REV ISS WEB NPH 12231 198-4 1071. - :: 작물분자육종연구실 ::cmb.snu.ac.kr/bod1/pds/research/cho.pdf · · 2014-01-24longitudinal and transverse provascular strands form

The rice narrow leaf2 and narrow leaf3 loci encode WUSCHEL-related 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

1Department 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

Authors for correspondence:Nam-Chon PaekTel: +82 2 8804524

Email: [email protected]

Gyung-Tae Kim

Tel: +82 51 2007519

Email: [email protected]

Received: 8 December 2012Accepted: 10 February 2013

New Phytologist (2013) 198: 1071–1084doi: 10.1111/nph.12231

Key words: auxin, lateral root, nal2 nal3,narrow-curly leaf, narrow-thin spikelet,OsWOX3A, rice (Oryza sativa), tiller.

Summary

� 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 transport-

related genes were significantly changed, leading to pinmutant-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 ofthe shoot apical meristem (SAM) by recruitment of a group offounder cells (Poethig & Szymkowiak, 1995; Dolan & Poethig,1998). SAM maintenance and meristematic cell fate in organprimordia are controlled by the interplay of homeodomaintranscription factors, including WUSCHEL (WUS), SHOOT-MERISTEMLESS (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 ofthe SAM (Reinhardt et al., 2000), and leaf protrusion occurs as aresult of increased cell division and cell expansion. During leafvascular formation in rice, provascular strands including the mid-rib extend longitudinally (Itoh et al., 2005). Thereafter, smalllongitudinal and transverse provascular strands form (Nishimuraet al., 2002), and bulliform cells differentiate on the adaxial sur-face. The adaxial–abaxial polarity of leaves is established by asym-metrical distribution of cell types. ASYMMETRIC LEAVES2

(AS2) and HD-ZIPIII family members act as adaxial-specific reg-ulators (McConnell et al., 2001; Iwakawa et al., 2002), whereasYABBY (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 theabaxial cell fate of aboveground lateral organs (Bowman, 2000).Recently, YAB genes were reported to have a wide function notonly in adaxial–abaxial polarity of leaves, but also in shootdevelopment (Sarojam et al., 2010).

In rice, YAB1 functions in stamen and carpel development(Jang et al., 2004). DROOPING LEAF (DL), a CRABS CLAW-related gene and a member of the YAB family, is required for car-pel specigenefication and leaf midrib development (Yamaguchiet al., 2004). YAB5/TONGARI-BOUSHI1 (TOB1) is involved inlateral organ development and maintenance of meristem organi-zation for spikelet development (Tanaka et al., 2012). Unlike theYAB members in Arabidopsis and maize, rice YAB1, YAB5 andDL do not show adaxial/abaxial-specific expression in lateralorgans (Yamada et al., 2011).

WUS-related homeobox (WOX) proteins regulate diverseaspects of Arabidopsis development (Haecker et al., 2004). For*These authors contributed equally to this work.

� 2013 The Authors

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instance, WOX2 and WOX8 regulate cell fate in the apical andbasal lineage of the proembryo (Breuninger et al., 2008). WOX3/PRESSED FLOWER (PRS) acts in lateral-axis expansion of lateralorgans (Matsumoto & Okada, 2001). WOX1 acts redundantlywith WOX3/PRS for cell proliferation in the blade outgrowthand margin development downstream of adaxial/abaxial polarityestablishment in Arabidopsis leaves (Nakata et al., 2012). WOX5functions in the maintenance of stem cells in shoot and root mer-istems (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 androot apices (Wu et al., 2005). Such WOX functions have beenalso reported in other plant species. In petunia, the WOX8/9homologue EVERGREEN regulates cymose inflorescencedevelopment (Rebocho et al., 2008), and the WOX1 homologueMAEWEST is required for laminar growth (Vandenbusscheet al., 2009). In Medicago truncatula, the WOX1 homologueSTENOFOLIA regulates leaf blade outgrowth and vascular pat-terning (Tadege et al., 2011).

In monocots, maize narrow sheath1 narrow sheath2 (ns1 ns2)double mutants display a narrow leaf sheath and margin-deletedphenotype in the lower portion of leaf blades. Positional cloningrevealed that ns1 and ns2 (hereafter ns) are redundant, duplicatedWOX3 genes, required to recruit founder cells from lateraldomains of the SAM and form lateral and marginal regions ofleaves (Scanlon et al., 1996; Nardmann et al., 2004). PRS, anArabidopsis NS homologue, recruits founder cells during lateralorgan development to form lateral domains of vegetative and flo-ral organs (Matsumoto & Okada, 2001; Shimizu et al., 2009). Inprs mutants, lateral stipules at the base of cauline leaves, sepalsand stamens are deleted because of failure to recruit lateral foun-der cells. The functions of PRS and NS are conserved to formmarginal regions of lateral organs through the recruitment offounder cells from SAM lateral domains. The rice genome con-tains three WOX3 genes, the duplicated OsWOX3A genes (alsotermed OsNS; Nardmann et al., 2007; GenBank accession num-ber: AB218893) on the short arms of chromosome 11 and 12,which are putative orthologues of ns1 and ns2 in maize and PRSin Arabidopsis, and OsWOX3B (LOC_Os05 g02730; GenBank:AM490244) on chromosome 5. OsWOX3A acts as a transcrip-tional repressor of YAB3 and OsWOX3A-overexpressing plantsexhibit twisted and knotted leaves. However, RNAi-mediateddownregulation of OsWOX3A produced no visible phenotype(Dai et al., 2007). OsWOX3B/DEP has been identified recentlyby positional cloning and functions in the regulation of trichomeformation in leaves and glumes (Angeles-Shim et al., 2012).

