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The rice narrow leaf2 and narrow leaf3 loci encode WUSCHELrelated homeobox 3A (OsWOX3A) and function in leaf, spikelet,
tiller and lateral root development
Sung-Hwan Cho1*, Soo-Cheul Yoo1*, Haitao Zhang1*, Devendra Pandeya1, Hee-Jong Koh1, Ji-Young Hwang2,
Gyung-Tae Kim2 and Nam-Chon Paek1
1
Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Korea; 2Department of
Molecular Biotechnology, Dong-A University, Busan, 604-714, Korea
Summary
Authors for correspondence:
Nam-Chon Paek
Tel: +82 2 8804524
Email: [email protected]
Gyung-Tae Kim
Tel: +82 51 2007519
Email: [email protected]
Received: 8 December 2012
Accepted: 10 February 2013
New Phytologist (2013) 198: 1071–1084
doi: 10.1111/nph.12231
Key words: auxin, lateral root, nal2 nal3,
narrow-curly leaf, narrow-thin spikelet,
OsWOX3A, rice (Oryza sativa), tiller.
In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ
development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double
mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets
and narrow-thin grains.
We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth
with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly
caused by marginal deformity in the lemma, gave rise to narrow-thin grains.
Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical
OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and
NS2 in maize and PRESSED FLOWER in Arabidopsis. OsWOX3A is expressed in the vascular
tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels
of several leaf development-associated genes were altered in nal2/3, and auxin transportrelated genes were significantly changed, leading to pin mutant-like phenotypes such as more
tillers and fewer lateral roots.
OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular
patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers
and lateral roots.
Introduction
Lateral organs of higher plants are initiated at the periphery of
the shoot apical meristem (SAM) by recruitment of a group of
founder cells (Poethig & Szymkowiak, 1995; Dolan & Poethig,
1998). SAM maintenance and meristematic cell fate in organ
primordia are controlled by the interplay of homeodomain
transcription factors, including WUSCHEL (WUS), SHOOTMERISTEMLESS (STM) and CLAVATA (Clark et al., 1996;
Gallois et al., 2002; Lenhard et al., 2002).
Leaf primordia develop in a radial pattern at the periphery of
the SAM (Reinhardt et al., 2000), and leaf protrusion occurs as a
result of increased cell division and cell expansion. During leaf
vascular formation in rice, provascular strands including the midrib extend longitudinally (Itoh et al., 2005). Thereafter, small
longitudinal and transverse provascular strands form (Nishimura
et al., 2002), and bulliform cells differentiate on the adaxial surface. The adaxial–abaxial polarity of leaves is established by asymmetrical distribution of cell types. ASYMMETRIC LEAVES2
*These authors contributed equally to this work.
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(AS2) and HD-ZIPIII family members act as adaxial-specific regulators (McConnell et al., 2001; Iwakawa et al., 2002), whereas
YABBY (YAB) and KANADI genes specify abaxial cell fate
(Siegfried et al., 1999; Kerstetter et al., 2001; Emery et al., 2003).
These genes exhibit polar expression patterns and determine the
abaxial cell fate of aboveground lateral organs (Bowman, 2000).
Recently, YAB genes were reported to have a wide function not
only in adaxial–abaxial polarity of leaves, but also in shoot
development (Sarojam et al., 2010).
In rice, YAB1 functions in stamen and carpel development
(Jang et al., 2004). DROOPING LEAF (DL), a CRABS CLAWrelated gene and a member of the YAB family, is required for carpel specigenefication and leaf midrib development (Yamaguchi
et al., 2004). YAB5/TONGARI-BOUSHI1 (TOB1) is involved in
lateral organ development and maintenance of meristem organization for spikelet development (Tanaka et al., 2012). Unlike the
YAB members in Arabidopsis and maize, rice YAB1, YAB5 and
DL do not show adaxial/abaxial-specific expression in lateral
organs (Yamada et al., 2011).
WUS-related homeobox (WOX) proteins regulate diverse
aspects of Arabidopsis development (Haecker et al., 2004). For
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instance, WOX2 and WOX8 regulate cell fate in the apical and
basal lineage of the proembryo (Breuninger et al., 2008). WOX3/
PRESSED FLOWER (PRS) acts in lateral-axis expansion of lateral
organs (Matsumoto & Okada, 2001). WOX1 acts redundantly
with WOX3/PRS for cell proliferation in the blade outgrowth
and margin development downstream of adaxial/abaxial polarity
establishment in Arabidopsis leaves (Nakata et al., 2012). WOX5
functions in the maintenance of stem cells in shoot and root meristems (Sarkar et al., 2007; Nardmann et al., 2009). WOX6/
PRETTY FEW SEEDS2 regulates ovule development (Park et al.,
2005). WOX9/STIMPY is required for early embryonic growth,
and the maintenance of cell division and growth in shoot and
root apices (Wu et al., 2005). Such WOX functions have been
also reported in other plant species. In petunia, the WOX8/9
homologue EVERGREEN regulates cymose inflorescence
development (Rebocho et al., 2008), and the WOX1 homologue
MAEWEST is required for laminar growth (Vandenbussche
et al., 2009). In Medicago truncatula, the WOX1 homologue
STENOFOLIA regulates leaf blade outgrowth and vascular patterning (Tadege et al., 2011).
In monocots, maize narrow sheath1 narrow sheath2 (ns1 ns2)
double mutants display a narrow leaf sheath and margin-deleted
phenotype in the lower portion of leaf blades. Positional cloning
revealed that ns1 and ns2 (hereafter ns) are redundant, duplicated
WOX3 genes, required to recruit founder cells from lateral
domains of the SAM and form lateral and marginal regions of
leaves (Scanlon et al., 1996; Nardmann et al., 2004). PRS, an
Arabidopsis NS homologue, recruits founder cells during lateral
organ development to form lateral domains of vegetative and floral organs (Matsumoto & Okada, 2001; Shimizu et al., 2009). In
prs mutants, lateral stipules at the base of cauline leaves, sepals
and stamens are deleted because of failure to recruit lateral founder cells. The functions of PRS and NS are conserved to form
marginal regions of lateral organs through the recruitment of
founder cells from SAM lateral domains. The rice genome contains three WOX3 genes, the duplicated OsWOX3A genes (also
termed OsNS; Nardmann et al., 2007; GenBank accession number: AB218893) on the short arms of chromosome 11 and 12,
which are putative orthologues of ns1 and ns2 in maize and PRS
in Arabidopsis, and OsWOX3B (LOC_Os05 g02730; GenBank:
AM490244) on chromosome 5. OsWOX3A acts as a transcriptional repressor of YAB3 and OsWOX3A-overexpressing plants
exhibit twisted and knotted leaves. However, RNAi-mediated
downregulation of OsWOX3A produced no visible phenotype
(Dai et al., 2007). OsWOX3B/DEP has been identified recently
by positional cloning and functions in the regulation of trichome
formation in leaves and glumes (Angeles-Shim et al., 2012).
