Supporting Information
Figures S1-S8, Tables S1-S5, and Methods S1
Fig. S1 Scanning electron microscopy of the adaxial and abaxial surfaces of nal2/3 leaves.
(a-d) Adaxial (a, c) and abaxial (b, d) surfaces of the wild-type (WT) and nal2/3 leaves in the
middle region of the fully expanded third leaf blade. BC, bulliform cell region; LV, large vein;
SV, small vein. Bars: 100 m.
1
Fig. S2 The shoot apex region of the nal2/3 mutants displayed reduced size of the shoot
apical meristem (SAM) and defective marginal structure in leaf primordia.
Sequential transverse sections through the shoot apex of 1-month-old seedlings were
examined. Bars: 100 m.
(a-e) 35 m (a), 25 m (b), 15 m (c) above the section (e) containing SAM in wild-type
(WT). (d) Magnified transverse sections in (c).
(f-j) 35 m (f), 25 m (g), 15 m (h) above the section (j) containing SAM in nal2/3. (i)
Magnified transverse sections in (h).
L4, fourth leaf; L5, fifth leaf; L6, sixth leaf; L7, seventh leaf; S, shoot apical meristem;
asterisks, the margins of overlapped leaves; arrowheads, the margin of the non-overlapped
leaves; dashed lines, the boundaries of the leaves.
2
Fig. S3 nal2/3 displayed normal morphology in pistil and stamen development.
Pistil and stamen developments were observed in wild-type (WT) and nal2/3 plants when
spikelets opened after heading.
L, lemma; Lo, lodicule; P, palea; Pi, pistil; S, stamen; Gl, glume. Bar: 2 mm.
3
Fig. S4 nal2/3 showed a significantly increased number of tillers at both active and maximum
tillering stages.
(a, b) The number of tillers of wild-type (WT) (a) and nal2/3 (b) plants 30 days after
germination. Asterisks represent tillers. Bars: 1 cm.
(c) Tiller number of WT and nal2/3 at the active tillering stage (30-days after germination)
and maximum tillering stage (90-days after germination). Values are shown as mean 
standard deviation obtained from 20 plants. Asterisks indicate statistically significant
differences compared with WT as determined by Student’s t-test (***, P < 0.001).
4
Fig. S5 Characterization of nal2 and nal3 mutant proteins and OsWOX3A homologs.
(a) Alignment of the mutated amino acids in nal2 and nal3. Black boxes show the changed
amino acids in nal2 or nal3.
(b) ClustalW amino acid sequence alignments of OsWOX3A with its homologs in maize and
Arabidopsis. NS1 and NS2 are narrow sheath1 and narrow sheath2 in maize, and PRS is
PRESSED FLOWER in Arabidopsis. Double and single lines represent a WOX3
homeodomain at the N-terminus and a WUSCHEL-box domain at the C-terminus,
respectively. Note that OsWOX3A has approximately 40 to 60 fewer amino acids than NS
and PRS, mainly in the middle region.
5
Fig. S6 Phylogenic tree of the WOX families.
Phylogenic tree was constructed by the neighbor-joining method, using the MEGA 5.1
software. Numbers at the branching points represent bootstrap values from 1,000 replicates.
GenBank accession numbers are shown in Table S5. Abbreviations are Arabidopsis thaliana
(At), Brachypodium distachyon (Bd), Oryza sativa (Os), and Zea mays (Zm).
6
Fig. S7 Complementation of nal2/3 by OsWOX3A.
(a) Leaf phenotype of transgenic lines containing the genomic OsWOX3A fragment. The
genomic OsWOX3A (2141-bp 5’-upstream, 612-bp ORF and 1078 bp 3’-downstream) was
transformed into nal2/3 calli, and 12 transgenic plants were obtained. All transgenic lines
rescued several defects in nal2/3. WT, wild-type; MR, midrib; LV, large vein; SV, small vein.
(b) Verification of true transformants. Genomic DNAs were extracted, and a 443-bp PCR
product for the OsWOX3A inside the T-DNA border regions of pC1300intC was amplified. M,
DNA marker; W, genomic DNA of wild-type as negative control; E, empty plasmid of
pC1300intC as negative control; V, recombinant plasmid of pC1300intC-OsWOX3A as
positive control; 1-12, genomic DNA of twelve transgenic plants in nal2/3 background.
Bar: (a) 1 mm.
7
Fig. S8 Endogenous free-IAA concentrations in the whole plants of 2-week-old wild-type
(WT) and nal2/3 using IAA ELISA analysis. Mean and SD values were obtained from three
replicates. n.s., no significant difference.