The plant hormone auxin (indole-3-acetic acid, IAA) playscritical roles in cell division, cell elongation, organ initiation, vas-cular differentiation and root initiation (Reinhardt et al., 2000;Benkova et al., 2003; Friml et al., 2003). Auxin is synthesized inthe young shoot apex and transported to maturing tissues. Direc-tional transport of auxin results from the asymmetric distributionof different auxin membrane carriers, that is, the influx carrierAUX1 family and the efflux carriers in the PIN-FORMED (PIN)family (Bennett et al., 1996; Galweiler et al., 1998; Xu et al.,

2005). The rice narrow leaf7 (nal7) mutant, a mutation inYUCCA8 (YUC8), involved in auxin synthesis, produces narrowand curly leaves throughout development (Fujino et al., 2008).The narrow and rolled leaf1 (nrl1), encoding the cellulosesynthase-like protein D4 (OsCslD4), regulates plant architectureand development of leaf veins and bulliform cells (Hu et al.,2010). Interestingly, NAL7/YUC8 levels are upregulated in nrl1mutants, suggesting the association of the nrl1 phenotype withauxin biosynthesis. Auxin maxima and polar transport triggerdevelopment of leaves and lateral roots from the initiation ofprimordia to the formation of the mature organ (Benkova et al.,2003). The narrow leaf1 (nal1) mutation affects polar auxintransport and vascular patterning (Qi et al., 2008). The Arabid-opsis pin mutants exhibit auxin transport defects and alterationsin shoot, flower and root development (Galweiler et al., 1998;Benkova et al., 2003; Reinhardt et al., 2003). Rice OsPIN1 andOsPIN2 are also involved in tiller and root development (Xuet al., 2005; Chen et al., 2012). Although auxin functions in leafdevelopment, vascular patterning and root initiation have beenreported (Reinhardt et al., 2003; Scarpella et al., 2006; Bilsbor-ough et al., 2011), auxin function in plant organ developmenthas not been fully elucidated.

In this study, we analysed the rice pleiotropic nal2 nal3(hereafter nal2/3) double mutant, which produces narrow-curlyleaves, opened spikelets, narrow-thin grains, more tillers andfewer lateral roots. Map-based cloning revealed that the nal2 andnal3 loci encode an identical OsWOX3A/OsNS protein. Ourhistological and molecular analyses in nal2/3 demonstrate aconserved role for OsWOX3A/NS/PRS in the regulation oflateral-axis outgrowth and margin development in leaves. Inaddition, our identification of OsWOX3A functions in thedevelopment of spikelet, tiller and root organs will inform furtherstudies of theWOX 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 DevelopmentAdministration, Korea. The nal2/3 mutant was backcrossed twicewith a Japanese japonica rice cv ‘Kinmaze’ and progressed for sev-eral generations. Kinmaze was used as the parental wild-typeplant in this study. The growth chamber conditions were 12-hlight (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 and50% ethanol overnight at 4°C, and dehydrated in a gradientseries of ethanol, cleared through a xylene series, then infiltratedthrough a series of Paraplast (Sigma) and finally embeddedin 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 weredried before staining with Toluidine blue O (Sigma) beforeobservation 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 mappingpopulation was generated by a cross of the nal2/3 mutant(japonica) and a tongil-type cv ‘Milyang23’ which has a closergenetic makeup to indica (Yoo et al., 2009). SSR markers wereobtained from information in GRAMENE, the Arizona Genom-ics Institute, and the Rice Genome Research Program. STS mark-ers on chromosomes 11 and 12 were identified by nucleotidesequence alignments between japonica and indica in the BACclones AL51300, BX000505, BX000500, BX000494 andBX000496 for high-resolution physical mapping. PCR primersequences for SSR and STS markers are listed in SupportingInformation Table S1.

Rice transformation and complementation

The recombinant pC1300intC binary vector (GenBank:AF294978) was prepared by inserting a 3831-bp genomic DNAsegment (2141-bp 5′-upstream, 612-bp ORF, and 1078-bp3′-downstream sequences ofOsWOX3A) for the complementationtest with nal2/3. The recombinant plasmid in an Agrobacteriumstrain, LBA4404, was introduced into the calli of mature embryosfrom nal2/3 mutant seeds (Jeon et al., 2000b). Transformantswere confirmed by PCR with primers in the pC1300intC vector(TC1) andOsWOX3A genomic fragment (TC2; Table S1).

RNA extraction, reverse transcription and quantitativereal-time PCR (qRT-PCR)

Total RNAs were extracted independently from wild-type andnal2/3 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 man-ufacturer’s instructions. For qRT-PCR, 2 lg of total RNA wasreverse-transcribed using the M-MLV reverse transcription kit(Promega). First-strand cDNAs equivalent to 50 ng of total RNAwere used for qRT-PCR. Each RT product was analysed using aLightCycler480 (Roche), with Universal SYBR Green MasterMix (Roche), and data analysis was conducted using the RocheOptical System software (v1.5). The qRT-PCR conditions wereas follows: activation at 95°C for 2 min, 40 cycles of denaturationat 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 ofqRT-PCR, we calculated the amplification efficiency by compar-ing the slope of the linear regression of Ct and log10 of genecopies. The expression of each gene was normalized againstUbiquitin. 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 todetermine statistically significant differences. The qRT-PCRprimer information is given in Table S1.

