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www.sciencemag.org/cgi/content/full/339/6120/704/DC1
Supplementary Materials for
Regulation of Flowering by Trehalose-6-Phosphate Signaling in Arabidopsis
thaliana
Vanessa Wahl,* Jathish Ponnu, Armin Schlereth, Stéphanie Arrivault, Tobias Langenecker,
Annika Franke, Regina Feil, John E. Lunn, Mark Stitt, Markus Schmid*
*To whom correspondence should be addressed. E-mail: vanessa.wahl@mpimp-golm.mpg.de (V.W.);
maschmid@tuebingen.mpg.de (M.S.)
Published 8 February 2013, Science 339, 704 (2013)
DOI: 10.1126/science.1230406
This PDF file includes:
Materials and Methods
Supplementary Text
Figs. S1 to S15
Tables S1 and S2
References
1
Materials and Methods
Plant material and growth conditions
All plants used in this work are of the Columbia (Col-0) accession. The tps1-2
GVG:TPS1 (referred to as ind-TPS1 #201 in the original publication) (18), ft-10, 35S:FT,
SUC2:FT, 35S:miR156, and 35:MIM156 lines have been described elsewhere
(Supplementary Text and Table S1). Genotypes were confirmed by PCR (Table S2). Plants
were grown in chambers at either 23°C, 65% relative humidity under a 2:1 mixture of Cool
White and Gro-Lux Wide Spectrum fluorescent lights, with a fluence rate of 125 to 175
μmol m-2
s-1
, or at 22°C, 65% relative humidity under Philips Fluors fluorescent lights
(F17T8/TL741), with a fluence rate of 130 to 160 µmol m-2
s-1
. Long-day (LD) and short-
day (SD) are defined as 16h light/8h dark and 8h light/16h dark, respectively.
Cloning
The generation of the amiRNA targeting TPS1 (amiR-TPS1) was done according to
the method described by Schwab and colleagues (31). Oligonucleotides that were used for
this purpose are listed in Table S2. The resulting EcoRI/BamHI fragment was introduced
into the Gateway® entry vector pJLBlue reverse to create pVW229 and recombined into a
pGREEN-II-based destination vector with a 35S promoter (pFK209)(10) using the
Gateway® LR clonase II Enzyme mix (Invitrogen, Carlsbad, CA) (pVW241). For
CLV3:TPS1 transgenic plants TPS1 was amplified from cDNA by a Pfu based PCR
reaction using gene specific oligonucleotides (Table S2), whereas an E. coli TPP, otsB SpeI
fragment was isolated from pDRIVE-otsB (15) to generate the CLV3:otsB transgenic line.
Both fragments were cloned into the Gateway® entry vector pJLBlue reverse to create
pVW099 and pVW406, respectively, and subsequently recombined into a destination
vector that harbors a CLV3 promoter (pFK202) resulting in pVW407 and pVW410.
RNA extraction, cDNA synthesis and qRT-PCR analysis
For the gene expression data reported in Fig. 1, Fig. 3, and figs. S3, S4, S6 and S12,
total RNA was isolated by phenol/chloroform extraction using TRIzol® Reagent (Life
Technologies, Darmstadt, Germany), followed by sodium acetate/glycogen-assisted ethanol
precipitation to improve RNA purity. First strand cDNA synthesis was carried out from
1µg of total RNA primed with oligo-(dT)18 using the RevertAid kit (Fermentas/Thermo
Scientific, St. Leon-Rot, Germany) in 20 µl reaction volume. The resulting single stranded
cDNA was diluted 5-fold and 4 µl were used as a template per reaction.
For the quantification of small RNAs (Fig. 3G), total RNA was reverse-transcribed
using a RevertAid kit (Fermentas/Thermo Scientific, St. Leon-Rot, Germany) following
the manufacturer’s protocol except that 1 µl of a 1:1 mixture of oligo-(dT)18 and the
miR156-specific stem-loop RT primer (G-30607;
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTC) was
used in the priming step. The stem-loop RT primer and the oligonucleotide pair used for
qRT-PCR analysis were designed according to Varkonyi-Gasic et al., 2007 (32).
qRT-PCR was performed using SYBR®
Green I (Life Technologies) reaction mixture
on an Opticon DNA engine (MJ Research/Biozym, Hessisch Oldendorf, Germany). The
thermal profile for the reaction was: 95°C for 5 min, 40 cycles of 95°C for 30 sec, 60°C for
30 sec and 72°C 20 sec, followed by 72°C for 7 min. Expression values were calculated by
2
the 2^- Cq method using TUBULIN (TUB2, AT5g62690) as a reference gene. Errors are
given as the lower (2^-( Cq-SD)) and upper (2^-( Cq+SD)) limit of the standard
deviation around the mean.