8
Table S1 Primers used in this study
A. Physical mapping markers
Locus
Contig
Location
Forward primer (5′→3′)
Reverse primer (5′→3′)
STS-1
AL513003
10.5 kb
TGCAGCTGATTCCGTTCCTC
GCTCTCCAATGTGGCTGCATG
STS-2
BX000505
113 kb
CCGCGATCGGATCTACCTATC
TCAGCACTGACGAGTCGAGC
STS-3
BX000500
13 kb
TGATGYGGTATGTCACACGGAC
CACATGGACCGATGGACGTG
STS-4
BX000494
61.4 kb
TTTGTGTGTCGACTGATCGAAC
CACAACGACATCCCTTAATCCC
STS-5
BX000494
28 kb
TGCTACGGTTGAACTGAAATTG
TTTATGCACCGCCTATCTAGG
STS-6
BX000496
101 kb
TGCTTATCCACATTGTCACATC
ATGGGACATACGGTTGCTG
RM19
AL928755
122.9kb
CAAAAACAGAGCAGATGAC
CTCAAGATGGACGCCAAGA
RM167
AC135794
130.1kb
GATCCAGCGTGAGGAACACGT
AGTCCGACCACAAGGTGCGTTGTC
RM229
AC150702
136.8kb
CACTCACACGAACGACTGAC
CGCAGGTTCTTGTGAAATGT
RM247
AL713906
90.7kb
TAGTGCCGATCGATGTAACG
CATATGGTTTTGACAAAGCG
RM277
AL845344
138.5kb
CGGTCAAATCATCACCTGAC
CAAGGCTTGCAAGGGAAG
RM286
BX000497
164.1kb
GGCTTCATCTTTGGCGAC
CCGGATTCACGAGATAAACTC
B. Gene-specific primers used for qRT-PCR
Gene
Forward primer (5'→3')
Reverse primer (5'→3')
ARF1
GCACTCCTTCTGCAAGATCC
GGCCCCTGTAGATGTGCTTA
ARF2
GTCCGTTCTGGCTCTCAATC
GGGAAGGCTGTGGAGATACA
ARF3
TCTCGTCCCTCTACGTGCTT
CCGATTACAACGGGAGCTTA
ARF4
TTCCTCCGTTTCAAGGAATG
AGCTGTGGTACCCCTGTTTG
ARF5
AAGGATGGGAAAGCTTGGTT
GAAATCCCATCTGGCTTCAA
YABBY1
TGGTGAATGTGCCAAACAAT
GCTTGGTGAAAAGGAGCAAG
YABBY2
AATTTTCGCGGTCAGTGTTC
AGTTTTCGCGGAAGCTCATA
YABBY3
ATCAAGGACGAAATCCAACG
GGCATCAGTCCAAAGTGGAT
YABBY4
ATCACATCAAAGGGGACCAA
CGAGTACGCAATGGCCTTAT
YABBY5
GAGCCTAATGACCGAACAGG
CTCTGCCGCTTCTCTGAAGT
YABBY6
TCTGTTCATCACTGGCTTCG
CGTGTTGCAGAAGTTGCAGT
YABBY7
GGAGACGATAGGGAGGGAAG
TGCTTGTTTGTGGGTGATGT
YUCCA1
ACCTCATCCTCGGTAACACG
TCTCCTTCACTGCTCCCACT
YUCCA2
GCCTGAAGGAGAAGGGAATC
AGCTCTCCAGGTAAGCCACA
YUCCA3
GTTGTGCTGCCAGAGATTGA
CCCTGGGCAAGATGTGTACT
YUCCA4
GGATGGCAGCACTGAAGAAT
AGAGACCACGCCTTGAGAAA
YUCCA5
ACCTCCTACGACGCCGCCATGATC
CTCCCAACACAGCGACGACAGAAC
YUCCA6
GTCATACTGGCCACCGGATA
CAGCATCTGAGGAGACACCA
YUCCA7
AAGGACGGGTACCCAAAGAC
TCTTCTTGGTTGGCTTCGTC
YUCCA8/NAL7
GTGGTGTGCATGTTCTACCG
TGGGCCTCTTGAGACCATAC
YUCCA9
AAGAGTGATGACGGGCTGAT
ATTGTTGTGGGTAGGCTTGC
NAL1
AGCTGACGGTGCATTTATCC
CCATCACAGTCCCAGTTGTG
NRL1
AAGAGGGACTTCCTCAAGAACAAG
TCGTACTCGCGCTTCACCTT
9
PIN1
AGTCAAGGGGAGGACAGGAT
TCCGTTTTACAAGGGTCAGC
PIN1b
TTCTGCACATTGCCATTGTT
AATGTGATGGGGAGAGCAAT
PIN1c
ATCGTGCAGGCAGCGTTGCC
ATGTAGTACACCAGCGTGAT
PIN2
TGTCAGATGCAGGGCTAGGAA
TGCCACAAGAAATGATCTTTGG
PIN3
TTGTTCAGGCGGCTCTACCA
AAGGAAATTGCTTACGCTGT
Ub
GTCTGATCTTCGCTGGCAAGCAGC
GCATACTGCTGTCCCACAGGAAACTG