Membrane protein extraction and immunoblot analysis

Membrane proteins were extracted from 2-wk-old rice seedlingsusing the ProteoExtract kit (M-PEK; Calbiochem, Darmstadt,Germany). Equal amounts of protein were subjected to SDS-PAGE and then transferred onto a polyvinylidene difluoride(PVDF) membrane (Amersham). The proteins were detected byimmunoblotting with anti-AtPIN1 (Santa Cruz Biotech, SantaCruz, CA, USA), anti-AtPIN2 (Agrisera, Vannas, Sweden) oranti-Lhcb1 (Agrisera) antibodies, and then stained with Coomas-sie Brilliant Blue (Sigma) for loading control. Quantification ofband 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 ofOsWOX3A expression

For GUS (b-glucuronidase) assays, a 2.1-kb 5′-upstream pro-moter region of OsWOX3A was amplified by genomic PCR(Table S1) and then subcloned into the pCAMBIA1301 plas-mids, fusing the NAL2/3 promoter to the GUS codingsequence (ProOsWOX3A:GUS). The construct was transformedinto rice calli as described (Jeon et al., 2000b). Nine indepen-dent transgenic lines were obtained after screening, and GUSactivity was detected histochemically as described (Jeffersonet al., 1987).

Subcellular localization of the OsWOX3-GFP fusion proteinin protoplasts

The ORF of OsWOX3, except for the stop codon, was amplifiedusing the primers ORF1 and ORF2 (Table S1), and subclonedinto pCAMLA-GFP, resulting in the 35S::OsWOX3-GFP. Maizemesophyll 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 ofwild-type, nal2, and nal3 (Table S1). Each cDNA fragment wasdigested with EcoRI and BamHI, and then inserted into thepGBKT7 vector as bait (Clontech, Mountain View, CA, USA).Bait clones were transformed into yeast strain AH109 with theempty prey vector pGADT7. The yeast cells containing thepGBKT7-p53 and pGADT7-T plasmids were used as a positivecontrol. The negative control AH109 cells were obtained byco-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/-Trpmedium. Transactivation activity was determined according tocell survival on SD/-Ade/-His/-Leu/-Trp medium.


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Effect of IAA on lateral root development

Surface-sterilized rice seeds were germinated on 10�7 M IAA0.5X MS medium with 0.8% phytoagar and grown vertically in agrowth chamber. At 14 d after germination, the length and num-ber 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 fullyexpanded third leaf blade were sampled. Sections were fixed inprimary fixative (2% paraformaldehyde, 2% glutaraldehyde),post-fixed with 1% osmium tetroxide, and dehydrated in a seriesof ethanol and propylene oxide, then finally embedded in Spurr’sresin. After polymerization, sections were observed with a scan-ning electron microscope (JSM 5410LV; JEOL, Tokyo, Japan).

Results

Phenotypic characterization of rice nal2 and nal3 (nal2/3)double mutants

The nal2/3 double mutant was named for its phenotype: theproduction of narrow leaf blades throughout development(Fig. 1a–c). These loci were also termed curly leaf2 (cul2) andcul3, respectively, in the GRAMENE database (http://www.gramene.org/) because the leaf blades exhibited both narrow andcurly phenotypes. The single nal2 or nal3 mutants did not showany defect in leaf morphology. Compared with the parental wild-type cv ‘Kinmaze’, the widths of leaf blades in nal2/3 were consis-tently 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 wasslightly reduced to c. 80% of wild-type (Fig. 1c; Table S2), andthe number of small veins (SVs) between adjacent LVs wasremarkably reduced to almost a half of wild-type. The number ofSVs between LVs was quite irregular in each leaf blade, and thevein distribution on the left and right side of the midrib wasdifferent. Sawtooth hairs at the leaf margins were significantlyreduced (Fig. 1d). Ligule, auricle, leaf sheath and stems were alsonarrower and thinner (Fig. 1e,f; Table S2). Despite the reductionin leaf width, the lengths of leaf blades and leaf sheaths, and plantheight were similar to wild-type (Table S2). These results indicatethat NAL2/3 function is mainly associated with vein patterningduring leaf development and lateral-axis expansion during shootorganogenesis.

Defects in lateral-axis outgrowth, margin development andvascular 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 ofmature leaf blades clearly showed reduced SV numbers betweenLVs in nal2/3 (Fig. 2b). The size of vascular bundles (VBs) wasreduced and simplified (Fig. 2c,e). The organization of xylem

and phloem were also altered (Fig. 2g,h). This result indicatesthat NAL2/3 acts in vascular patterning during leaf development.

Bulliform cells, located between adjacent veins on the adaxialside of leaf blades, were more abundant and bigger, and wereirregular in shape, compared with their spherical form in wild-type (Fig. 2a–f). In addition, there were significantly fewer longi-tudinal furrows (the zone of bulliform cells) on the adaxial sidedue to fused SVs (Fig. S1c). Therefore, the upward curling dur-ing leaf elongation appears to be associated with abnormal devel-opment of bulliform cells. Moreover, the mesophyll cell regionsbetween veins and bulliform cells were narrower and thicker(Fig. 2d,f). Interestingly, nal2/3 clearly lacked leaf margin

(a) (c)

(d)

(e) (f)

(b)

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 thefield. (b) Narrow leaf blades in nal2/3 curl towards the adaxial side duringleaf elongation and thus appear much narrower than their actual width(see Supporting Information Table S2). (c) Enlarged adaxial side leaf bladesin (b). MR, midrib; LV, large vein; SV, small vein. (d) Defectivedevelopment of sawtooth hairs at the leaf margins of nal2/3. (e, f) nal2/3also produces markedly narrow leaf sheath (LS), ligule (L), auricle (A) andcollar (C), and therefore a narrow-thin stem. Two-month-old plants werephotographed. Bars: (a) 10 cm; (b) 1 cm; (c) 2 mm; (d) 0.1mm; (e, f) 1 cm.



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structures; the edges of leaf blades were blunt (Fig. 2g,h) and saw-tooth hairs at the margins of leaf blades were nearly absent orpoorly developed (Fig. 1d), indicating that adaxial–abaxial pat-terning was compromised in nal2/3 mutants. nal2/3 mutant havefewer cell files in the margins of leaf blades without a change incell size (Fig. 2g,h), indicating that cell proliferation of the mar-gin of leaf blades was reduced. Defective leaf margin structureswere also observed in the leaf sheaths. The overlapped margins ofthe leaf sheaths were much shorter, the leaf sheaths were nar-rower, and there were fewer vasculatures in each leaf sheath innal2/3 mutants (Fig. 2i,j). Taken together, these results indicatethat NAL2/3 regulates lateral-axis outgrowth, margin develop-ment and vascular patterning during leaf organ formation.