For the gene expression data reported in figs. S5 and S13, total RNA was isolated
using a modified phenol/chloroform extraction method according to Wan and Wilkins (33).
DNA digestion was performed on 9 µg RNA in a total reaction volume of 50 µl using 2
units of TURBO DNA-free™ (Ambion/Life Technologies, Darmstadt, Germany) according
to the manufacturer’s instructions. Reactions were incubated at 37°C for 45 min and
inactivated by addition of 0.2 volume of DNase inactivation reagent and incubation for 5
min at room temperature. The reactions were centrifuged at 10000 x g for 2 min, and the
supernatants were transferred to new tubes. Completeness of DNA digestion was confirmed
by qRT-PCR using primers for an intronic region of the At5g65080 gene. cDNA was
synthesized from the entire DNase digested RNA (50 µl) using SuperScript™III Reverse
Transcriptase (Invitrogen/Life Technologies, Darmstadt, Germany). The RNA was
denaturated at 65°C for 5 min, followed by incubation on ice for at least 1 min prior to
adding the remaining reagents. Reactions were incubated at 50°C for 45 min, then at 55°C
for 15 min, before being deactivated by heating at 70°C for 15 min. The relative cDNA
concentration and quality were determined using primers against POLYUBIQUITIN10
(UBI10, At4g05320) (34) and primers against the 3´ and 5´ regions of GAPDH
(At1g13440), respectively. Samples with Cq 5´GAPDH - Cq 3´GAPDH values > 2 were
excluded from further analyses. qRT-PCR was performed on two to four biological
replicates with four technical replicates each. TUBULIN (TUB2, At5g62690), SAND
family protein (SAND, At2g28390), POLYUBIQUITIN10 (UBI10, At4g05320) and
PROTEIN PHOSPHATASE 2A (PDF2, At1g13320) were used as reference genes
(reference gene index, RGI) (34). qRT-PCR was performed on an ABI Prism 7900 HT fast
real time PCR system (Applied Biosystems/Life Technologies, Darmstadt, Germany) in
384 well PCR plates with a total reaction volume of 5 µl (1 µl forward primer, 1 µl reverse
primer (0.5 µM, each), 0.5 µl cDNA and 2.5 µl Power SYBR® Green-PCR Master Mix
(Applied Biosystems/Life Technologies, Darmstadt, Germany) using the following
program: 50°C for 2 min, 95°C for 5 min, 40 cycles of 95°C for 15 sec and 60°C for 60 sec,
followed by 95°C for 15 sec, 60°C for 15 sec, 95°C for 15 sec. Steps 5, 6 and 7 were
introduced to record a dissociation or melting curve of each product in order to detect non-
specific amplification. Expression values were calculated by the 2^- Cq method using the
geometric mean of the Cq-values of the RGI as a reference. Deviation from the mean is
given as the lower (2^-( Cq-SD)) and upper (2^-( Cq+SD)) limit of the standard
deviation.