The plant hormone auxin (indole-3-acetic acid, IAA) plays
critical roles in cell division, cell elongation, organ initiation, vascular differentiation and root initiation (Reinhardt et al., 2000;
Benkova et al., 2003; Friml et al., 2003). Auxin is synthesized in
the young shoot apex and transported to maturing tissues. Directional transport of auxin results from the asymmetric distribution
of different auxin membrane carriers, that is, the influx carrier
AUX1 family and the efflux carriers in the PIN-FORMED (PIN)
family (Bennett et al., 1996; Galweiler et al., 1998; Xu et al.,
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2005). The rice narrow leaf7 (nal7) mutant, a mutation in
YUCCA8 (YUC8), involved in auxin synthesis, produces narrow
and curly leaves throughout development (Fujino et al., 2008).
The narrow and rolled leaf1 (nrl1), encoding the cellulose
synthase-like protein D4 (OsCslD4), regulates plant architecture
and development of leaf veins and bulliform cells (Hu et al.,
2010). Interestingly, NAL7/YUC8 levels are upregulated in nrl1
mutants, suggesting the association of the nrl1 phenotype with
auxin biosynthesis. Auxin maxima and polar transport trigger
development of leaves and lateral roots from the initiation of
primordia to the formation of the mature organ (Benkova et al.,
2003). The narrow leaf1 (nal1) mutation affects polar auxin
transport and vascular patterning (Qi et al., 2008). The Arabidopsis pin mutants exhibit auxin transport defects and alterations
in shoot, flower and root development (Galweiler et al., 1998;
Benkova et al., 2003; Reinhardt et al., 2003). Rice OsPIN1 and
OsPIN2 are also involved in tiller and root development (Xu
et al., 2005; Chen et al., 2012). Although auxin functions in leaf
development, vascular patterning and root initiation have been
reported (Reinhardt et al., 2003; Scarpella et al., 2006; Bilsborough et al., 2011), auxin function in plant organ development
has not been fully elucidated.
In this study, we analysed the rice pleiotropic nal2 nal3
(hereafter nal2/3) double mutant, which produces narrow-curly
leaves, opened spikelets, narrow-thin grains, more tillers and
fewer lateral roots. Map-based cloning revealed that the nal2 and
nal3 loci encode an identical OsWOX3A/OsNS protein. Our
histological and molecular analyses in nal2/3 demonstrate a
conserved role for OsWOX3A/NS/PRS in the regulation of
lateral-axis outgrowth and margin development in leaves. In
addition, our identification of OsWOX3A functions in the
development of spikelet, tiller and root organs will inform further
studies of the WOX family.
Materials and Methods
Plant materials and growth conditions
The nal2 nal3 (nal2/3) double recessive mutant of Oryza sativa L.
japonica rice was previously obtained from Kyushu University,
Japan, and has been maintained by the Rural Development
Administration, Korea. The nal2/3 mutant was backcrossed twice
with a Japanese japonica rice cv ‘Kinmaze’ and progressed for several generations. Kinmaze was used as the parental wild-type
plant in this study. The growth chamber conditions were 12-h
light (500 lmol m 2 s 1) at 30°C and 12-h dark at 20°C.
Histological analysis of leaves and spikelets
Samples were fixed in 3.7% formaldehyde, 5% acetic acid and
50% ethanol overnight at 4°C, and dehydrated in a gradient
series of ethanol, cleared through a xylene series, then infiltrated
through a series of Paraplast (Sigma) and finally embedded
in 100% Paraplast at 55–60°C or in Technovit 7100 resin
(Heraeus Kulzer GmbH, Frankfurt, Germany) for thin sections.
Then, 4–12 lm-thick microtome sections were mounted on glass
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slides. The sections were deparaffinized in 100% xylene and were
dried before staining with Toluidine blue O (Sigma) before
observation on an Axioscope2 microscope (Zeiss).
Genetic and physical mapping of nal2 and nal3
For map-based cloning of the nal2 and nal3 loci, an F2 mapping
population was generated by a cross of the nal2/3 mutant
(japonica) and a tongil-type cv ‘Milyang23’ which has a closer
genetic makeup to indica (Yoo et al., 2009). SSR markers were
obtained from information in GRAMENE, the Arizona Genomics Institute, and the Rice Genome Research Program. STS markers on chromosomes 11 and 12 were identified by nucleotide
sequence alignments between japonica and indica in the BAC
clones AL51300, BX000505, BX000500, BX000494 and
BX000496 for high-resolution physical mapping. PCR primer
sequences for SSR and STS markers are listed in Supporting
Information Table S1.
Rice transformation and complementation
The recombinant pC1300intC binary vector (GenBank:
AF294978) was prepared by inserting a 3831-bp genomic DNA
segment (2141-bp 5′-upstream, 612-bp ORF, and 1078-bp
3′-downstream sequences of OsWOX3A) for the complementation
test with nal2/3. The recombinant plasmid in an Agrobacterium
strain, LBA4404, was introduced into the calli of mature embryos
from nal2/3 mutant seeds (Jeon et al., 2000b). Transformants
were confirmed by PCR with primers in the pC1300intC vector
(TC1) and OsWOX3A genomic fragment (TC2; Table S1).
RNA extraction, reverse transcription and quantitative
real-time PCR (qRT-PCR)
Total RNAs were extracted independently from wild-type and
nal2/3 using the RNA extraction kit (iNtRON Biotechnology,
Seoul, Korea), and were treated with RNAse-free DNase (Ambion)
to remove possible contaminating genomic DNA as per the manufacturer’s instructions. For qRT-PCR, 2 lg of total RNA was
reverse-transcribed using the M-MLV reverse transcription kit
(Promega). First-strand cDNAs equivalent to 50 ng of total RNA
were used for qRT-PCR. Each RT product was analysed using a
LightCycler480 (Roche), with Universal SYBR Green Master
Mix (Roche), and data analysis was conducted using the Roche
Optical System software (v1.5). The qRT-PCR conditions were
as follows: activation at 95°C for 2 min, 40 cycles of denaturation
at 95°C for 15 s and annealing/extension at 60°C for 60 s,
followed by melt analysis ramping to 95°C. For the efficiency of
qRT-PCR, we calculated the amplification efficiency by comparing the slope of the linear regression of Ct and log10 of gene
copies. The expression of each gene was normalized against
Ubiquitin. The relative expression of each gene in WT and nal2/
3 plants was analysed using the 2 DDCT method as described
(Livak & Schmittgen, 2001), and Student’s t-test was used to
determine statistically significant differences. The qRT-PCR
primer information is given in Table S1.