In order to examine the nal2/3 phenotype in early leaf develop-ment, transverse sections of the shoot apex region were examined.The width of leaf blades was reduced in the L5 and L6 primordiaof nal2/3 (Fig. S2); the margins of wild-type leaves overlappedbut nal2/3 leaves were not. In sequential transverse sections of theSAM, we found the SAM was smaller in nal2/3, compared withwild-type (Fig. S2d,e,i,j). These results indicate that NAL2/3 is

involved in lateral domain development at early leaf developmen-tal stages, and the reduced size of SAM in nal2/3 is probably asso-ciated with the narrow leaf production from the leaf primordialstage.

Defective lemma and palea morphogenesis during spikeletdevelopment in nal2/3

In rice, the spikeletes are normally opened at fertilization stage(Fig. S3) and closed after fertilization and throughout grain fill-ing to produce normally shaped grains. However, the spikelets ofnal2/3 were not closed after fertilization and remained open dur-ing grain filling, resulting in narrow-thin grains (Fig. 3a,b; TableS2). Transverse sections of spikelets in nal2/3 showed that thewidths 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 alllemmas was increased in nal2/3 because two more VBs werelocated at the end of the lemma margins (Fig. 3e,f). In addition,the lemma and palea margins were not fully developed (Fig. 3c,

(a) (b)

(c) (d) (e) (f)

(g) (h)

(i) (j)

Fig. 2 Defective organ development innal2/3 leaf blades and leaf sheaths. (a–h)Transverse sections through fully expandedleaf blades in 3-month-old rice (Oryza sativa)plants. (a, b) The number of small veins (SVs)between large veins (LVs) is significantlyreduced in nal2/3. (c–f) Enlarged LVs (c, e)and SVs (d,f) in (a, b). Between veins, largerand more numerous bulliform cells (BCs) areconstantly observed on the adaxial surface ofnal2/3. (g, h) Transverse sections throughthe marginal regions. (i, j) Transverse sectionsthrough the stem regions (leaf sheaths andleaf blades) in the 2-month-old plants of WT(i) and nal2/3 (j). Red dotted lines representthe 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, leafsheath. Bars: (a, b) 100 lm; (c, e) 50 lm;(d, f) 20 lm; (g, h) 10 lm; (i, j) 1 mm.





e). In particular, the size and number of fibrous sclerenchyma atthe marginal region of the lemma in nal2/3 were significantlyreduced to 31% and 28% of those in wild-type, respectively(Table S3). Cell size and cell number in the other regions oflemma were also reduced in nal2/3 (Fig. 3e,f). Thus, it appearsthat the reduced widths of lemma and palea and marginal abnor-mality in nal2/3 cause a failure in the reattachment at the junc-tion of lemma and palea after fertilization (Fig. 3a,e). Theseresults indicate that NAL2/3 also functions in lemma and paleamorphogenesis during floral organ development in rice.

In this respect, nal2/3 mutation negatively affected severalagronomic traits of rice; fertility, grain weight, the length of pani-cles, and the number of spikelets per panicle were significantlyreduced (Table S4). Interestingly, tiller number was considerably

increased in both active and maximum tillering stages in nal2/3plants (Table S4; Fig. S4), whereas the number of panicles perplant was significantly reduced in the mutant (Table S4), indicat-ing that the production of unproductive tillers increases in nal2/3. Taken together, our phenotypic analysis of the field-grownnal2/3 plants suggests that nal2/3 mutation negatively affectsboth vegetative and reproductive organ development with asevere loss of grain yield.

Map-based cloning of nal2 and nal3 loci

In order to identify the nal2 and nal3 loci, genetic mapping wasperformed using an F2 mapping population generated from across of nal2/3 (japonica) and the wild-type indica cv‘Milyang23’. The segregation ratio of 10 000 F2 individuals wasconsistent with the expected ratio of 15 (9388 wild-type): 1 (612mutant), a typical Mendelian segregation ratio for unlinkedduplicate genes. Using 612 F2 individuals displaying the mutantphenotype, we found that nal2 was closely linked to an SSRmarker RM286 on the short arm of chromosome 11 (Fig. 4a).The 384-kb candidate region was annotated by the Rice GenomeResearch Program (http://rgp.dna.affrc.go.jp/). Among the anno-tated genes in the first BAC clone BX072548, one highly likelycandidate gene, OsWOX3A/OsNS (Os11 g01130), was found(Fig. S5a). Next, the nal3 locus was mapped onto the short armof chromosome 12 using the SSR markers RM19, RM247 andRM277 (Fig. 4b). Using six sequence tagged site (STS) markers(Table S1), the nal3 locus was further narrowed down to a149-kb interval from STS 6 to the end of the chromosome. Inthe two BAC clones in this interval (GenBank: BX000503 andBX000496), we found an identical OsWOX3A gene(Os12 g01120) that has the same nucleotide sequence as the can-didate gene for nal2 on chromosome 11. This finding was notsurprising because the first 3 Mb on the short-arm ends of chro-mosomes 11 and 12 are completely duplicated (The Rice Chro-mosomes 11 and 12 Sequencing Consortia, 2005). OsWOX3Ahas 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 analy-sis revealed that OsWOX3A/OsNS belongs to the same clade asZmNS1, ZmNS2 and AtWOX3/PRS (Fig. S6; Table S5). How-ever, OsWOX3A shares only 66% aa sequence identity withmaize NS1 and NS2 (hereafter NS), and 42% with ArabidopsisWOX3/PRS, mainly because the middle sequences ofOsWOX3A are quite dissimilar to those of NS and PRS.

