULIN (TUB) mRNA of 2 week old seedling leaves of two
independent transgenic IR64 lines constitutively expressing pZmUbi::OsTPP7 (OX1 and OX2),
KHO, NIL-AG1 and IR64. The transcript is absent in KHO and NIL-AG1, which contain native
OsTPP7 alleles, but present in OX lines which are constitutively expressing OsTPP7.
d, Determination of selected transcript levels by quantitative RT-PCR on RNA isolated from
IR64 (white bars) and NIL-AG1 (grey bars) embryo-coleoptile tissue of seedlings grown for 4
days in the dark and under submergence (H2O) or aerobic conditions (AIR). POLYUBIQUTIN
served as the reference transcript as levels of UBIQITIN and TUBULIN mRNAs were less
consistent. Data represent mean ± s.e.m. of four independent experiments (n=4). Significant
differences between genotypes and treatments were determined by ANOVA and are denoted by
different letters, p≤0.05.
26 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
26
OsTPP7 = LOC_Os09g20390, CIPK15 = LOC_Os11g02240 (upstream regulator of SnRK119),
SNRK1A = LOC_Os05g45420 (global integrator of energy signaling28-30), MYBS1 =
LOC_Os01g34060 (α-amylase-activating transcription factor20, regulated by SnRK129), EXP3 =
LOC_Os10g40720 and EXP11 = LOC_Os02g44108 (expansins, proposed to be involved in cell
elongation20), GH17 = LOC_Os01g71860 (Family 17 hydrolase, proposed to be involved in cell
wall loosening), ADH1 = LOC_Os11g10480 (alcohol dehydrogenase 1, key fermentative
enzyme20, involved in hypoxia tolerance), RAmy3D = LOC_Os08g36910 (α-amylase shown to
be involved in hypoxia tolerance20)
27
Starch
Maltose
NIL-‐AG1
T6PSnRK1TPP
Endosperm EmbryoScutellum
Starch
Maltose
α-‐amylases
IR64
T6P TrehaloseSnRK1TPP
HexosesSucrose
T6P/Sucrose ColeoptileMYBS1
α-‐amylases (3)
Sucrose (5) Hexoses (6)
Trehalose (1)
Elongation Growth (2)
MYBS1 (4)
OsTPP7
T6P/Sucrose
NATURE PLANTS | www.nature.com/natureplants 27
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
26
OsTPP7 = LOC_Os09g20390, CIPK15 = LOC_Os11g02240 (upstream regulator of SnRK119),
SNRK1A = LOC_Os05g45420 (global integrator of energy signaling28-30), MYBS1 =
LOC_Os01g34060 (α-amylase-activating transcription factor20, regulated by SnRK129), EXP3 =
LOC_Os10g40720 and EXP11 = LOC_Os02g44108 (expansins, proposed to be involved in cell
elongation20), GH17 = LOC_Os01g71860 (Family 17 hydrolase, proposed to be involved in cell
wall loosening), ADH1 = LOC_Os11g10480 (alcohol dehydrogenase 1, key fermentative
enzyme20, involved in hypoxia tolerance), RAmy3D = LOC_Os08g36910 (α-amylase shown to
be involved in hypoxia tolerance20)
27
Starch
Maltose
NIL-‐AG1
T6PSnRK1TPP
Endosperm EmbryoScutellum
Starch
Maltose
α-‐amylases
IR64
T6P TrehaloseSnRK1TPP
HexosesSucrose
T6P/Sucrose ColeoptileMYBS1
α-‐amylases (3)
Sucrose (5) Hexoses (6)
Trehalose (1)
Elongation Growth (2)
MYBS1 (4)
OsTPP7
T6P/Sucrose
28 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
28
Supplementary Figure 7 | Proposed mechanism of OsTPP7 function in enhancing anaerobic
germination
Blue ovals indicate upregulated metabolites or transcripts associated with presence of OsTPP7
(green oval). Orange oval indicated downregulated metabolite ratio associated with presence of
OsTPP7. Solid arrows indicate steps or interactions supported by the literature. Dashed arrows
indicate interactions for which there is no experimental evidence in the literature. White tringles
indicate flux of sugars. Bold outlines indicate increased flux.