C. Primers for transformation verification of OsWOX3A genomic DNA in nal2/3 mutant
Product
Primer (5'→3')
TC1
ACGACAGGTTTCCCGACTGG
TC2
CCAATGCACTGAAGGATGTTG
D. Primers for transcriptional activation analysis in yeast GAL4 system
Product
Primer (5'→3')
Y-F
GAATTCATGCCTCAGACCCCTTCG
Y-R333
GGATCCGTGGTGTAGCTGCATCAC
Y-F316
GAATTCGTGATGCAGCTACACCAC
Y-R
GGATCCTTAATTGGTGGAGGTGGAG
E. Primers for OsWOX3 promoter genomic DNA for OsWOX3Apro:GUS construction
Product
Primer (5'→3')
WOX3Apro:GUS-F
AGATCCACTCATCAGTACCTTTG
WOX3Apro:GUS-R
TCTCTAAAGTCTTCGATTCAGAAC
F. Primers for OsWOX3 cDNA for OsWOX3A-GFP construction
Product
Primer (5'→3')
ORF1
TCTAGAATGCCTCAGACCCCTTCGAC
ORF2
GGATCCATTGGTGGAGGTGGAGC
10
Table S2 Morphological characteristics of nal2/3
Parameter
Wild-type
nal2/3
Leaf sheath length (cm)
25.7  4.5 (10)
23.1  2.3 (10) n.s.
Leaf blade length (cm)
40.4  1.8 (10)
38.7  3.6 (10) n.s.
Leaf blade width (mm)
10.1  1.5 (10)
6.1  0.3 (10) ***
Numbers of LVs
5.7  0.4 (10)
4.6  0.8 (10) **
Number of SVs between two adjacent LVs
4.7  0.3 (10)
2.7  0.6 (10) ***
Major diameter of main stem (mm)
5.65  0.21 (10)
3.80  0.28 (10) ***
Minor diameter of main stem (mm)
3.15  0.07 (10)
2.15  0.07 (10) ***
Spikelet length (mm)
7.4  0.1 (20)
7.5  0.4 (20) ***
Spikelet width (mm)
3.5  0.1 (20)
3.5  0.3 (20) ***
Spikelet thickness (mm)
2.3  0.1 (20)
2.2  0.3 (20) ***
Extended width of lemma (mm)a
3.90  0.29 (4)
2.25  0.13 (4) ***
2.44  0.09 (4)
1.24  0.09 (4) ***
0.65  0.06 (8)
0.44  0.07 (8) ***
Grain length (mm)
5.3  0.1 (20)
5.8  0.3 (20) ***
Grain width (mm)
3.0  0.1 (20)
2.2  0.4 (20) **
Grain thickness (mm)
2.1  0.0 (20)
1.7  0.1 (20) ***
Lateral root length (mm) b
8.89  2.69 (10)
8.14  3.54 (10) n.s.
Extended width of palea (mm) a
Length between veins of lemma (mm)
a
Means and standard deviations were obtained from the longest third leaf blades at heading
stage grown in natural conditions and fully developed spikelets and grains after harvest
(sampling numbers in parentheses). Leaf widths and the number of large veins and small
veins were measured in the broadest middle region.
a
Lemma and palea widths were measured in the transverse sections of middle region of
young spikelets immediately after heading.
b
Lateral root length on the primary root (10 plants) was measured
Asterisks indicate significant difference determined by Student’s t-test (**, P<0.01; ***,
P<0.001). n.s. represents no significant difference.