In order to determine the lesions in nal2/3, we sequenced 20RT-PCR products from nal2/3 mutant leaves. We found twoclasses of cDNAs with different OsWOX3A mutations, which weexpected to represent the two different loci, nal2 and nal3(Figs 4c, S5a). Seven cDNAs had a change from G to A at the66th base of the open reading frame (ORF), causing a singleamino acid substitution from Met (ATG) to Ile (ATA) (desig-nated nal2). In the other 13 cDNAs, several deletion, insertionand point mutations occurred (designated nal3). To confirm thatthese OsWOX3A mutations were responsible for the nal2/3

(a) (b)

(c) (d)

(e) (f)

Fig. 3 nal2/3 exhibits opened spikelets due to abnormal development ofpalea and lemma, giving rise to narrow-thin grains. (a, b) Comparison ofrice (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 spikeletswere 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) Asterisksindicate vascular bundles. (d, f) Black arrowheads indicate the junction oflemma 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.



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phenotype, we next complemented the mutant phenotype. Forcomplementation, the 3831-bp genomic DNA of OsWOX3A (seethe Materials and Methods section) was expressed in nal2/3plants. 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 fragmentcomplemented the nal2/3 mutation.

OsWOX3A is a nuclear protein that is expressed in thevasculatures of various organs

The subcellular localization of an OsWOX3A-GFP fusion wasexamined in maize leaf protoplasts, which revealed thatOsWOX3A is a nuclear protein (Fig. 5a,b). We also used qRT-PCR to measure organ/tissue-specific expression of OsWOX3A.OsWOX3A was ubiquitously expressed, and expressed at high lev-els in the shoot base (SB; including the SAM) and young panicle(Fig. 5c). Unexpectedly, it was also expressed in roots at similarlevels to leaf blades. To examine the spatial expression ofOsWOX3A in each organ, we stained for b-glucuronidase (GUS)activity in the transgenic plants containing the ProOsWOX3A:GUSreporter gene (see the Materials and Methods section). In

agreement with the qRT-PCR results, OsWOX3A was expressedin both large and small VBs of the leaf blade and leaf sheath,especially phloem tissues, and the shoot base and the vascular cyl-inder of roots (Fig. 5d–i). GUS activity was also observed in thelongitudinal 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 vasculaturesfor the rice organ development.

Altered expression of leaf development-associated genes innal2/3

Leaf development is regulated by several groups of genes; forexample, auxin synthesis-related genes such as YUC genes (Fujinoet al., 2008), auxin transport-associated genes (Benkova et al.,2003), and YAB genes (Dai et al., 2007). To further understandthe regulatory functions of OsWOX3A in leaf organogenesis, wemeasured mRNA levels of leaf development-associated genes inthe SB region by qRT-PCR (Fig. 6a). We measured auxin signaltransduction, transport and biosynthesis-related genes, such asAUXIN RESPONSE FACTOR (ARF), PIN-FORMED (PIN) andYUC family genes, respectively, as well as leaf development-

(a)

(c)

(b)

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 SSRmarker, RM286, and the short-arm end of chromosome 11. A BAC clone, BX072548, of the region contained a candidate gene,OsWOX3A. (b) Map-based 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 nal3mutations inOsWOX3A.OsWOX3A hasone 612-bp exon. Black boxes represent the changed nucleotides in nal2 and nal3 loci. Asterisks indicate changes in nucleotides that alter amino acids innal2 and nal3 (Fig. S5a).





related genes, such as NAL1, NRL1 and YAB family genes. Wefound that in nal2/3, ARF1, ARF4, YAB4, YAB7, YUC8/NAL7and PIN1 were significantly downregulated, and that YAB1-3,YUC1, YUC4 and PIN2 were upregulated. Expression levels ofYAB5, NRL1 and PIN3 were slightly reduced. However, theexpression levels of ARF2-3, ARF5, YAB6, YUC2-3, YUC6-7,YUC9 and NAL1 were not altered (Fig. 6a). The qRT-PCRresults suggest that OsWOX3A modulates the transcription levelsof several auxin- and leaf development-related genes.

The expression levels of ARF genes are affected by the amountof free IAA (Waller et al., 2002; Xing et al., 2011). YUC genes,encoding flavin monooxygenase-like enzymes, function in theauxin biosynthesis pathway (Yamamoto et al., 2007; Fujino et al.,2008). Thus, we examined endogenous free auxin concentrationsin wild-type and nal2/3 seedlings with an IAA-ELISA kit (seeMethods S1) and found no significant difference in endogenousfree IAA concentrations (Fig. S8), indicating that the alteredexpression of some of ARF and YUC genes did not affect theendogenous free IAA content in nal2/3. Because IAA concentra-tions were not altered in nal2/3 mutant, we hypothesized thatcompromised auxin transport caused the mutant phenotype.Immunoblot analysis using the anti-PIN1 antibody (a-PIN1)and a-PIN2 revealed that PIN1 was significantly reducedwhereas the PIN2 concentration was slightly increased in nal2/3

(Fig. 6b), consistent with their expression levels. These resultssuggest that the nal2/3 phenotype may be related to altered auxintransport in addition to the transcriptional activity of leaf devel-opment-related genes.

Altered expression ofOsPIN1 andOsPIN2may reducelateral root numbers in nal2/3

Overexpression of OsPIN1 significantly increased the lateral root(LR) number in the transgenic rice (Xu et al., 2005), whereasOsPIN2-overexpressing plants displayed reduced LR density(Chen et al., 2012). Because the expression levels of OsPIN1 andOsPIN2 were altered in nal2/3, we further characterized the pinmutant-associated phenotypes such as the number of lateral oradventitious roots. Consistent with OsPIN expression, LR num-ber was significantly reduced in nal2/3 (Fig. 7c,d,g). However,adventitious root number was not different between wild-typeand nal2/3 (Fig. 7g). The results indicate that altered expressionof OsPIN1 and OsPIN2 negatively affects LR development innal2/3.