In the OsTPP7-lacking IR64 background carbon fluxes from starch to hexoses for fermentative
energy production and supply of carbon for growth related macromolecules is limited, which is
at least in part due feedback inhibition of starch mobilization in source by sucrose levels in
sink17,19. This is assumed to be partially mediated through SnRK1 and T6P levels9, 29. T6P
contents are tightly correlated to sucrose contents22, resulting in stable T6P:sucrose ratios25.
In the OsTPP7-containing NIL-AG1 and AG1-1 lines, T6P conversion to trehalose is enhanced
as evident from higher trehalose (1) contents (Fig. 2c). This is speculated to partially alleviate
feedback inhibition of sugar fluxes from source to sink, allowing for increased carbon utilization
for energy and macromolecule production, ultimately enhancing coleoptile elongation (2) (Fig.
1b and c, Supplementary Fig. 3c).
Higher α-amylase activities (3) (Fig. 1c, Supplementary Fig. 3d) due to increased MYBS1
expression (4) (Supplementary Fig. 6d) lead to higher sucrose (5) (Fig. 2c) and hexose (6) (Fig.
2d) contents in the embryo-coleoptile. Higher sucrose contents are thought to translate to
moreT6P25, which we speculated to be buffered by T6P breakdown via OsTPP7 (Supplementary
Fig. 5) leading to T6P contents similar to those observed in IR64 (Fig 2a).
29
IR64NIL-AG1
IR42PSB Rc82
NSIC Rc222KHO
FR13A
a
IR42
IR64-AG1
FR13A
IR64
c
b
NATURE PLANTS | www.nature.com/natureplants 29
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
28
Supplementary Figure 7 | Proposed mechanism of OsTPP7 function in enhancing anaerobic
germination
Blue ovals indicate upregulated metabolites or transcripts associated with presence of OsTPP7
(green oval). Orange oval indicated downregulated metabolite ratio associated with presence of
OsTPP7. Solid arrows indicate steps or interactions supported by the literature. Dashed arrows
indicate interactions for which there is no experimental evidence in the literature. White tringles
indicate flux of sugars. Bold outlines indicate increased flux.
In the OsTPP7-lacking IR64 background carbon fluxes from starch to hexoses for fermentative
energy production and supply of carbon for growth related macromolecules is limited, which is
at least in part due feedback inhibition of starch mobilization in source by sucrose levels in
sink17,19. This is assumed to be partially mediated through SnRK1 and T6P levels9, 29. T6P
contents are tightly correlated to sucrose contents22, resulting in stable T6P:sucrose ratios25.
In the OsTPP7-containing NIL-AG1 and AG1-1 lines, T6P conversion to trehalose is enhanced
as evident from higher trehalose (1) contents (Fig. 2c). This is speculated to partially alleviate
feedback inhibition of sugar fluxes from source to sink, allowing for increased carbon utilization
for energy and macromolecule production, ultimately enhancing coleoptile elongation (2) (Fig.
1b and c, Supplementary Fig. 3c).
Higher α-amylase activities (3) (Fig. 1c, Supplementary Fig. 3d) due to increased MYBS1
expression (4) (Supplementary Fig. 6d) lead to higher sucrose (5) (Fig. 2c) and hexose (6) (Fig.
2d) contents in the embryo-coleoptile. Higher sucrose contents are thought to translate to
moreT6P25, which we speculated to be buffered by T6P breakdown via OsTPP7 (Supplementary
Fig. 5) leading to T6P contents similar to those observed in IR64 (Fig 2a).
29
IR64NIL-AG1
IR42PSB Rc82
NSIC Rc222KHO
FR13A
a
IR42
IR64-AG1
FR13A
IR64
c
b
30 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
30
Supplementary Figure 8 | Genotype and performance of NIL-AG1 under direct seeding
Pan-genome similarities of the tested lines to IR64 determined using 4037 single nucleotide
polymorphism (SNP) markers across the genome are: 99.0% for NIL-AG1, 89.7% for IR42,
87.8% for PSB Rc82, 85.3% for NSIC Rc222, 77.5% for KHO, and 67.5% for FR13A. a,
Graphical genotype of 10.6-13.7 Mb region of chromosome 9. A single SNP at 12.33 Mb
between the KHO allele in NIL-AG1 at the qAG-9-2 locus is indicated in red. b, Performance of
NIL-AG1 compared to the original parent IR64, donor KHO, and FR13A under AG stress with
dry seeds sown underwater in the greenhouse. c, Comparison of direct seeded IR64, IR42 and
FR13A under AG stress in the field condition.