11
Table S3 Cell size and cell number in the marginal region of lemma of wild-type and nal2/3
plants
Parameter
Wild-type
nal2/3
Cell size (mm2)
40.98  5.75 (n=4)
12.91  3.33 (n=4)***
Cell number
149.25  15.50 (n=4)
41.50  5.32 (n=4)***
Means and standard deviations were obtained from the transverse sections of middle region
of young stage sample. At least 134 cells of wild-type and 33 cells for nal2/3 were measured
per lemma sample. Asterisks indicate significant difference determined by Student’s t-test
(***, P<0.001).
Table S4 Agronomic traits in nal2/3
Parameter
Wild-type
nal2/3
Culm length (cm)
74.3 ± 3.1 (10)
70.7 ± 2.2 (10) **
Panicle length (cm)
22.3 ± 1.9 (10)
19.1 ± 1.7 (10) **
Number of panicles/plant
17.9 ± 1.9 (10)
12.9 ± 1.9 (10) **
Number of spikelets/panicle
117.1 ± 8.3 (10)
83.7 ± 9.3 (10) ***
Fertility (%)
92.0 ± 2.5 (10)
54.9 ± 9.2 (10) ***
1000 grain weight (g)
23.7 ± 2.1 (10)
19.1 ± 2.0 (10) **
Grain yield/plant (g)
45.8 ± 6.8 (10)
13.9 ± 3.9 (10) ***
Number of tillers
22.5 ± 3.1 (10)
31.1 ± 2.8 (10) ***
Means and standard deviations were obtained from field-grown plants or fully developed
grains (sampling numbers in parentheses). Asterisks indicate significant difference
determined by Student’s t-test (**, P<0.01; ***, P<0.001).
12
Table S5 Accession numbers of WOX proteins in the phylogenic tree (Fig. S6)
Name of Protein
Accession No.
Name of Protein
Accession No.
AtWOX1
AAP37133
OsWOX3B/DEP
CAM32354
AtWOX2
AAP37131
OsWOX4
CAJ84142
AtWOX3/PRS
AAP37135
OsWOX5
NP_915421
AtWOX4
AAP37134
OsWOX6
ABF95709
AtWOX5
AAP37136
OsWOX7
NP_916815
AtWOX6
AAP37137
OsWOX8
NP_915983
AtWOX7
NP_196196
QHB
BAB84412
AtWOX8
AAP37138
OsWOX10
BAD05582
AtWOX9
AAP37139
OsWOX11
BAF22586
AtWOX10
NP_173494
ZmNS1
Q70UV1
AtWOX11
AAP37140
ZmNS2
Q6S3I3
AtWOX12
AAP37141
ZmWOX2A
CAJ84159
AtWOX13
AAP37142
ZmWOX2B
CAJ84170
AtWOX14
NP_173493
ZmWOX3A
CAM32346
BdNS
CAM32355
ZmWOX3B
CAM33396
BdWOX2
CAM32356
ZmWOX4
CAJ84160
BdWOX3
CAM32357
ZmWOX5A
CAJ84161
BdWOX4
CAM32358
ZmWOX5B
CAJ84162
BdWOX5
CAM32359
ZmWOX9A
CAJ84163
BdWOX9
CAM32360
ZmWOX9B
CAJ84164
BdWOX11/12A
CAM32363
ZmWOX9C
CAJ84165
BdWOX11/12B
CAM32364
ZmWOX11/12A
CAJ84167
BdWOX13
CAM32361
ZmWOX11/12B
CAJ84167
OsWOX1
CAE04846
ZmWOX13A
CAJ84168
OsWOX2
Q5W7C3
ZmWOX13B
CAJ84169
OsWOX3A/NS
BAE48302
13
Methods S1. Quantification of endogenous IAA
To quantify endogenous IAA, 1 g of plant tissues were ground in liquid nitrogen and crude
IAA was extracted overnight at -20°C in 10 ml of 80 % methanol. IAA was purified by
acidifying the samples to pH 2.7 with 1 M HCl, applying them at a rate of 1 ml/min to a 500
mg C18 Bond Elute SPE column (Agilent Technologies) conditioned with 1 ml methanol and
1 ml 1% acetic acid, washing with 1 ml 10% methanol in 1% acetic acid, and then eluting
with 1 ml methanol. After evaporation to dryness, each sample was methylated by adding 0.2
ml 2-propanol, 1 ml methyl chloride, and 5 l trimethylsilyl-diazomethane in hexane and
then incubated for 30 min at room temperature. Five μl of 2 M acetic acid in hexane was
added to destroy excessive diazomethane in each sample, which was subsequently evaporated
to dryness. After trimethylation, IAA was quantified using IAA ELISA quantitation kit
(Agrisera, Sweden) according to the manufacturer’s instructions.
14