A previous report indicated that in OsPIN1-RNAi rice, polarauxin distribution was compromised, and this was partially com-plemented by exogenous IAA (NAA; Xu et al., 2005). To furthertest whether reduced number of LRs in nal2/3 was caused by

(a) (c)

(f)

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(g) (h) (i)

(e)

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Fig. 5 Nuclear localization of OsWOX3A andexpression profiles ofOsWOX3A. (a, b)Nuclear localization of OsWOX3A-GFP inmaize protoplasts. Bright-field image (a), andoverlays of the green and red images (b), areshown. The GFP signal is indicated in green,and chlorophyll autofluorescence is shown inred. (c)OsWOX3A expression in varioustissues. Total RNAs were isolated from leafblade (LB), leaf sheath (LS), shoot base (SB)and root (RT) tissues in 2-wk-old rice (Oryzasativa) plants, and young panicle (YP) tissuesjust after heading in the main stem. qRT-PCRanalysis was performed to measure theirrelative mRNA levels, which were normalizedto the transcript levels of Ubiquitin (Ub).Mean and SD values were obtained fromthree biological replicates. This experimentwas repeated twice with similar results. (d–j)Histochemical analysis ofOsWOX3A

expression by GUS assay (see the Materialsand Methods section). GUS activity wasdetected in the large vein (LV) and small vein(SV) of leaf blade (d) and leaf sheath (e),especially in the phloem (PH) but not inxylem (X) tissues of vascular bundle (f),meristem tissues in the shoot base (g),vascular cylinder (VC) of primary root (h) andits transverse section (i) from 2-wk-oldplants, and longitudinal veins (V) of a youngspikelet just after heading in the main stem(j). Bars: (d, f), 500 lm; (e), 20 lm; (g, h),200 lm; (i, j), 1 mm.



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failure of endogenous IAA distribution, we grew nal2/3 plants inthe MS medium containig 10�7 M IAA for 2 wk. As expected,exogenous IAA treatment partially rescued the reduced LR num-ber in the mutant, to c. 90% of wild-type (Fig. 7b,e–g), suggest-ing that altered expression of OsPIN1 and OsPIN2 in nal2/3compromised the precise distribution of endogenous IAA, as pre-viously observed in other OsPIN mutants (Xu et al., 2005; Chenet al., 2012).

In order to further understand OsWOX3A function in LR for-mation, we analysed OsWOX3A expression during LR develop-ment histochemically using transgenic rice containing theProOsWOX3A:GUS transgene (Fig. 8). OsWOX3A was expressed atthe base region of LR from primordial to early developmentalstages (Fig. 8a–c). After emergence of LR primordia, OsWOX3Aexpression was detected only in the vascular cylinder during LRelongation (Fig. 8d,e). This result supports the idea thatOsWOX3A is involved in development of LR primordia.

OsWOX3A acts as a transcriptional activator

The qRT-PCR results suggested that in addition to the repressionof YAB3 (Dai et al., 2007), OsWOX3A may also be involved inthe transcriptional upregulation of several leaf development-asso-ciated genes (Fig. 6a). To test whether OsWOX3A can act as atranscriptional activator, we used the yeast GAL4 system inwhich a transactivating protein fused to the GAL4 DNA bindingdomain activates a HIS reporter gene, enabling recombinantyeast to survive on His-deficient medium. We found that theyeast cells expressing OsWOX3A fused to a DNA-bindingdomain (DB:OsWOX3A) grew vigorously on the His-deficientmedium, indicating that OsWOX3A has transactivation activity.However, the mutated nal2 (DB:nal2) and nal3 (DB:nal3) pro-teins lost the activity completely (Fig. 9, first panel). Both N-ter-minal and C-terminal fragments of OsWOX3A also showedtranscriptional activation in yeast. However, the C-terminal

fragment of nal2 and the N- or C-terminal fragments of nal3 didnot activate the reporter gene (Fig. 9, second and third panels),suggesting that both the N-terminal WOX3 homeodomain andthe C-terminal WUS-box domain are required for the transacti-vation activity of OsWOX3A.

Discussion

Here, we examined the pleiotropic phenotype of nal2/3 doublemutant rice, and used map-based cloning to identify NAL2 andNAL3 on chromosomes 11 and 12, respectively. They encode anidentical OsWOX3A/OsNS protein that is homologous to NS ofmaize and PRS of Arabidopsis, all of which belong to the sameWOX3 subfamily (Figs S5b, S6; Zhang et al., 2007). Both NSand PRS play an important role in the recruitment of foundercells for margin development of lateral organ primordia in mono-cots and eudicots, respectively (Nardmann et al., 2004). How-ever, the functions of NS/PRS homologues in other plant speciesremain unknown. Our histological and molecular studies showthat in rice, OsWOX3A is involved in differentiation of lemmaand palea in spikelets, and tiller and LR production, in additionto lateral-axis expansion in leaves.