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Membrane protein extraction and immunoblot analysis
Membrane proteins were extracted from 2-wk-old rice seedlings
using the ProteoExtract kit (M-PEK; Calbiochem, Darmstadt,
Germany). Equal amounts of protein were subjected to SDSPAGE and then transferred onto a polyvinylidene difluoride
(PVDF) membrane (Amersham). The proteins were detected by
immunoblotting with anti-AtPIN1 (Santa Cruz Biotech, Santa
Cruz, CA, USA), anti-AtPIN2 (Agrisera, Vännäs, Sweden) or
anti-Lhcb1 (Agrisera) antibodies, and then stained with Coomassie Brilliant Blue (Sigma) for loading control. Quantification of
band intensity on the immunoblots was performed using Image J
(v1.36) software according to the instructions (http://rsb.info.
nih.gov/ij/docs/menus/analyze.html#gels).
Histochemical analysis of OsWOX3A expression
For GUS (b-glucuronidase) assays, a 2.1-kb 5′-upstream promoter region of OsWOX3A was amplified by genomic PCR
(Table S1) and then subcloned into the pCAMBIA1301 plasmids, fusing the NAL2/3 promoter to the GUS coding
sequence (ProOsWOX3A:GUS). The construct was transformed
into rice calli as described (Jeon et al., 2000b). Nine independent transgenic lines were obtained after screening, and GUS
activity was detected histochemically as described (Jefferson
et al., 1987).
Subcellular localization of the OsWOX3-GFP fusion protein
in protoplasts
The ORF of OsWOX3, except for the stop codon, was amplified
using the primers ORF1 and ORF2 (Table S1), and subcloned
into pCAMLA-GFP, resulting in the 35S::OsWOX3-GFP. Maize
mesophyll protoplasts were transfected by PEG transformation
(Sheen, 2001; Yoo et al., 2007). At 20 h after transformation,
GFP signals were observed by confocal microscopy (MRC-1024;
BioRad).
Transcriptional activation in the yeast GAL4 system
The OsWOX3A ORF was divided into two parts: N-terminal
(1–333 bp) and C-terminal (316–612 bp). Full-length (primers:
Y-F and Y-R), N-terminal (Y-F and Y-R333) and C-terminal
(Y-F316 and Y-R) fragments were amplified from the cDNAs of
wild-type, nal2, and nal3 (Table S1). Each cDNA fragment was
digested with EcoRI and BamHI, and then inserted into the
pGBKT7 vector as bait (Clontech, Mountain View, CA, USA).
Bait clones were transformed into yeast strain AH109 with the
empty prey vector pGADT7. The yeast cells containing the
pGBKT7-p53 and pGADT7-T plasmids were used as a positive
control. The negative control AH109 cells were obtained by
co-transforming the empty pGBKT7 and pGADT7 plasmids.
Cotransformed cells were identified by spotting on SD/-Leu/
-Trp medium, then replica-plated on SD/-Ade/-His/-Leu/-Trp
medium. Transactivation activity was determined according to
cell survival on SD/-Ade/-His/-Leu/-Trp medium.
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Effect of IAA on lateral root development
(c)
(a)
Surface-sterilized rice seeds were germinated on 10 7 M IAA
0.5X MS medium with 0.8% phytoagar and grown vertically in a
growth chamber. At 14 d after germination, the length and number of lateral roots were measured.
Scanning electron microscopy
In order to examine the adaxial and abaxial surfaces of leaf blades,
1.5 mm transverse sections of the middle region of a fully
expanded third leaf blade were sampled. Sections were fixed in
primary fixative (2% paraformaldehyde, 2% glutaraldehyde),
post-fixed with 1% osmium tetroxide, and dehydrated in a series
of ethanol and propylene oxide, then finally embedded in Spurr’s
resin. After polymerization, sections were observed with a scanning electron microscope (JSM 5410LV; JEOL, Tokyo, Japan).
(b)
(d)
Results
Phenotypic characterization of rice nal2 and nal3 (nal2/3)
double mutants
The nal2/3 double mutant was named for its phenotype: the
production of narrow leaf blades throughout development
(Fig. 1a–c). These loci were also termed curly leaf2 (cul2) and
cul3, respectively, in the GRAMENE database (http://www.
gramene.org/) because the leaf blades exhibited both narrow and
curly phenotypes. The single nal2 or nal3 mutants did not show
any defect in leaf morphology. Compared with the parental wildtype cv ‘Kinmaze’, the widths of leaf blades in nal2/3 were consistently narrow from early seedling to fully mature stages (Fig. 1a,
b; Table S2). In addition, the nal2/3 leaves curled upward
(Fig. 1b). The number of large veins (LVs) in the leaf blades was
slightly reduced to c. 80% of wild-type (Fig. 1c; Table S2), and
the number of small veins (SVs) between adjacent LVs was
remarkably reduced to almost a half of wild-type. The number of
SVs between LVs was quite irregular in each leaf blade, and the
vein distribution on the left and right side of the midrib was
different. Sawtooth hairs at the leaf margins were significantly
reduced (Fig. 1d). Ligule, auricle, leaf sheath and stems were also
narrower and thinner (Fig. 1e,f; Table S2). Despite the reduction
in leaf width, the lengths of leaf blades and leaf sheaths, and plant
height were similar to wild-type (Table S2). These results indicate
that NAL2/3 function is mainly associated with vein patterning
during leaf development and lateral-axis expansion during shoot
organogenesis.
Defects in lateral-axis outgrowth, margin development and
vascular patterning during leaf development
We performed a histological analysis to define the effects of nal2/
3 on leaf organ development in more detail. Cross-sections of
mature leaf blades clearly showed reduced SV numbers between
LVs in nal2/3 (Fig. 2b). The size of vascular bundles (VBs) was
reduced and simplified (Fig. 2c,e). The organization of xylem
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(e)
(f)
Fig. 1 Phenotype of the rice (Oryza sativa) nal2/3 double mutant. (a)
Three-month-old rice plants of wild-type (WT) and nal2/3 grown in the
field. (b) Narrow leaf blades in nal2/3 curl towards the adaxial side during
leaf elongation and thus appear much narrower than their actual width
(see Supporting Information Table S2). (c) Enlarged adaxial side leaf blades
in (b). MR, midrib; LV, large vein; SV, small vein. (d) Defective
development of sawtooth hairs at the leaf margins of nal2/3. (e, f) nal2/3
also produces markedly narrow leaf sheath (LS), ligule (L), auricle (A) and
collar (C), and therefore a narrow-thin stem. Two-month-old plants were
photographed. Bars: (a) 10 cm; (b) 1 cm; (c) 2 mm; (d) 0.1 mm; (e, f) 1 cm.
and phloem were also altered (Fig. 2g,h). This result indicates
that NAL2/3 acts in vascular patterning during leaf development.