RNA in situ hybridization and histological staining
For RNA in situ hybridization plants were grown under LD or SD at 22°C. Meristem
samples were collected by removing all but the smallest leaves (vegetative and transition
meristem) or flowers (inflorescence meristem), fixed, dehydrated and embedded into wax
using an automated embedding system (ASP300S, Leica, Wetzlar, Germany). Sections of
8-10 µm thickness were prepared using a rotary microtome (Leica RM2265; Leica,
Wetzlar, Germany). Probes were synthesized with the DIG RNA Labeling Kit (Roche,
Mannheim, Germany) on PCR products of whole ORFs of the target genes, which had been
cloned into the pGEM®-T Easy vector (Promega, Madison, WI; Table S2 for oligo
3
sequences). For the otsB and the LFY probes the pDRIVE-otsB and a cloned LFY cDNA
were used as templates (15, 35). RNA in situ hybridizations were carried out as described
(36). Briefly, sections were incubated with Histoclear (Biozym Scientific GmbH, Hessisch
Oldendorf, Germany) and processed through an ethanol series. Proteinase K (Roche,
Mannheim, Germany) treated slides were hybridized at 55°C overnight. Slides were
blocked with 1% blocking reagent (Roche, Mannheim, Germany) in 1×TBS/0.1% Triton
X-100. Anti-DIG antibody (Roche, Mannheim, Germany) was used for immunological
detection and an NBT/BCIP stock solution (Roche, Mannheim, Germany) was diluted 1:50
in 10% polyvinyl alcohol (PVA) in TNM-50 for the colorimetric detection. For toluidine
blue staining reactions the sections were incubated with Histoclear and processed through
an ethanol series. The slides were then incubated in a 0.01% toluidine blue/sodium borate
solution for 1 min before they were briefly washed with water and incubated in 80%
ethanol for 2 min. Histological sections were imaged with an Olympus BX-51 microscope
equipped with a DC View III digital camera.
Microarray hybridization and analysis
RNA was extracted from dissected apices from Col-0 and tps1-2 GVG:TPS1 plants
grown for 20 days in SD and plants that had been induced to flower by shifting them to LD
for 5 days. All samples were collected at zeitgeber (ZT) 0-2 h to minimize differences in
expression of circadian or diurnally regulated genes. Apices were snap frozen in liquid
nitrogen and were stored at -80°C prior to total RNA extraction using the Plant RNeasy
Mini kit (Qiagen, Hilden, Germany). The MessageAmpTm
II-Biotin Enhanced, Single
Round aRNA Amplification Kit (Ambion/Life Technologies, Darmstadt, Germany) was
used in all subsequent steps to synthesize biotinylated cRNA according to the
manufacturer’s instructions. A total of 15 μg biotinylated cRNA was fragmented and the
quality was assessed by gel electrophoresis, using 2 μl of the fragmented cRNA.
Fragmented cRNA samples were hybridized to GeneChip Arabidopsis ATH1 arrays
(Affymetrix, Santa Clara, USA) according to the manufacturer's instructions. Washing and
staining of the GeneChip arrays were done following the EukGe-WS2_v4 protocol on an
Affymetrix GenChip Fluidics Station 450. The array chips were scanned using an
Affymetrix GeneChip Scanner 3000. Array data (.CEL files) were normalized using the
gcRMA package (37) implemented in R and differentially expressed transcripts were
identified by RankProducts analysis (38).
Metabolite measurements
For metabolite measurements whole rosettes of LD- or SD-grown plants were
harvested at the end of the night (EN) and/or at the end of the day (ED). Meristems were
manually excised using diamond knives (Scimed, Isselburg, Germany). T6P was extracted
with methanol/chloroform and measured using enzymatically authenticated standards and a
[2H]T6P internal standard to correct for ion suppression and matrix effects as previously
described (15). Sucrose was measured as described in (39) using an assay adapted to
microtitre plates (40).
For protein measurements the residual non-aqueous phase from the
methanol/chloroform extraction was evaporated to dryness in vacuo and the pellet
resuspended in 400 µl of 0.1 M NaOH. Protein content was assayed using the Bradford
method (41) with BSA as standard. At least two different volumes of extract (ranging
4
between 3-20 µl) were assayed, with duplicate aliquots for each volume, and the total
protein content in the extract was calculated by linear regression through zero.
Supplementary Text
Mutant alleles and transgenic lines used in this work
Two independent TPS1 mutant alleles (tps1-1 and tps1-2) have been isolated from a
transposon tagged mutant population by Eastmond and colleagues (16). Both mutants
displayed homozygous embryo lethality late during seed development, indicating that
functional TPS1 is required for embryogenesis (16). However, using a rescue construct that
facilitates dexamethasone-inducible overexpression of TPS1 from the constitutive
UBIQUITIN10 promoter, homozygous tps1-2 mutants could be recovered (18). Four
independent tps1-2 rescue lines (#201, #202, #211 and #213) that all carried single copies
of the inducible TPS1 (ind-TPS1) rescue construct were isolated and studied in more detail
(18). The homozygous mutant plants displayed retarded growth and did not initiate
flowering, unless dexamethasone was applied to induce TPS1 expression (18). In this work
we used progeny of the stable line ind-TPS1 #201, to which we refer as tps1-2 GVG:TPS1.