OsWOX3A in lamina outgrowth and vascular patterning

The nal2/3 mutation has a wide range of morphological defectsin 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 leafblades with upward curling (Fig. 1b,c), and the widths of all leafblades are consistently narrower, starting from the primordialstage (Fig. S2). In addition, nal2/3 has reduced leaf margin struc-tures (Fig. 1d) and defects in distribution and arrangement ofLVs and SVs (Figs 1c, 2a–h). In rice, SVs are formed later than

(a) (b)

Fig. 6 Altered mRNA and protein concentrations for genes associated with leaf development in rice (Oryza sativa) nal2/3. (a) Relative abundance ofmRNAs 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 forqRT-PCR. The expression level of each gene was normalized to Ubiquitin in each RNA sample. The relative abundance of gene expression in nal2/3wasdetermined by dividing by the expression level of each gene in wild-type (WT). YUCCA5was not amplified fromWT or nal2/3. Mean and standarddeviation (SD) values were obtained from three independent samples. Asterisks indicate statistically significant differences compared with WT asdetermined 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. Purifiedmembrane proteins were immunoblotted with anti-AtPIN1 and anti-AtPIN2 antibodies, and then an anti-Lhcb1 antibody as a loading control. Themembrane was stained with Coomassie Brilliant Blue (CBB) to show equal loading. Two independent experiments were performed and similar results wereobtained (right panel). The relative intensities of protein bands were calculated using Image J and are indicated on the bottom of each gel image.





LVs during leaf development (Itoh et al., 2005). The short inter-val between LVs in nal2/3 (Fig. 2b) suggests that there is notenough space to generate as many SVs as in wild-type. In addi-tion, narrower and thicker mesophyll cell regions between SVsand bulliform cells (Fig. 2b,f) indicate that there is a defect inlateral cell proliferation between veins in nal2/3. To compensatefor the reduction of lateral cell number in nal2/3, newly dividedmesophyll cells may be deposited vertically during leaf elonga-tion, resulting in increased leaf thickness (Tsukaya, 2008). Dur-ing leaf initiation, leaf primordia are also narrower and thicker

(Fig. S2), suggesting that there are fewer leaf founder cells innal2/3. This result indicates that NAL2/3 genes are required forcell proliferation in lateral region, which promote outgrowth ofleaf blade, from the leaf primordial stage. In addition, nal2/3clearly 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 innal2/3 mutants. Similar defective phenotypes were observed inArabidopsis prs and wox1 double mutants in which leaf bladeoutgrowth was compromised due to the reduced cell proliferationin lateral region, resulting in production of narrow and thickleaves (Vandenbussche et al., 2009; Nakata et al., 2012). Thedouble mutant also showed defects in margin-specific tissues andadaxial/abaxial patterning, which are caused by loss-of-functionmutation of middle domain-specific WOX genes in early-devel-oping leaves (Nakata et al., 2012). These results suggest thatNAL2/3 may work in the similar manner with Arabidopsis WOXgenes for leaf development. In maize, ns mutant has narrow leafsheaths with a margin-deleted phenotype in the lower portion ofthe leaf blades (Scanlon et al., 1996). Although NS andOsWOX3A have similar functions in lateral-axis development ofleaf primordia, the phenotypic defects in their mature leaves arequite different, mainly due to their structural differences in theupper and lower portions in leaf blades. In addition, SAM sizewas also reduced in nal2/3, as observed in sequential transversesections (Fig. S2), suggesting that NAL2/3 genes may be requiredfor the maintenance of SAM size. The Arabidopsis yab quadruplemutants showed defects in apical dominance, reduced SAM aswell as loss of leaf expansion with polarity defects (Sarojam et al.,2010). These results indicate that YAB gene activity is requiredfor both shoot development and marginal domain establishmentin leaf blade, and altered expression of YAB genes in nal2/3 mayhave caused defects in development of SAM as well as leaf expan-sion.

Recently, OsWOX3B/DEP, a homologue of OsWOX3A, hasbeen reported to regulate the formation of bristle-type trichomesin the leaves and glumes (Angeles-Shim et al., 2012). Althoughthey belong to the same subfamily of OsWOX proteins (Fig. S6),the biological function of OsWOX3B/DEP was considerably dif-ferent from that of OsWOX3A, suggesting that rice WOX3 geneswithin the same subfamily may have diversified to have separatebiological functions. Together, our results suggest thatOsWOX3A plays an important role in promoting cell prolifera-tion in leaf width during leaf initiation and outgrowth, which is aconserved function of WOX3 among species. This can beexplained by the specific expression of OsWOX3A/OsNS in thelateral margins of leaf primordia as described previously(Nardmann et al., 2007).

OsWOX3A regulates lemma and palea development inspikelets

Defects in seed development were not reported for maize nsmutants or Arabidopsis prs mutants; however, the rice nal2/3mutant produces abnormally narrow-thin grains with openedspikelets. Lemma and palea in the outmost layer of the spikelet

(a) (b)

(c) (d) (e) (f)

(g)

Fig. 7 Reduced numbers of lateral roots in nal2/3were partially rescuedby treatment with exogenous IAA (indole-3-acetic acid, auxin). Rice(Oryza sativa) seeds were germinated and grown on regular MS mediumfor 2 d and then uniformly germinated seeds were transferred to theshown plates. 2-wk-old seedlings were observed. (a, b) Root morphologyof wild-type (WT) (a) and nal2/3 (b) seedlings grown on agar plateswithout (-IAA) or with a supplement of 0.1 lM IAA. (c, d) Magnified viewof WT (c) and nal2/3 (d) lateral roots on the primary root in (a) redrectangles. (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 numberof lateral and adventitious roots of WT (grey bars) and nal2/3 (white bars)seedlings grown with or without IAA. The photo for adventitious rootnumbers is not shown. LR, lateral root; AR, adventitious root. Mean andSD values were obtained from 15 plants for each line. Asterisks indicatestatistically significant differences compared with WT as determined byStudent’s t-test (**, P < 0.01; ***, P < 0.001). Bars: (a, b) 1 cm; (c–f)2 mm.