Bulliform cells, located between adjacent veins on the adaxial
side of leaf blades, were more abundant and bigger, and were
irregular in shape, compared with their spherical form in wildtype (Fig. 2a–f). In addition, there were significantly fewer longitudinal furrows (the zone of bulliform cells) on the adaxial side
due to fused SVs (Fig. S1c). Therefore, the upward curling during leaf elongation appears to be associated with abnormal development of bulliform cells. Moreover, the mesophyll cell regions
between veins and bulliform cells were narrower and thicker
(Fig. 2d,f). Interestingly, nal2/3 clearly lacked leaf margin
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(b)
(a)
(c)
Fig. 2 Defective organ development in
nal2/3 leaf blades and leaf sheaths. (a–h)
Transverse sections through fully expanded
leaf blades in 3-month-old rice (Oryza sativa)
plants. (a, b) The number of small veins (SVs)
between large veins (LVs) is significantly
reduced in nal2/3. (c–f) Enlarged LVs (c, e)
and SVs (d,f) in (a, b). Between veins, larger
and more numerous bulliform cells (BCs) are
constantly observed on the adaxial surface of
nal2/3. (g, h) Transverse sections through
the marginal regions. (i, j) Transverse sections
through the stem regions (leaf sheaths and
leaf blades) in the 2-month-old plants of WT
(i) and nal2/3 (j). Red dotted lines represent
the margins of overlapping leaves. M,
mesophyll cell; VBS, vascular bundle sheath;
X, xylem; PH, phloem; ABS, abaxial side;
ADS, adaxial side; SH, sawtooth hair; LS, leaf
sheath. Bars: (a, b) 100 lm; (c, e) 50 lm;
(d, f) 20 lm; (g, h) 10 lm; (i, j) 1 mm.
(e)
(g)
(h)
(i)
(j)
structures; the edges of leaf blades were blunt (Fig. 2g,h) and sawtooth hairs at the margins of leaf blades were nearly absent or
poorly developed (Fig. 1d), indicating that adaxial–abaxial patterning was compromised in nal2/3 mutants. nal2/3 mutant have
fewer cell files in the margins of leaf blades without a change in
cell size (Fig. 2g,h), indicating that cell proliferation of the margin of leaf blades was reduced. Defective leaf margin structures
were also observed in the leaf sheaths. The overlapped margins of
the leaf sheaths were much shorter, the leaf sheaths were narrower, and there were fewer vasculatures in each leaf sheath in
nal2/3 mutants (Fig. 2i,j). Taken together, these results indicate
that NAL2/3 regulates lateral-axis outgrowth, margin development and vascular patterning during leaf organ formation.
In order to examine the nal2/3 phenotype in early leaf development, transverse sections of the shoot apex region were examined.
The width of leaf blades was reduced in the L5 and L6 primordia
of nal2/3 (Fig. S2); the margins of wild-type leaves overlapped
but nal2/3 leaves were not. In sequential transverse sections of the
SAM, we found the SAM was smaller in nal2/3, compared with
wild-type (Fig. S2d,e,i,j). These results indicate that NAL2/3 is
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(d)
(f)
involved in lateral domain development at early leaf developmental stages, and the reduced size of SAM in nal2/3 is probably associated with the narrow leaf production from the leaf primordial
stage.
Defective lemma and palea morphogenesis during spikelet
development in nal2/3
In rice, the spikeletes are normally opened at fertilization stage
(Fig. S3) and closed after fertilization and throughout grain filling to produce normally shaped grains. However, the spikelets of
nal2/3 were not closed after fertilization and remained open during grain filling, resulting in narrow-thin grains (Fig. 3a,b; Table
S2). Transverse sections of spikelets in nal2/3 showed that the
widths of lemma and palea were significantly reduced to 58%
and 51%, respectively, compared with those of wild-type
(Fig. 3c–f; Table S2). Noticeably, the number of VBs in all
lemmas was increased in nal2/3 because two more VBs were
located at the end of the lemma margins (Fig. 3e,f). In addition,
the lemma and palea margins were not fully developed (Fig. 3c,
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(b)
(a)
(c)
(e)
(d)
(f)
Fig. 3 nal2/3 exhibits opened spikelets due to abnormal development of
palea and lemma, giving rise to narrow-thin grains. (a, b) Comparison of
rice (Oryza sativa) wild-type (WT) and nal2/3 spikelets and grains. (c, e)
Transverse section of young spikelets in WT and nal2/3, showing five (c)
and seven (e) vascular bundles in the lemma, respectively. The spikelets
were sampled just after emergence of panicles in the main stem. (d, f)
Magnified junction zones between lemma and palea (c, e), showing one
(d) and two (f) vascular bundles in (c) and (e), respectively. (c–f) Asterisks
indicate vascular bundles. (d, f) Black arrowheads indicate the junction of
lemma and palea. L, lemma; P, palea; G, grain; NSC, nonsilicified cell; SC,
silicified cell; SPC, spongy parenchymatous cell; FS, fibrous sclerenchyma.
Bars: (a, b) 3 mm; (c, e) 100 lm; (d, f) 50 lm.
e). In particular, the size and number of fibrous sclerenchyma at
the marginal region of the lemma in nal2/3 were significantly
reduced to 31% and 28% of those in wild-type, respectively
(Table S3). Cell size and cell number in the other regions of
lemma were also reduced in nal2/3 (Fig. 3e,f). Thus, it appears
that the reduced widths of lemma and palea and marginal abnormality in nal2/3 cause a failure in the reattachment at the junction of lemma and palea after fertilization (Fig. 3a,e). These
results indicate that NAL2/3 also functions in lemma and palea
morphogenesis during floral organ development in rice.
In this respect, nal2/3 mutation negatively affected several
agronomic traits of rice; fertility, grain weight, the length of panicles, and the number of spikelets per panicle were significantly
reduced (Table S4). Interestingly, tiller number was considerably
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increased in both active and maximum tillering stages in nal2/3
plants (Table S4; Fig. S4), whereas the number of panicles per
plant was significantly reduced in the mutant (Table S4), indicating that the production of unproductive tillers increases in nal2/
3. Taken together, our phenotypic analysis of the field-grown
nal2/3 plants suggests that nal2/3 mutation negatively affects
both vegetative and reproductive organ development with a
severe loss of grain yield.
Map-based cloning of nal2 and nal3 loci
In order to identify the nal2 and nal3 loci, genetic mapping was
performed using an F2 mapping population generated from a
cross of nal2/3 (japonica) and the wild-type indica cv
‘Milyang23’. The segregation ratio of 10 000 F2 individuals was
consistent with the expected ratio of 15 (9388 wild-type): 1 (612
mutant), a typical Mendelian segregation ratio for unlinked
duplicate genes. Using 612 F2 individuals displaying the mutant
phenotype, we found that nal2 was closely linked to an SSR
marker RM286 on the short arm of chromosome 11 (Fig. 4a).