Later three weak tps1 alleles (tps1-11, tps1-12, and tps1-13) were isolated that were all late
flowering (17). Since also the 35S:amiR-TPS1 lines we have established in this work show
a similar phenotype we can safely conclude, that reduction of TPS1 function delays
flowering.
FT is a potent promoter of flowering (1) and loss of FT function has been shown to
delay flowering. ft-10 is a strong loss-of-function allele isolated from the GABI-Kat
collections of T-DNA insertion mutants (42, 43). Plants homozygous for the T-DNA
insertion in the FT gene flower late under LD (43). The late flowering phenotype of ft-10
could be rescued using a construct containing 5.7-kb of the FT upstream sequence fused to
the FT cDNA (44), indicating that loss of FT function was causing the delay in flowering.
The previously published transgenic lines used in this work (Table S1) originate from
large populations of independent T1 plants that all showed the same phenotype.
Furthermore, all these transgenes behave dominantly in genetic cross, making it unlikely
that the phenotypes observed are due to inadvertently knocking out genes by the T-DNA
insertion. The 35S:FT and SUC2:FT lines used in this work originate from populations of
29 independently obtained T1 plants each that were all early flowering (10). Early
flowering in response to FT misexpression from either the constitutive 35S promoter or the
phloem companion cell-specific SUC2 promoter has also been demonstrated by several
other laboratories (Table S1, Supporting references). Delayed flowering and changes in the
plastochron in response to miR156 overexpression have been described by two laboratories
independently of each other (45, 46). The 35S:miR156 line used in this work has been
selected from a population of more than 60 independently obtained T1 lines (45) that all
showed the typical miR156 overexpression phenotype. Conversely, early flowering can be
induced by overexpression of miR156 mimicry constructs from the 35S promoter (28, 29).
Examples of publications using these lines are listed in Table S1 (Supporting references).
5
Fig. S1.
Flowering time of transgenic lines established in this study. Histograms of flowering time
of (A) Col-0 wild-type and of independent (B) 35S:amiR-TPS1, (C) CLV3:TPS1, and (D)
CLV3:otsB T1 lines are shown. Flowering time is given as the number of leaves on the
main shoot. Total leaf number of individual plants used to generate the stable transgenic
lines analyzed in subsequent generations (Table 1) is indicated. Shaded areas indicate the
range of leaf number of Col-0.
6
Fig. S2.
Effect of 35:amiR-TPS1 on TPS1 mRNA levels in Arabidopsis thaliana rosettes.
Expression of TPS1 is strongly reduced in Col-0 (light grey) and 35S:amiR-TPS1 #6 (dark
grey) plants grown in LD. qRT-PCR was performed on whole rosettes harvested at ZT = 16
h (end of day, ED) 4, 6, 8, 10, 12, 14, 19 and 24 days after germination (DAG). Error bars,
upper and lower limit of the S.D. of four biological replicates with four technical repetitions
each.
7
Fig. S3.
T6P and sucrose content in the 35S:amiR-TPS1 line. (A and B) T6P and (C and D) sucrose
content in whole rosettes of Col-0 and 35S:amiR-TPS1 plants grown on MS medium
without sucrose under LD (16h light / 8h dark) conditions harvested 14 days after
germination at ZT = 4 h (4 h after lights on), normalized to (A and C) total protein
concentration or (B and D) fresh weight. Error bars, S.D.; significance was calculated using
Student’s t-test: ** p<0.01, *** p<0.001 based on four biological replicates.
8
Fig. S4.
Floral transition is delayed in the 35S:amiR-TPS1 lines. Emergence of flower primordia
was scored by RNA in situ hybridization with a LFY probe on longitudinal sections of wild-
type (Col-0) and in three independent 35S:amiR-TPS1 lines (#2, #5 and #6). Sections were
prepared from apices of plants grown under LD and harvested every second day from 4 to
14 days after germination (DAG) and every fifth day thereafter. Black stars indicate
meristem summit. Size bar, 100 µm.
