Development 143: doi:10.1242/dev.129833: Supplementary information
SUPPLEMENTARY MATERIALS AND METHODS
Growth conditions and plant materials
Arabidopsis plants were grown under continuous light at 23 °C on soil. Seeds for cna–2
phb–13 phv–11 er–2 (Prigge et al., 2005) and gWUS-GFP3 (Tucker et al., 2008) were
kind gifts from Steven Clark and Shinobu Takada, respectively. TSC:GFP seeds (Müller
and Sheen 2008) were obtained from the Nottingham Arabidopsis Stock Centre. For
making bel1–6 cna–2 phb–13 phv–11 er–2, female flowers of cna–2 phb–13 phv–11 er–
2 were pollinated with bel1-6 pollen (Robinson-Beers et al., 1992). All mutants used for
this study were homozygous for er–2.
Genotyping of bel1–6
In the bel1–6 allele (Robinson-Beers et al., 1992), a C-to-A transversion at poition +707
from the start codon caused a change of glutamine into translational stop. A 965
BEL1F2 and BEL1R5. The BEL1 fragment is not cut by AflII (New England Biolabs),
while the bel1–6 fragment is cut into 710 and 225 bp fragments.
Genotyping of er–2
The "A" residue at position +4923 from the start codon is deleted in er–2. The resulting
amino acid sequence is 168-residues shorter than the wild-type ER. A pair of PCR
Development • Supplementary information
bp-fragment containing this mutation site was amplified by PCR with primer pair
Development 143: doi:10.1242/dev.129833: Supplementary information
primers (er2_dCAPS_F and er2_dCAPS_R) was designed in the flanking region of this
deletion site to amplify 314 bp fragments of er–2, er or ER. The PCR products were
digested with Tsp45I (New England Biolabs) to yield 200 and 114 bp fragments in er or
ER, but the restriction site is absent in er–2.
Genotyping of cna–2, phb–13 and phv–11
To make bel1–6 cna–2 phb–13 phv–11 er–2 or cna–2 phb–13 phv–11 er–2 carrying a
marker construct, mutant or WT alleles of HD-ZIPIII genes were genotyped following
Prigge et al. (2005).
Construction of pCNA: amiRWUS
amiRWUS was generated following the protocol of Web MicroRNA Designer 3
(http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). To make amiRWUS fragment, three
containing miR319a sequence as PCR template: a) miR*-wus-a (IV), miR-wus-a (II)
and miR*-wus-s (III); b) miR-wus-s (I). Overlap PCR was performed to make
amiRWUS using the products of these reactions as precursors. Arms for seamless
assembly were added to amiRWUS by PCR with primer pair of amiRWUS_12FW and
amiRWUS_12RV.
Development • Supplementary information
precursor fragments were amplified with the following pairs of primers using pRS300
Development 143: doi:10.1242/dev.129833: Supplementary information
pCNA (-4046 to -319) was amplified by pCNA_12FW and pCNA_12RV from
Col-0 genomic DNA. Transcriptional terminator of nopaline synthase (NOS-ter) was
amplifyied using NOSter_12FW and NOSter_12RV on pBI121. Linearized pMLBarT
with appropriate arms for seamless assembly was prepared by PCR with a primer pair
of pMLBarT_12FW and pMLBarT_12RV (Fig. S1B).
pCNA, amiRWUS and NOS-ter fragments were assembled and cloned into
pMLBarT using GeneArt Seamless PLUS Cloning and Assembly Kit (Life
Technologies). bel1–6/+ cna–2 phb–13 phv–11 er–2 plants were transformed with the
construct by floral dip method (Clough and Bent, 1998). Six independent lines were
obtained by BASTA selection on T0 population.
Expression of amiRWUS was confirmed by RT-PCR with a primer pair of
amiRWUS_12FW and amiRWUS_12RV (Fig. S1E). The PCR amplicons were directly
Constructions of pWUS:CNA-δmiRNA and pWUS:CNA
pWUS (-4001 to 4) was amplified using pWUS_26FW and pWUS_26RV so as to
include the region between -3308 and -2114 which is essential for WUS expression in
ovules (Bäurle and Laux, 2005). CNA-δmiRNA is designed to have T-to-A transition at
1284 and G-to-A transition at 1287 (from the start codon of mRNA, respectively)
following Emery et al. (2003). To make CNA-δmiRNA fragments, three precursor
Development • Supplementary information
sequenced to check if the amplicons represented amiRWUS (Fig. S1F).