The 384-kb candidate region was annotated by the Rice Genome
Research Program (http://rgp.dna.affrc.go.jp/). Among the annotated genes in the first BAC clone BX072548, one highly likely
candidate gene, OsWOX3A/OsNS (Os11 g01130), was found
(Fig. S5a). Next, the nal3 locus was mapped onto the short arm
of chromosome 12 using the SSR markers RM19, RM247 and
RM277 (Fig. 4b). Using six sequence tagged site (STS) markers
(Table S1), the nal3 locus was further narrowed down to a
149-kb interval from STS 6 to the end of the chromosome. In
the two BAC clones in this interval (GenBank: BX000503 and
BX000496), we found an identical OsWOX3A gene
(Os12 g01120) that has the same nucleotide sequence as the candidate gene for nal2 on chromosome 11. This finding was not
surprising because the first 3 Mb on the short-arm ends of chromosomes 11 and 12 are completely duplicated (The Rice Chromosomes 11 and 12 Sequencing Consortia, 2005). OsWOX3A
has a single 612-bp exon and encodes a 203-amino acid (aa)
protein containing a WUS homeodomain at the N-terminus
(4–68 aa) and a WUS-box domain at the C-terminus
(172–179 aa; Haecker et al., 2004; Fig. S5b). Phylogenetic analysis revealed that OsWOX3A/OsNS belongs to the same clade as
ZmNS1, ZmNS2 and AtWOX3/PRS (Fig. S6; Table S5). However, OsWOX3A shares only 66% aa sequence identity with
maize NS1 and NS2 (hereafter NS), and 42% with Arabidopsis
WOX3/PRS, mainly because the middle sequences of
OsWOX3A are quite dissimilar to those of NS and PRS.
In order to determine the lesions in nal2/3, we sequenced 20
RT-PCR products from nal2/3 mutant leaves. We found two
classes of cDNAs with different OsWOX3A mutations, which we
expected to represent the two different loci, nal2 and nal3
(Figs 4c, S5a). Seven cDNAs had a change from G to A at the
66th base of the open reading frame (ORF), causing a single
amino acid substitution from Met (ATG) to Ile (ATA) (designated nal2). In the other 13 cDNAs, several deletion, insertion
and point mutations occurred (designated nal3). To confirm that
these OsWOX3A mutations were responsible for the nal2/3
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Fig. 4 Map-based cloning of nal2 and nal3. (a) Map-based cloning of nal2. The nal2 locus was mapped to a 384-kb genomic region between an SSR
marker, RM286, and the short-arm end of chromosome 11. A BAC clone, BX072548, of the region contained a candidate gene, OsWOX3A. (b) Mapbased cloning of nal3. The nal3 locus was mapped to a 149-kb genomic region between STS-6 and the short-arm end of chromosome 12. OsWOX3A,
identical to the nal2 candidate gene, was found in two BAC clones, BX000503 and BX000496. (c) nal2 and nal3 mutations in OsWOX3A. OsWOX3A has
one 612-bp exon. Black boxes represent the changed nucleotides in nal2 and nal3 loci. Asterisks indicate changes in nucleotides that alter amino acids in
nal2 and nal3 (Fig. S5a).
phenotype, we next complemented the mutant phenotype. For
complementation, the 3831-bp genomic DNA of OsWOX3A (see
the Materials and Methods section) was expressed in nal2/3
plants. All transgenic lines produced normally shaped leaves (Fig.
S7) and spikelets (data not shown) in the nal2/3 background,
showing that the introduced genomic OsWOX3A fragment
complemented the nal2/3 mutation.
OsWOX3A is a nuclear protein that is expressed in the
vasculatures of various organs
The subcellular localization of an OsWOX3A-GFP fusion was
examined in maize leaf protoplasts, which revealed that
OsWOX3A is a nuclear protein (Fig. 5a,b). We also used qRTPCR to measure organ/tissue-specific expression of OsWOX3A.
OsWOX3A was ubiquitously expressed, and expressed at high levels in the shoot base (SB; including the SAM) and young panicle
(Fig. 5c). Unexpectedly, it was also expressed in roots at similar
levels to leaf blades. To examine the spatial expression of
OsWOX3A in each organ, we stained for b-glucuronidase (GUS)
activity in the transgenic plants containing the ProOsWOX3A:GUS
reporter gene (see the Materials and Methods section). In
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agreement with the qRT-PCR results, OsWOX3A was expressed
in both large and small VBs of the leaf blade and leaf sheath,
especially phloem tissues, and the shoot base and the vascular cylinder of roots (Fig. 5d–i). GUS activity was also observed in the
longitudinal veins of spikelets in young panicles at heading stage
(Fig. 5j). These results support the hypothesis, based on the nal2/
3 phenotype, that OsWOX3A is mainly expressed in vasculatures
for the rice organ development.
Altered expression of leaf development-associated genes in
nal2/3
Leaf development is regulated by several groups of genes; for
example, auxin synthesis-related genes such as YUC genes (Fujino
et al., 2008), auxin transport-associated genes (Benkova et al.,
2003), and YAB genes (Dai et al., 2007). To further understand
the regulatory functions of OsWOX3A in leaf organogenesis, we
measured mRNA levels of leaf development-associated genes in
the SB region by qRT-PCR (Fig. 6a). We measured auxin signal
transduction, transport and biosynthesis-related genes, such as
AUXIN RESPONSE FACTOR (ARF), PIN-FORMED (PIN) and
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related genes, such as NAL1, NRL1 and YAB family genes. We
found that in nal2/3, ARF1, ARF4, YAB4, YAB7, YUC8/NAL7
and PIN1 were significantly downregulated, and that YAB1-3,
YUC1, YUC4 and PIN2 were upregulated. Expression levels of
YAB5, NRL1 and PIN3 were slightly reduced. However, the
expression levels of ARF2-3, ARF5, YAB6, YUC2-3, YUC6-7,
YUC9 and NAL1 were not altered (Fig. 6a). The qRT-PCR
results suggest that OsWOX3A modulates the transcription levels
of several auxin- and leaf development-related genes.
The expression levels of ARF genes are affected by the amount
of free IAA (Waller et al., 2002; Xing et al., 2011). YUC genes,
encoding flavin monooxygenase-like enzymes, function in the
auxin biosynthesis pathway (Yamamoto et al., 2007; Fujino et al.,
2008). Thus, we examined endogenous free auxin concentrations
in wild-type and nal2/3 seedlings with an IAA-ELISA kit (see
Methods S1) and found no significant difference in endogenous
free IAA concentrations (Fig. S8), indicating that the altered
expression of some of ARF and YUC genes did not affect the
endogenous free IAA content in nal2/3. Because IAA concentrations were not altered in nal2/3 mutant, we hypothesized that
compromised auxin transport caused the mutant phenotype.
Immunoblot analysis using the anti-PIN1 antibody (a-PIN1)
and a-PIN2 revealed that PIN1 was significantly reduced
whereas the PIN2 concentration was slightly increased in nal2/3
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(j)
Fig. 5 Nuclear localization of OsWOX3A and
expression profiles of OsWOX3A. (a, b)
Nuclear localization of OsWOX3A-GFP in
maize protoplasts. Bright-field image (a), and
overlays of the green and red images (b), are
shown. The GFP signal is indicated in green,
and chlorophyll autofluorescence is shown in
red. (c) OsWOX3A expression in various
tissues. Total RNAs were isolated from leaf
blade (LB), leaf sheath (LS), shoot base (SB)
and root (RT) tissues in 2-wk-old rice (Oryza
sativa) plants, and young panicle (YP) tissues
just after heading in the main stem. qRT-PCR
analysis was performed to measure their
relative mRNA levels, which were normalized
to the transcript levels of Ubiquitin (Ub).