9
Fig. S5.
Diurnal expression of GIGANTEA (GI) and TWIN SISTER OF FT (TSF) in tps1-2
GVG:TPS1. (A) Expression of GI and (B) TSF in 12- to 14-day-old LD grown Col-0
(closed circles) and tps1-2 GVG:TPS1 (open diamonds) rosettes. Expression was
determined by qRT-PCR using three biological replicates with three technical repetitions
for each sample. Shaded areas indicate dark periods. Error bars, upper and lower limit of
the S.D.
10
Fig. S6.
Photoperiod pathway genes in 35S:amiR-TPS1 #6 throughout development. Expression of
(A) GIGANTEA (GI), (B) CONSTANS (CO), (C) FLOWERING LOCUS T (FT), and (D)
TWIN SISTER OF FT (TSF) was determined by qRT-PCR in LD-grown Col-0 (light grey)
and 35S:amiR-TPS1 (dark grey). Whole rosettes were harvested at ZT = 16 h (end of day,
ED) 4, 6, 8, 10, 12, 14, 19 and 24 days after germination (DAG). Error bars indicate the
upper and lower limit of the S.D. of four biological replicates with four technical repetitions
each.
11
Fig. S7.
FLOWERING LOCUS T (FT) expression upon dexamethasone (DEX) application. 13-days-
old LD-grown Col-0 and tps1-2 GVG:TPS1 plants were treated with 1 µM dexamethasone
(+DEX) or mock treated (-DEX) and FT expression was measured (A) 24 h and (B) 48 h
after induction in rosettes leaves harvested at ZT=16 h (end of day, ED). Error bars indicate
the upper and lower limit of S.D. of three biological replicates with three technical
repetitions each.
12
Fig. S8.
TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) expression at the shoot apex.
Toluidine blue staining of shoot apices (A-D). RNA in situ hybridization of LFY (E-H) and
TPS1 (I-L). Longitudinal sections were prepared from tissue of plants grown under SD
conditions for 30 days and then transferred to LD to synchronously induce flowering.
Samples were harvested 0 (A, E, I), 3 (B, F, J), 5 (C, G, K) and 7 (D, H, L) days after the
shift. Black stars indicate meristem summit; scale bar, 100 µm.
13
Fig. S9.
CLV3:TPS1 grown under SD conditions. (A) Wild-type (Col-0). (B) CLV3:TPS1 #7. (C)
CLV3:TPS1 #15. Pictures were taken 50 days after germination. Scale bar, 1 cm.
14
Fig. S10.
Mean expression of photoperiod pathway genes at the shoot meristem. Total RNA was
isolated from manually dissected shoot apical meristems of 21-day-old SD-grown Col-0
(light grey) and tps1-2 GVG:TPS1 (dark grey) plants, converted into biotinlyated cRNA
and hybridized to Affymetrix Arabidopsis Genome ATH1 arrays. The transcripts for two
important photoperiod pathway genes, FT and SOC1, were not detectable by microarray in
meristems of SD-grown plants. Minimum and maximum values obtained by hybridization
of two biological replicates, from which the mean expression was calculated, are indicated.
15
Fig. S11.
Mean expression of flowering regulators: ambient temperature and vernalization. RNA was
isolated from manually dissected meristems of 21-day-old SD-grown Col-0 (light grey) and
tps1-2 GVG:TPS1 (dark grey) plants, converted into biotinylated cRNA and hybridized to
Affymetrix Arabidopsis Genome ATH1 arrays. The maximum and minimum values of two
biological replicates, from which the mean expression was calculated, are indicated.
16
Fig. S12.
Mean expression of gibberellic acid biosynthesis and signaling genes at the shoot apical
meristem of 21-day-old SD-grown Col-0 (light grey) and tps1-2 GVG:TPS1 (dark grey)
plants. Expression was determined by hybridization to Affymetrix Arabidopsis Genome
ATH1 arrays. The minimum and maximum values obtained by hybridization biological
duplicate, from which the mean expression was calculated, are indicated.