Development 143: doi:10.1242/dev.129833: Supplementary information
fragments were amplified by using following pairs of primers: dCNA_A_F1 and
dCNA_A_R1, dCNA_B_F1 and dCNA_B_R1, and dCNA_C_F1 and dCNA_C_R1.
These fragments were connected by overlap PCR with dCNA_A_F1 and dCNA_C_R1.
CNA or CNA-δmiRNA, as well as pWUS and Nos-ter fragments, were cloned into
pMLBarT using GeneArt Seamless PLUS Cloning and Assembly Kit.
GUS staining in pWUS>>uidA plants
pWUS:LhG4 driver line was crossed with OP:uidA responder line to make
pWUS>>uidA plants. Seeds for these lines were kindly provided by John Bowman.
GUS staining was performed as previously described (Meister et al., 2002).
qRT-PCR analysis
Total RNAs were extracted from gynoecia containing stage 2 ovules. Contaminating
Scientific). qRT-PCR analyses were performed using One Step SYBR PrimeScript
RT-PCR Kit (TAKARA) on an ABI PRISM 7900HT Real-Time PCR System (Applied
Bioystems). Primers for WUS and Protein Phosphatae 2A subunit (AtPP2A;
At1g13320) were designed by Primer 3 (http://bioinfo.ut.ee/primer3/), while those for
GFP were followed Han et al. (2014). WUS and GFP expression levels were
normalized to those of AtPP2A (Czechowski et al., 2005).
Development • Supplementary information
DNA was digested with DNase by using TURBO DNA-free Kit (Thermo Fisher
Development 143: doi:10.1242/dev.129833: Supplementary information
References
Bäurle, I. and Laux, T. (2005). Regulation of WUSCHEL transcription in the stem cell
niche of the Arabidopsis shoot meristem. Plant Cell 17, 2271–2280.
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., Scheible, W-R. (2005).
Genome-wide identification and testing of superior reference genes for transcript
normalization in Arabidopsis. Plant Physiol. 139, 5–17.
Han, Y., Zhang, C., Yang, H. and Jiao, Y. (2014). Cytokinin pathway mediates
APETALA1 function in the establishment of determinate floral meristems in Arabidopsis.
Proc. Natl. Acad. Sci. USA 111, 6840–6845.
Meister, R.J., Kotow, L.M. and Gasser, C.S. (2002). SUPERMAN attenuates positive
INNER NO OUTER autoregulation to maintain polar development of Arabidopsis ovule
Development • Supplementary information
outer integuments. Development 129, 4281–4289.
Fi
g.S1.(
A)qRTPCRanal
y
s
esonWUS i
nWT(
c
ol
)andc
naphbphv.(
B)Cons
t
r
uc
t
us
edf
ormak
i
ngc
naphbphvpCNA:
ami
RWUSpl
ant
s
.(
C,D)GFPs
i
gnal
si
n
pCNA:
GFPpl
ant
s
.pCNApr
omot
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pr
es
s
i
ont
hr
oughoutt
hec
hal
az
al
ar
eai
nc
l
udi
ng
t
woi
nt
egument
s
.Sc
al
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s
,100μm.(
C)ov
ul
es
,(
D)s
hootapex
.(
E)Ex
pr
es
s
i
onof
ami
RWUS(
422bp)i
nc
naphbphvpCNA:
ami
RWUSpl
ant
sanal
y
s
edbyRTPCR.
(
F)
Nuc
l
eot
i
des
equenc
esof422bpr
agment
si
nFi
g.S1E.Onl
yt
hes
equenc
esar
oundt
he
t
ar
get
r
ec
ogni
t
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ons
i
t
e(
box
ed)ar
ei
ndi
c
at
ed.A422bpbandex
c
i
s
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r
om eac
hl
anes
wasdi
r
ec
t
l
yus
edf
ort
hes
equenc
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ng.Not
et
hatmi
R319as
equenc
esar
eal
s
odet
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ed,
whi
c
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sus
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boneofami
RWUS,bec
aus
epr
i
mer
sar
edes
i
gnedi
nt
he
bac
k
bonepar
t
.