Mean and SD values were obtained from
three biological replicates. This experiment
was repeated twice with similar results. (d–j)
Histochemical analysis of OsWOX3A
expression by GUS assay (see the Materials
and Methods section). GUS activity was
detected in the large vein (LV) and small vein
(SV) of leaf blade (d) and leaf sheath (e),
especially in the phloem (PH) but not in
xylem (X) tissues of vascular bundle (f),
meristem tissues in the shoot base (g),
vascular cylinder (VC) of primary root (h) and
its transverse section (i) from 2-wk-old
plants, and longitudinal veins (V) of a young
spikelet just after heading in the main stem
(j). Bars: (d, f), 500 lm; (e), 20 lm; (g, h),
200 lm; (i, j), 1 mm.
(Fig. 6b), consistent with their expression levels. These results
suggest that the nal2/3 phenotype may be related to altered auxin
transport in addition to the transcriptional activity of leaf development-related genes.
Altered expression of OsPIN1 and OsPIN2 may reduce
lateral root numbers in nal2/3
Overexpression of OsPIN1 significantly increased the lateral root
(LR) number in the transgenic rice (Xu et al., 2005), whereas
OsPIN2-overexpressing plants displayed reduced LR density
(Chen et al., 2012). Because the expression levels of OsPIN1 and
OsPIN2 were altered in nal2/3, we further characterized the pin
mutant-associated phenotypes such as the number of lateral or
adventitious roots. Consistent with OsPIN expression, LR number was significantly reduced in nal2/3 (Fig. 7c,d,g). However,
adventitious root number was not different between wild-type
and nal2/3 (Fig. 7g). The results indicate that altered expression
of OsPIN1 and OsPIN2 negatively affects LR development in
nal2/3.
A previous report indicated that in OsPIN1-RNAi rice, polar
auxin distribution was compromised, and this was partially complemented by exogenous IAA (NAA; Xu et al., 2005). To further
test whether reduced number of LRs in nal2/3 was caused by
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Fig. 6 Altered mRNA and protein concentrations for genes associated with leaf development in rice (Oryza sativa) nal2/3. (a) Relative abundance of
mRNAs of leaf development-related genes in the shoot base (SB) of nal2/3 seedlings. Ten-day-old plants grown in the growth chamber were used for
qRT-PCR. The expression level of each gene was normalized to Ubiquitin in each RNA sample. The relative abundance of gene expression in nal2/3 was
determined by dividing by the expression level of each gene in wild-type (WT). YUCCA5 was not amplified from WT or nal2/3. Mean and standard
deviation (SD) values were obtained from three independent samples. Asterisks indicate statistically significant differences compared with WT as
determined by Student’s t-test (*, P < 0.05; ***, P < 0.001). (b) Protein abundances of OsPIN1 and OsPIN2 in 14-d-old WT and nal2/3 seedlings. Purified
membrane proteins were immunoblotted with anti-AtPIN1 and anti-AtPIN2 antibodies, and then an anti-Lhcb1 antibody as a loading control. The
membrane was stained with Coomassie Brilliant Blue (CBB) to show equal loading. Two independent experiments were performed and similar results were
obtained (right panel). The relative intensities of protein bands were calculated using Image J and are indicated on the bottom of each gel image.
failure of endogenous IAA distribution, we grew nal2/3 plants in
the MS medium containig 10 7 M IAA for 2 wk. As expected,
exogenous IAA treatment partially rescued the reduced LR number in the mutant, to c. 90% of wild-type (Fig. 7b,e–g), suggesting that altered expression of OsPIN1 and OsPIN2 in nal2/3
compromised the precise distribution of endogenous IAA, as previously observed in other OsPIN mutants (Xu et al., 2005; Chen
et al., 2012).
In order to further understand OsWOX3A function in LR formation, we analysed OsWOX3A expression during LR development histochemically using transgenic rice containing the
ProOsWOX3A:GUS transgene (Fig. 8). OsWOX3A was expressed at
the base region of LR from primordial to early developmental
stages (Fig. 8a–c). After emergence of LR primordia, OsWOX3A
expression was detected only in the vascular cylinder during LR
elongation (Fig. 8d,e). This result supports the idea that
OsWOX3A is involved in development of LR primordia.
OsWOX3A acts as a transcriptional activator
The qRT-PCR results suggested that in addition to the repression
of YAB3 (Dai et al., 2007), OsWOX3A may also be involved in
the transcriptional upregulation of several leaf development-associated genes (Fig. 6a). To test whether OsWOX3A can act as a
transcriptional activator, we used the yeast GAL4 system in
which a transactivating protein fused to the GAL4 DNA binding
domain activates a HIS reporter gene, enabling recombinant
yeast to survive on His-deficient medium. We found that the
yeast cells expressing OsWOX3A fused to a DNA-binding
domain (DB:OsWOX3A) grew vigorously on the His-deficient
medium, indicating that OsWOX3A has transactivation activity.
However, the mutated nal2 (DB:nal2) and nal3 (DB:nal3) proteins lost the activity completely (Fig. 9, first panel). Both N-terminal and C-terminal fragments of OsWOX3A also showed
transcriptional activation in yeast. However, the C-terminal
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fragment of nal2 and the N- or C-terminal fragments of nal3 did
not activate the reporter gene (Fig. 9, second and third panels),
suggesting that both the N-terminal WOX3 homeodomain and
the C-terminal WUS-box domain are required for the transactivation activity of OsWOX3A.
Discussion
Here, we examined the pleiotropic phenotype of nal2/3 double
mutant rice, and used map-based cloning to identify NAL2 and
NAL3 on chromosomes 11 and 12, respectively. They encode an
identical OsWOX3A/OsNS protein that is homologous to NS of
maize and PRS of Arabidopsis, all of which belong to the same
WOX3 subfamily (Figs S5b, S6; Zhang et al., 2007). Both NS
and PRS play an important role in the recruitment of founder
cells for margin development of lateral organ primordia in monocots and eudicots, respectively (Nardmann et al., 2004). However, the functions of NS/PRS homologues in other plant species
remain unknown. Our histological and molecular studies show
that in rice, OsWOX3A is involved in differentiation of lemma
and palea in spikelets, and tiller and LR production, in addition
to lateral-axis expansion in leaves.