31
Supplementary Table 1| Gene ontology enrichment (GO) analysis of differentially
regulated genes between a native promoter Os TPP7 transgenic line (AG1-1) and the
corresponding null segregant line (entries can be found in Supplementary Table 3)
GO analyzed with AgriGO: http://bioinfo.cau.edu.cn/agriGO/Most significantly differentially up regulated genes (FDR <0.05) (n=62); too few down regualted genes for GOGO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0005618 C cell wall 6 31 1179 34296 0.00059 0.018GO:0030312 C external encapsulating structure 6 31 1189 34296 0.00061 0.018GO:0008361 P regulation of cell size 3 31 567 34296 0.014 0.36GO:0016049 P cell growth 3 31 567 34296 0.014 0.36GO:0090066 P regulation of anatomical structure size 3 31 567 34296 0.014 0.36GO:0065008 P regulation of biological quality 4 31 863 34296 0.0074 0.36GO:0032535 P regulation of cellular component size 3 31 567 34296 0.014 0.36GO:0005576 C extracellular region 3 31 730 34296 0.028 0.55GO:0040007 P growth 3 31 736 34296 0.029 0.59GO:0005975 P carbohydrate metabolic process 4 31 1439 34296 0.04 0.71GO:0008289 F lipid binding 2 31 346 34296 0.039 0.71
Most Significantly differentially up regulated genes (|FC| > 2, p < 0.05 (n=192)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0030312 C external encapsulating structure 22 166 1189 34296 9.90E-‐08 6.40E-‐06GO:0005618 C cell wall 22 166 1179 34296 8.50E-‐08 6.40E-‐06GO:0006950 P response to stress 50 166 4660 34296 3.00E-‐08 8.80E-‐06GO:0050896 P response to stimulus 60 166 6928 34296 1.50E-‐06 0.00023GO:0009628 P response to abiotic stimulus 30 166 3022 34296 0.00013 0.013GO:0015979 P photosynthesis 8 166 324 34296 0.00022 0.016GO:0009607 P response to biotic stimulus 17 166 1404 34296 0.00053 0.032GO:0005773 C vacuole 18 166 1723 34296 0.0019 0.083GO:0005488 F binding 91 166 15115 34296 0.0035 0.21
Most Significantly differentially down regulated genes (|FC| > 2, p < 0.05 (n=80)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0016787 F hydrolase activity 17 58 4293 34296 0.00055 0.015GO:0003824 F catalytic activity 35 58 13508 34296 0.001 0.015GO:0005623 C cell 50 58 22048 34296 0.00018 0.016GO:0005102 F receptor binding 2 58 69 34296 0.0065 0.063GO:0009991 P response to extracellular stimulus 5 58 393 34296 0.00056 0.099GO:0007154 P cell communication 5 58 512 34296 0.0018 0.16GO:0008289 F lipid binding 3 58 346 34296 0.021 0.16GO:0006950 P response to stress 16 58 4660 34296 0.0038 0.19GO:0009628 P response to abiotic stimulus 12 58 3022 34296 0.0042 0.19GO:0009605 P response to external stimulus 5 58 681 34296 0.006 0.21GO:0050896 P response to stimulus 20 58 6928 34296 0.008 0.23GO:0005773 C vacuole 8 58 1723 34296 0.0083 0.38GO:0005829 C cytosol 11 58 3345 34296 0.023 0.7
NATURE PLANTS | www.nature.com/natureplants 31
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
30
Supplementary Figure 8 | Genotype and performance of NIL-AG1 under direct seeding
Pan-genome similarities of the tested lines to IR64 determined using 4037 single nucleotide
polymorphism (SNP) markers across the genome are: 99.0% for NIL-AG1, 89.7% for IR42,
87.8% for PSB Rc82, 85.3% for NSIC Rc222, 77.5% for KHO, and 67.5% for FR13A. a,
Graphical genotype of 10.6-13.7 Mb region of chromosome 9. A single SNP at 12.33 Mb
between the KHO allele in NIL-AG1 at the qAG-9-2 locus is indicated in red. b, Performance of
NIL-AG1 compared to the original parent IR64, donor KHO, and FR13A under AG stress with
dry seeds sown underwater in the greenhouse. c, Comparison of direct seeded IR64, IR42 and
FR13A under AG stress in the field condition.