17
Fig. S13.
Expression of integrator genes in tps1-2 GVG:TPS1. Expression of (A) SOC1 and (B) FUL
was determined by qRT-PCR in RNA extracted from 10-day-old whole rosettes and leaves
of 20- to 40-day-old LD-grown Col-0 (light grey) and tps1-2 GVG:TPS1 (dark grey) plants
(DAG, days after germination). Note that flowering in wild-type Col-0 occurs at about 10
days in LD (fig. S4). Error bars, upper and lower limit of the S.D. of the mean of three
biological replicates with two technical repetitions each.
18
Fig. S14.
Expression of integrator genes in 35S:amiR-TPS1. Expression of (A) SOC1 and (B) FUL
was determined by qRT-PCR in LD-grown Col-0 (light grey) and 35S:amiR-TPS1 (dark
grey). Whole rosettes were harvested at ED at 4, 6, 8, 10, 12, 14, 19 and 24 days after
germination (DAG). Note that flowering in wild-type Col-0 occurs at about 10 days in LD
(fig. S4). Error bars indicate the upper and lower limit of the S.D. of four biological
replicates with four technical repetitions each.
19
Fig. S15.
A minimal model of the dual role of TPS1/T6P in the regulation of flowering time.
Signaling from the T6P pathway is absolutely essential for expression of FT and TSF in the
phloem companion cells even under otherwise inductive photoperiod. In addition,
TPS1/T6P signaling regulates the expression of SPL genes at the shoot apical meristem
both directly and via miR156. Together, these events ensure that plants make the transition
to flowering only after day length exceeds a certain threshold and carbohydrates are
available to support the energy-demanding processes of flowering and seed production.
Solid lines, direct interactions; dashed line, indirect interactions; transport of FT protein
(florigen) and sucrose from leaves to the shoot apical meristem is indicated.
20
Table S1.
Mutants and transgenic lines used in this study.
Line Source Supporting References Comment
tps1-2 GVG:TPS1 (18) ind-TPS1 #201 (18)
ft-10 (42, 43) (9, 10, 44, 47-49)
35S:FT (10) (4, 5, 48, 49)
SUC2:FT (10) (8, 9, 47, 49)
35S:miR156 (45) (25, 27, 46, 48, 49)
35S:MIM156 (28, 29) (25, 27, 49)
35S:amiR-TPS1 this study
CLV3:TPS1 this study
CLV3:otsB this study
21
Table S2.
Oligonucleotides used in this study.
Gene (AGI) Oligo Sequence (5’ -> 3’) Comment
Oligonucleotides used for cloning
amiR-TPS1
G-6761
G-6762
G-6763
G-6764
GATCTTCGGGGACCTCAAGTGGTTCTCTCTTTTGTATTCC
GAACCACTTGAGGTCCCCGAAGATCAAAGAGAATCAATGA
GAACAACTTGAGGTCGCCGAAGTTCACAGGTCGTGATATG
GAACTTCGGCGACCTCAAGTTGTTCTACATATATATTCCT
TPS1
At1g78580
G-3908
G-3909
GCGGGTACCATGCCTGGAAATAAGTACAACTG
CGCTCTAGATTAAGGTGAGGAAGTGGTGTCAG
Oligonucleotides used for genotyping