OsWOX3A in lamina outgrowth and vascular patterning
The nal2/3 mutation has a wide range of morphological defects
in development of various organs in rice (Figs 1–3, 7; Tables S2,
S4). Especially, it profoundly reduces leaf width, similar to nal1,
nal7, and nrl1 mutants in rice (Fujino et al., 2008; Qi et al.,
2008; Hu et al., 2010). The nal2/3 mutant produces narrow leaf
blades with upward curling (Fig. 1b,c), and the widths of all leaf
blades are consistently narrower, starting from the primordial
stage (Fig. S2). In addition, nal2/3 has reduced leaf margin structures (Fig. 1d) and defects in distribution and arrangement of
LVs and SVs (Figs 1c, 2a–h). In rice, SVs are formed later than
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Fig. 7 Reduced numbers of lateral roots in nal2/3 were partially rescued
by treatment with exogenous IAA (indole-3-acetic acid, auxin). Rice
(Oryza sativa) seeds were germinated and grown on regular MS medium
for 2 d and then uniformly germinated seeds were transferred to the
shown plates. 2-wk-old seedlings were observed. (a, b) Root morphology
of wild-type (WT) (a) and nal2/3 (b) seedlings grown on agar plates
without (-IAA) or with a supplement of 0.1 lM IAA. (c, d) Magnified view
of WT (c) and nal2/3 (d) lateral roots on the primary root in (a) red
rectangles. (c–f) PR, primary root. (e, f) Magnified view of WT and nal2/3
lateral roots on the primary root in (b) red rectangles. (g) (c–f) The number
of lateral and adventitious roots of WT (grey bars) and nal2/3 (white bars)
seedlings grown with or without IAA. The photo for adventitious root
numbers is not shown. LR, lateral root; AR, adventitious root. Mean and
SD values were obtained from 15 plants for each line. Asterisks indicate
statistically significant differences compared with WT as determined by
Student’s t-test (**, P < 0.01; ***, P < 0.001). Bars: (a, b) 1 cm; (c–f)
2 mm.
LVs during leaf development (Itoh et al., 2005). The short interval between LVs in nal2/3 (Fig. 2b) suggests that there is not
enough space to generate as many SVs as in wild-type. In addition, narrower and thicker mesophyll cell regions between SVs
and bulliform cells (Fig. 2b,f) indicate that there is a defect in
lateral cell proliferation between veins in nal2/3. To compensate
for the reduction of lateral cell number in nal2/3, newly divided
mesophyll cells may be deposited vertically during leaf elongation, resulting in increased leaf thickness (Tsukaya, 2008). During leaf initiation, leaf primordia are also narrower and thicker
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(Fig. S2), suggesting that there are fewer leaf founder cells in
nal2/3. This result indicates that NAL2/3 genes are required for
cell proliferation in lateral region, which promote outgrowth of
leaf blade, from the leaf primordial stage. In addition, nal2/3
clearly lacked leaf margin structures; blunt leaf blades (Fig. 2g,h)
and fewer sawtooth hairs at the margins of leaf blades (Fig. 1d),
indicating that adaxial–abaxial patterning was compromised in
nal2/3 mutants. Similar defective phenotypes were observed in
Arabidopsis prs and wox1 double mutants in which leaf blade
outgrowth was compromised due to the reduced cell proliferation
in lateral region, resulting in production of narrow and thick
leaves (Vandenbussche et al., 2009; Nakata et al., 2012). The
double mutant also showed defects in margin-specific tissues and
adaxial/abaxial patterning, which are caused by loss-of-function
mutation of middle domain-specific WOX genes in early-developing leaves (Nakata et al., 2012). These results suggest that
NAL2/3 may work in the similar manner with Arabidopsis WOX
genes for leaf development. In maize, ns mutant has narrow leaf
sheaths with a margin-deleted phenotype in the lower portion of
the leaf blades (Scanlon et al., 1996). Although NS and
OsWOX3A have similar functions in lateral-axis development of
leaf primordia, the phenotypic defects in their mature leaves are
quite different, mainly due to their structural differences in the
upper and lower portions in leaf blades. In addition, SAM size
was also reduced in nal2/3, as observed in sequential transverse
sections (Fig. S2), suggesting that NAL2/3 genes may be required
for the maintenance of SAM size. The Arabidopsis yab quadruple
mutants showed defects in apical dominance, reduced SAM as
well as loss of leaf expansion with polarity defects (Sarojam et al.,
2010). These results indicate that YAB gene activity is required
for both shoot development and marginal domain establishment
in leaf blade, and altered expression of YAB genes in nal2/3 may
have caused defects in development of SAM as well as leaf expansion.
Recently, OsWOX3B/DEP, a homologue of OsWOX3A, has
been reported to regulate the formation of bristle-type trichomes
in the leaves and glumes (Angeles-Shim et al., 2012). Although
they belong to the same subfamily of OsWOX proteins (Fig. S6),
the biological function of OsWOX3B/DEP was considerably different from that of OsWOX3A, suggesting that rice WOX3 genes
within the same subfamily may have diversified to have separate
biological functions. Together, our results suggest that
OsWOX3A plays an important role in promoting cell proliferation in leaf width during leaf initiation and outgrowth, which is a
conserved function of WOX3 among species. This can be
explained by the specific expression of OsWOX3A/OsNS in the
lateral margins of leaf primordia as described previously
(Nardmann et al., 2007).
OsWOX3A regulates lemma and palea development in
spikelets
Defects in seed development were not reported for maize ns
mutants or Arabidopsis prs mutants; however, the rice nal2/3
mutant produces abnormally narrow-thin grains with opened
spikelets. Lemma and palea in the outmost layer of the spikelet
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Fig. 8 OsWOX3A expression during lateral root formation. Two-week-old transgenic rice (Oryza sativa) plants expressing ProOsWOX3A:GUS were stained
to observe OsWOX3A expression at various developmental stages. (a–c) GUS expression in lateral root (LR) initiation stages (a) and early developmental
stages (b, c). Note that ProOsWOX3A:GUS was highly expressed at the base of lateral root primordia. Arrowheads indicate GUS signal detected at the base
of LR. (d, e) GUS expression in later stages of LR development. Note that ProOsWOX3A:GUS signals were only detected in the vasculatures. Asterisk
indicates the absence of GUS signal at the base of LR. Bars, 50 lm.
maintain proper meristem organization in spikelet development.
It thus appears that OsWOX3A is required for the positive regulation by YAB5/TOB1 during spikelet development, because YAB5
expression was slightly reduced in nal2/3 (Fig. 6a), and
OsWOX3A is highly expressed in the young panicle (Fig. 5c),
especially in the longitudinal veins of developing spikelets
(Fig. 5j).