31
Supplementary Table 1| Gene ontology enrichment (GO) analysis of differentially
regulated genes between a native promoter Os TPP7 transgenic line (AG1-1) and the
corresponding null segregant line (entries can be found in Supplementary Table 3)
GO analyzed with AgriGO: http://bioinfo.cau.edu.cn/agriGO/Most significantly differentially up regulated genes (FDR <0.05) (n=62); too few down regualted genes for GOGO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0005618 C cell wall 6 31 1179 34296 0.00059 0.018GO:0030312 C external encapsulating structure 6 31 1189 34296 0.00061 0.018GO:0008361 P regulation of cell size 3 31 567 34296 0.014 0.36GO:0016049 P cell growth 3 31 567 34296 0.014 0.36GO:0090066 P regulation of anatomical structure size 3 31 567 34296 0.014 0.36GO:0065008 P regulation of biological quality 4 31 863 34296 0.0074 0.36GO:0032535 P regulation of cellular component size 3 31 567 34296 0.014 0.36GO:0005576 C extracellular region 3 31 730 34296 0.028 0.55GO:0040007 P growth 3 31 736 34296 0.029 0.59GO:0005975 P carbohydrate metabolic process 4 31 1439 34296 0.04 0.71GO:0008289 F lipid binding 2 31 346 34296 0.039 0.71
Most Significantly differentially up regulated genes (|FC| > 2, p < 0.05 (n=192)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0030312 C external encapsulating structure 22 166 1189 34296 9.90E-‐08 6.40E-‐06GO:0005618 C cell wall 22 166 1179 34296 8.50E-‐08 6.40E-‐06GO:0006950 P response to stress 50 166 4660 34296 3.00E-‐08 8.80E-‐06GO:0050896 P response to stimulus 60 166 6928 34296 1.50E-‐06 0.00023GO:0009628 P response to abiotic stimulus 30 166 3022 34296 0.00013 0.013GO:0015979 P photosynthesis 8 166 324 34296 0.00022 0.016GO:0009607 P response to biotic stimulus 17 166 1404 34296 0.00053 0.032GO:0005773 C vacuole 18 166 1723 34296 0.0019 0.083GO:0005488 F binding 91 166 15115 34296 0.0035 0.21
Most Significantly differentially down regulated genes (|FC| > 2, p < 0.05 (n=80)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0016787 F hydrolase activity 17 58 4293 34296 0.00055 0.015GO:0003824 F catalytic activity 35 58 13508 34296 0.001 0.015GO:0005623 C cell 50 58 22048 34296 0.00018 0.016GO:0005102 F receptor binding 2 58 69 34296 0.0065 0.063GO:0009991 P response to extracellular stimulus 5 58 393 34296 0.00056 0.099GO:0007154 P cell communication 5 58 512 34296 0.0018 0.16GO:0008289 F lipid binding 3 58 346 34296 0.021 0.16GO:0006950 P response to stress 16 58 4660 34296 0.0038 0.19GO:0009628 P response to abiotic stimulus 12 58 3022 34296 0.0042 0.19GO:0009605 P response to external stimulus 5 58 681 34296 0.006 0.21GO:0050896 P response to stimulus 20 58 6928 34296 0.008 0.23GO:0005773 C vacuole 8 58 1723 34296 0.0083 0.38GO:0005829 C cytosol 11 58 3345 34296 0.023 0.7
32 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
32
Supplementary Table 2| Grain quality parameters of NIL-AG1 compared to IR64
Entries Length
(mm)
Width
(mm)
Chalkiness
(%)
ACa
(%)
GT GC
(mm)
BR
(%)
MR
(%)
HR
(%)
IR64 6.95 2.27 1.9 24.3 I 78 78.9 70.3 64.3
NIL-AG1 6.96 2.33 2.5 26.1 I 80 79.3 70.4 63.0
aAC=amylose content, GT=gelatinization temperature, GC=gel consistency, BR=brown rice,
MR=milled rice, HR=head rice, I=intermediate
Supplementary Table 3| RNA-seq analysis of the AG1-1 trasngenic (TPP7::TPP7 in IR64)
and IR64 coleoptile-embryo tissue after 4 d germination in darkness
Supplementary Table 4| List of primers
33
Supplementary References
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construction and analysis of 12 deep-coverage large-insert BAC libraries that represent
the 10 genome types of the genus Oryza. Genome Res. 16, 140-147 (2006).