TPS1
At1g78580
G-22756
G-22758
GACACTTGGTTTCTTGATATGTCCTG
GCTGTCTTGGATACTGAACCAGT
tps1-2
At1g78580
G-19968
G-22758
GAGCGTCGGTCCCCACACTTCTATAC
GCTGTCTTGGATACTGAACCAGT
35S:MIM156 G-0474
G-2225
AGAACACGGGGGACGAGCT
CGCATATCTCATTAAAGCAGG
FT
At1g65480
P-0230
P-0231
AGGGTTGCTAGGACTTGGAACA
ATATTGATGAATCTCTGTTGTGG
ft-10
At1g65480
P-0230
G-2081
AGGGTTGCTAGGACTTGGAACA
CCCATTTGGACGTGAATGTAGACAC
Oligonucleotides used for qRT-PCR (Figs. 1, 3; Figs. S5, S7, S13)
TUB2
AT5g62690
N-0078
N-0079
GAGCCTTACAACGCTACTCTGTCTGTC
CACCAGACATAGTAGCAGAAATCAAG
165 bp
E = 2.014
GI
At1g22770
G-30970
G-30971
AGCAGTGGTCGACGGTTTATC
ATGGGTATGGAGCTTTGGTTC
141 bp
E = 1.936
CO
At5g15840
G-30962
G-30963
CACTACAACGACAATGGTTCC
GGTCAGGTTGTTGCTCTACTG
142 bp
E = 1.988
FT
At1G65480
G-30966
G-30967
CCCTGCTACAACTGGAACAAC
CACCCTGGTGCATACACTG
135 bp
E = 1.980
TSF
At4g20370
G-33510
G-33511
TGCCACCACTGGAAATGCC
CGTTTGTCTTCCGAGTTGCC
113 bp
E = 2.056
SPL3
At2g33810
G-30976
G-30977
CTCATGTTCGGATCTCTGGTC
TTTCCGCCTTCTCTCGTTGTG
134 bp
E = 1.917
SPL4
At1g53160
G-30978
G-30979
CTCTCAGGACTTAACCAACGC
CAGAGCTCTTCCTTCTTCGC
130 bp
E = 2.120
SPL5
At3g15270
G-31000
G-31001
AAGGCATCTGCTGCGACTGTTG
TCCTCCTCCTCTCATTGTGTCC
139 bp
E = 2.027
miR156 G-30606
G-30608 GTGCAGGGTCCGAGGT
GCGGCGGTGACAGAAGAGAGT 60 bp
E = 1.917
SOC1 At2g45660
G-30998
G-30999
AAACGAGAAGCTCTCTGAAAAG
AAGAACAAGGTAACCCAATGAAC
145 bp
E = 2.035
FUL
At5g60910
G-00654
G-00655
TTGCAAGATCACAACAATTCGCTTCTC
GAGAGTTTGGTTCCGTCAACGACGATG
204 bp
E = 1.926
Oligonucleotides used for qRT-PCR (Figs. S2, S6, S14)
UBI
At4g05320
P-0348
P-0349
CACACTCCACTTGGTCTTGCGT
TGGTCTTTCCGGTGAGAGTCTTCA
71 bp
E = 1.982
22
TUB2
At5g62690
P-0344
P-0345
GAGCCTTACAACGCTACTCTGTCTGTC
ACACCAGACATAGTAGCAGAAATCAAG
167 bp
E = 1.965
SAND
At2g28390
P-0346
P-0347
AACTCTATGCAGCATTTGATCCACT
TGATTGCATATCTTTATCGCCATC
61 bp
E = 1.976
PDF2
At1g13320
P-0350
P-0351
TAACGTGGCCAAAATGATGC
GTTCTCCACAACCGCTTGGT
61 bp
E = 1.982
TPS1
At1g78580
P-0596
P-0597
GAAACTCAAGACGTCCTTCACCAG
TCTAGCATTGGTGCGAGTACGAC
107 bp
E = 1.989
GI
At1g22770
P-0422
P-0423
AACCAAAGCTCCATACCCATCA
GCGTTGAAGAATCGATAGGACG
92 bp
E = 1.976
CO
At5g15840
P-370
P-371
AACAGCTTCACACCCAAGAACG
GGTCAGGTTGTTGCTCTACTGTCC
53 bp
E = 1.968
FT
At1G65480
P-414
P-415
TGGAACAACCTTTGGCAATGAG
CGACACGATGAATTCCTGCAG
71 bp
E = 1.954
TSF
At4g20370
P-570
P-571
GAAATGCCTTTGGCAATGAGGT
CCGGAACAATACCAACACAATACG
86 bp
E = 1.958
SOC1
At2g45660
P-532
P-533
TTGAGCAGCTCAAGCAAAAGGA
TCCCCACTTTTCAGAGAGCTTCTC
68 bp
E = 1.975
FUL
At5g60910
P-416
P-417
GTAACCTCCTCCAGAGATGGCTTT
ACGTAACATCCAAGCCGGAAG
105 bp
E = 1.970
E, primer pair efficiency.
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