Development • Supplementary information
Development 143: doi:10.1242/dev.129833: Supplementary information
Fi
g.S2.Expr
essi
onofWUSi
nWT,cnaphbphvandbel
1–6ovul
es.(
A,B)
GUSst
ai
ni
ngi
npWUS>>ui
dAWTpl
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satst
ages2–I
I
I(
A)and2–I
V(
B)
.(
CF)
WTovul
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t
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ansgeneatst
age2I
I
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sual
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f
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r
ansgeneat
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sual
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obeonbel
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,J)andmer
ged(
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nner
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nt
egument
;n,nucel
l
us.Scal
ebar
s,25µm i
nAH;50µm i
nI
K.
Development • Supplementary information
Development 143: doi:10.1242/dev.129833: Supplementary information
Development 143: doi:10.1242/dev.129833: Supplementary information
Table S1. Primers
Name
Sequence
Description/Purpose
BEL1F2
BEL1R5
er2_dCAPS_F
er2_dCAPS_R
miR-wus-s (I)
CAAGCCAAGGTCTTTCCCTTTC
ATAAATATGGAGGACCACTA
CTGTAGTCCAAGGATCATTCACAG
AGCTGAGAGAGATAGGCAAGAAAG
gaTAATTCCCGTTATTGAAGCTTtctctcttttgt
attcc
gaAAGCTTCAATAACGGGAATTAtcaaaga
gaatcaatga
gaAAACTTCAATAACCGGAATTTtcacaggtc
gtgatatg
gaAAATTCCGGTTATTGAAGTTTtctacatata
tattcct
GAGGAGTACAAACACACGCTCGG
GAACGATCCATGGCGATGCCTTAA
ACCTGCAGATTGATGCCCTGAGATT
TGTGTTTGTACTCCTCAGCAAAACTC
TCGCCATGGATCGTTCAAACATTTGG
GCGGCCGCCCCGATCTAGTAACATAGA
T
ACCTGCAGATTGATGCCCTGAGATT
TGTGTTTGTACTCCTCAGCAAAACTC
ACCTGCAGAAGTACATTTTAAATCTAAC
CTTAA
ATTGCCATGTGTGTTTGATTCGACT
Genotyping
Genotyping
Genotyping
Genotyping
Making pCNA:amiRWUS
miR*-wus-s (III)
miR*-wus-a (IV)
amiRWUS_12FW
amiRWUS_12RV
pCNA_12FW
pCNA_12RV
NOSter_12FW
NOSter_12RV
pMLBarT_12FW
pMLBarT_12RV
pWUS_26FW
pWUS_26Rv
dCNA_A_F1
dCNA_A_R1
dCNA_B_F1
dCNA_B_R1
dCNA_C_F1
dCNA_C_R1
SR_AtPP2AF1
SR_AtPP2AR1
SR_WUSF1
SR_WUSR1
ATGGCAATGTCTTGCAAGGATGG
AATGGAATCTGGTCCAGGCTTCATTCCA
G
CTGGAATGAAGCCTGGACCAGATTCCAT
T
GGTTCCACCATTGGCAGTTGGCAACAC
GTGTTGCCAACTGCCAATGGTGGAACC
ATCTCACACAAAGGACCAATTGATG
ACGAATTTCCTGATGTACGCTT
TACTCTCCAGTGCCTGTCTTCA
GTCAACGTTAAACTTAACCAAGAC
CATTAACAACACCACATTCAGTACC
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pCNA:amiRWUS
Making pWUS:CNA-δmiRNA
and pWUS:CNA
Making pWUS:CNA-δmiRNA
and pWUS:CNA
Making pWUS:CNA-δmiRNA
Making pWUS:CNA-δmiRNA
Making pWUS:CNA-δmiRNA
Making pWUS:CNA-δmiRNA
Making pWUS:CNA-δmiRNA
Making pWUS:CNA-δmiRNA
qRT-PCR
qRT-PCR
qRT-PCR
qRT-PCR
Development • Supplementary information
miR-wus-a (II)