Fig. 9 The N- and C-terminal regions of OsWOX3A showed transactivation
activity. Yeast cells co-transformed with the bait clones containing fulllength, N-terminal (His-rich domain) or C-terminal (Gln-rich domain)
region of OsWOX3A (WT) and empty prey clone survived in the highly
stringent SD/-Ade/-His/-Leu/-Trp media ( 4), but those with the fulllength or N-terminal region of nal2 or nal3 protein did not survive. Yeast
cells with the C-terminal region of nal2 protein survived, but not those of
nal3 protein in the ( 4) media. PC, pGBKT7-53 and pGADT7-T vectors
(positive control); NC, empty pGBKT7 and pGADT7 vectors (negative
control).
are remarkably narrower and thinner (Fig. 3a,b; Table S2), resulting in opened spikelets during grain filling (Fig. 3a). Additionally, a reduction in cell size and number, and extra VBs were
observed in the margins of the lemma (Fig. 3e,f; Table S3). The
Arabidopsis prs mutants showed repressed growth of the lateral
sepals (Matsumoto & Okada, 2001). Lemma and palea in rice
are presumed to be equivalent to sepals in eudicot flowers
(Ferrario et al., 2004). In this respect, defects in floral organ
development of nal2/3 are similar to those of prs mutants, in
which marginal cells of the adaxial and abaxial sepals disappear.
Unlike prs, however, nal2/3 mutants show no defect in other
floral organs (Fig. S3). This suggests that WOX3 functions in
flower development differ among rice, maize and Arabidopsis,
mainly due to differences in morphology.
Unlike the reduced VBs in leaves, in the margin of the lemma
in nal2/3 we observed two more VBs and reduced size and number of cells (Fig. 3e,f). It is not certain that the regulatory roles of
OsWOX3A differ in leaf and spikelet development. In lemma
development, the rice MADS-box genes OsMADS1 and
OsMAD34 act as important regulators (Jeon et al., 2000a; Prasad
et al., 2005; Gao et al., 2010). Recently, Tanaka et al. (2012)
showed that lemma and palea were reduced in the spikelets of rice
yab5/tob1 mutants; they suggested that YAB5/TOB1 functions to
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OsWOX3A is involved in formation of tillers and LRs,
possibly by regulating OsPIN1 and OsPIN2 expression
Among the leaf development-associated genes, OsWOX3A probably acts as a negative regulator of YAB1, YAB2, YAB3, YUC1,
YUC4 and OsPIN2 (Dai et al., 2007). However, it appears that
OsWOX3A also acts as a positive regulator of several leaf development-associated genes, especially those involved in auxin synthesis and transport, such as ARF1, ARF4, YUC8/NAL7 and
OsPIN1 (Fig. 6a), although it remains to be determined whether
OsWOX3A binds to their promoter regions directly as a transcriptional activator (Fig. 9).
Auxin plays important roles in controlling cell identity, cell
division, initiation of lateral organs, and formation of leaf
margins and vasculature (Scanlon, 2003; Scarpella et al., 2006;
Grieneisen et al., 2007). The ARFs are all transcription factors
that bind to auxin-response elements in the promoters of early
auxin-responsive genes (Tiwari et al., 2003). Although YUC and
ARF transcript levels were altered, endogenous concentrations of
free IAA were not significantly changed in nal2/3 (Fig. S8). The
auxin efflux carrier PIN1 facilitates polar transport of auxin in
Arabidopsis (Paponov et al., 2005; Kleine-Vehn et al., 2009)
and in rice (Xu et al., 2005; Qi et al., 2008). Nonpolarized
localization of PIN1 causes abnormal distribution of the auxin
maxima required for vascular networking in Arabidopsis
(Shirakawa et al., 2009). In rice, repression of OsPIN1 by RNAi
caused defects in adventitious roots and increased the number
of tillers. Overexpression of OsPIN1 increased LR number (Xu
et al., 2005) and overexpression of OsPIN2 increased tiller
number and reduced LR number (Chen et al., 2012). In nal2/3,
tiller number increased (Fig. S4) and LR number decreased,
possibly due to both downregulation of OsPIN1 and upregulation
of OsPIN2. Furthermore, the reduced LR number in nal2/3 was
rescued by exogenous IAA treatment, similar to the case of
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OsPIN1-RNAi plants (Xu et al., 2005). This strongly suggests
that increased tillers and reduced LRs in nal2/3 seem to be
attributable to altered expression of OsPIN1 and OsPIN2. In
Arabidopsis, AtPIN1 is expressed in the inner layer cells of LR
primordia; however, AtPIN2 is detected in the outer cells only
after primordium emergence (Benkova et al., 2003). Precise regulation of spatial expressions of AtPIN1 and AtPIN2 is crucial
in LR development, which is based on balanced auxin supply to
the tip and its PIN2-dependent retrieval (Benkova et al., 2003).
Notably, OsWOX3A expression was detected at the base region
of LR primordia during LR emergence (Fig. 8). Therefore, it
can be speculated that OsWOX3A modulates LR organogenesis
by regulating OsPIN1 and OsPIN2.
The YUC genes, whose expression was altered in nal2/3
(Fig. 6a), are involved in auxin biosynthesis and thereby control
leaf blade outgrowth and margin development (Wang et al.,
2011). The PRS and WOX1 are required for promoting cell proliferation in outgrowth of leaf blade, maybe due to activated cell
division via auxin and cytokinin (Nakata et al., 2012). In the ns
mutants of maize, microarray data showed that TIR1 and ARFGAP, auxin signalling- and transport-related genes are downregulated (Zhang et al., 2007). In addition, STF of Medicago
truncatula and LAM1 of tobacco, the Arabidopsis WOX1 orthologues, also affect auxin concentrations (Tadege et al., 2011),
strongly suggesting that the WOX family acts in the regulation of
auxin biosynthesis, signalling or transport.
Our physiological and molecular data suggest that OsWOX3A
regulates the transcription of genes involved in auxin synthesis,
signalling and/or polar transport for lateral cell proliferation during vegetative and reproductive organ development (Table S2,
Table S4). Further identification of as-yet-unknown regulatory
genes downstream of OsWOX3A will provide further insights
into the function of the WOX3 subfamily.
Acknowledgements
This work was supported by a grant from the Next-Generation
BioGreen 21 Program (Plant Molecular Breeding Center No.
PJ008128), Rural Development Administration, Republic of
Korea.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Scanning electron microscopy of the adaxial and abaxial
surfaces of nal2/3 leaves.
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.
Fig. S3 nal2/3 displayed normal morphology in pistil and stamen
development.
Fig. S4 nal2/3 showed a significantly increased number of tillers
at both active and maximum tillering stages.
Fig. S5 Characterization of nal2 and nal3 mutant proteins and
OsWOX3A homologues.
Fig. S6 Phylogenic tree of the WOX families.
Fig. S8 Endogenous free-IAA concentrations in the whole plants
of 2-wk-old wild-type (WT) and nal2/3 using IAA ELISA analysis.
Table S1 Primers used in this study
Table S2 Morphological characteristics of nal2/3
Table S3 Cell size and cell number in the marginal region of
lemma of wild-type and nal2/3 plants
Table S4 Agronomic traits in nal2/3
Table S5 Accession numbers of WOX proteins in the phylogenic
tree (Fig. S6)
Methods S1 Quantification of endogenous IAA.
Please note: Wiley-Blackwell are not responsible for the content
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authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
Fig. S7 Complementation of nal2/3 by OsWOX3A.
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