42. Church, G. M. & Gilbert, W. Genomic sequencing. Proceedings of the National Academy
of Sciences 81, 1991-1995 (1984).
43. Liu, Y. G., Mitsukawa, N., Oosumi, T. & Whittier, R. F. Efficient isolation and mapping
of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.
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44. Cervera, M. in Transgenic Plants: Methods and Protocols Vol. 286 Methods in
Molecular Biology (ed Leandro Peña) 203-213 (Humana Press, 2004).
45. Fukao, T., Yeung, E. & Bailey-Serres, J. The submergence tolerance gene SUB1A delays
leaf senescence under prolonged darkness through hormonal regulation in rice. Plant
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46. Pfaffl, M. W., Horgan, G. W. & Dempfle, L. Relative expression software tool (REST)
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method. Nat Protoc 3, 1101-1108 (2008).
NATURE PLANTS | www.nature.com/natureplants 33
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124
32
Supplementary Table 2| Grain quality parameters of NIL-AG1 compared to IR64
Entries Length
(mm)
Width
(mm)
Chalkiness
(%)
ACa
(%)
GT GC
(mm)
BR
(%)
MR
(%)
HR
(%)
IR64 6.95 2.27 1.9 24.3 I 78 78.9 70.3 64.3
NIL-AG1 6.96 2.33 2.5 26.1 I 80 79.3 70.4 63.0
aAC=amylose content, GT=gelatinization temperature, GC=gel consistency, BR=brown rice,
MR=milled rice, HR=head rice, I=intermediate
Supplementary Table 3| RNA-seq analysis of the AG1-1 trasngenic (TPP7::TPP7 in IR64)
and IR64 coleoptile-embryo tissue after 4 d germination in darkness
Supplementary Table 4| List of primers
33
Supplementary References
41. Ammiraju, J. S. S. et al. The Oryza bacterial artificial chromosome library resource:
construction and analysis of 12 deep-coverage large-insert BAC libraries that represent
the 10 genome types of the genus Oryza. Genome Res. 16, 140-147 (2006).
42. Church, G. M. & Gilbert, W. Genomic sequencing. Proceedings of the National Academy
of Sciences 81, 1991-1995 (1984).
43. Liu, Y. G., Mitsukawa, N., Oosumi, T. & Whittier, R. F. Efficient isolation and mapping
of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.
Plant J. 8, 457-463 (1995).
44. Cervera, M. in Transgenic Plants: Methods and Protocols Vol. 286 Methods in
Molecular Biology (ed Leandro Peña) 203-213 (Humana Press, 2004).
45. Fukao, T., Yeung, E. & Bailey-Serres, J. The submergence tolerance gene SUB1A delays
leaf senescence under prolonged darkness through hormonal regulation in rice. Plant
Physiol. 160, 1795-1807 (2012).
46. Pfaffl, M. W., Horgan, G. W. & Dempfle, L. Relative expression software tool (REST)
for group-wise comparison and statistical analysis of relative expression results in real-
time PCR. Nucleic Acids Res. 30, e36 (2002).
47. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-
time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408
(2001).
48. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T)
method. Nat Protoc 3, 1101-1108 (2008).
34 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124
34
49. Mustroph, A. et al. Profiling translatomes of discrete cell populations resolves altered
cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 106, 18843-
18848 (2009).
50. Horan, K. et al. Annotating genes of known and unknown function by large-scale
coexpression analysis. Plant Physiol.147, 41-57 (2008).