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are remarkably narrower and thinner (Fig. 3a,b; Table S2), result-ing in opened spikelets during grain filling (Fig. 3a). Addition-ally, a reduction in cell size and number, and extra VBs wereobserved in the margins of the lemma (Fig. 3e,f; Table S3). TheArabidopsis prs mutants showed repressed growth of the lateralsepals (Matsumoto & Okada, 2001). Lemma and palea in riceare presumed to be equivalent to sepals in eudicot flowers(Ferrario et al., 2004). In this respect, defects in floral organdevelopment of nal2/3 are similar to those of prs mutants, inwhich marginal cells of the adaxial and abaxial sepals disappear.Unlike prs, however, nal2/3 mutants show no defect in otherfloral organs (Fig. S3). This suggests that WOX3 functions inflower development differ among rice, maize and Arabidopsis,mainly due to differences in morphology.

Unlike the reduced VBs in leaves, in the margin of the lemmain nal2/3 we observed two more VBs and reduced size and num-ber of cells (Fig. 3e,f). It is not certain that the regulatory roles ofOsWOX3A differ in leaf and spikelet development. In lemmadevelopment, the rice MADS-box genes OsMADS1 andOsMAD34 act as important regulators (Jeon et al., 2000a; Prasadet al., 2005; Gao et al., 2010). Recently, Tanaka et al. (2012)showed that lemma and palea were reduced in the spikelets of riceyab5/tob1 mutants; they suggested that YAB5/TOB1 functions to

maintain proper meristem organization in spikelet development.It thus appears that OsWOX3A is required for the positive regula-tion by YAB5/TOB1 during spikelet development, because YAB5expression was slightly reduced in nal2/3 (Fig. 6a), andOsWOX3A is highly expressed in the young panicle (Fig. 5c),especially in the longitudinal veins of developing spikelets(Fig. 5j).

OsWOX3A is involved in formation of tillers and LRs,possibly by regulatingOsPIN1 andOsPIN2 expression

Among the leaf development-associated genes, OsWOX3A proba-bly acts as a negative regulator of YAB1, YAB2, YAB3, YUC1,YUC4 and OsPIN2 (Dai et al., 2007). However, it appears thatOsWOX3A also acts as a positive regulator of several leaf devel-opment-associated genes, especially those involved in auxin syn-thesis and transport, such as ARF1, ARF4, YUC8/NAL7 andOsPIN1 (Fig. 6a), although it remains to be determined whetherOsWOX3A binds to their promoter regions directly as a tran-scriptional activator (Fig. 9).

Auxin plays important roles in controlling cell identity, celldivision, initiation of lateral organs, and formation of leafmargins and vasculature (Scanlon, 2003; Scarpella et al., 2006;Grieneisen et al., 2007). The ARFs are all transcription factorsthat bind to auxin-response elements in the promoters of earlyauxin-responsive genes (Tiwari et al., 2003). Although YUC andARF transcript levels were altered, endogenous concentrations offree IAA were not significantly changed in nal2/3 (Fig. S8). Theauxin efflux carrier PIN1 facilitates polar transport of auxin inArabidopsis (Paponov et al., 2005; Kleine-Vehn et al., 2009)and in rice (Xu et al., 2005; Qi et al., 2008). Nonpolarizedlocalization of PIN1 causes abnormal distribution of the auxinmaxima required for vascular networking in Arabidopsis(Shirakawa et al., 2009). In rice, repression of OsPIN1 by RNAicaused defects in adventitious roots and increased the numberof tillers. Overexpression of OsPIN1 increased LR number (Xuet al., 2005) and overexpression of OsPIN2 increased tillernumber 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 upregulationof OsPIN2. Furthermore, the reduced LR number in nal2/3 wasrescued by exogenous IAA treatment, similar to the case of

(a) (b) (c) (d) (e)

Fig. 8 OsWOX3A expression during lateral root formation. Two-week-old transgenic rice (Oryza sativa) plants expressing ProOsWOX3A:GUS were stainedto observeOsWOX3A expression at various developmental stages. (a–c) GUS expression in lateral root (LR) initiation stages (a) and early developmentalstages (b, c). Note that ProOsWOX3A:GUS was highly expressed at the base of lateral root primordia. Arrowheads indicate GUS signal detected at the baseof LR. (d, e) GUS expression in later stages of LR development. Note that ProOsWOX3A:GUS signals were only detected in the vasculatures. Asteriskindicates the absence of GUS signal at the base of LR. Bars, 50 lm.

Fig. 9 The N- and C-terminal regions of OsWOX3A showed transactivationactivity. Yeast cells co-transformedwith the bait clones containing full-length, N-terminal (His-rich domain) or C-terminal (Gln-rich domain)region of OsWOX3A (WT) and empty prey clone survived in the highlystringent SD/-Ade/-His/-Leu/-Trp media (�4), but those with the full-length or N-terminal region of nal2 or nal3 protein did not survive. Yeastcells with the C-terminal region of nal2 protein survived, but not those ofnal3 protein in the (�4) media. PC, pGBKT7-53 and pGADT7-T vectors(positive control); NC, empty pGBKT7 and pGADT7 vectors (negativecontrol).





OsPIN1-RNAi plants (Xu et al., 2005). This strongly suggeststhat increased tillers and reduced LRs in nal2/3 seem to beattributable to altered expression of OsPIN1 and OsPIN2. InArabidopsis, AtPIN1 is expressed in the inner layer cells of LRprimordia; however, AtPIN2 is detected in the outer cells onlyafter primordium emergence (Benkova et al., 2003). Precise reg-ulation of spatial expressions of AtPIN1 and AtPIN2 is crucialin LR development, which is based on balanced auxin supply tothe tip and its PIN2-dependent retrieval (Benkova et al., 2003).Notably, OsWOX3A expression was detected at the base regionof LR primordia during LR emergence (Fig. 8). Therefore, itcan be speculated that OsWOX3A modulates LR organogenesisby regulating OsPIN1 and OsPIN2.

The YUC genes, whose expression was altered in nal2/3(Fig. 6a), are involved in auxin biosynthesis and thereby controlleaf blade outgrowth and margin development (Wang et al.,2011). The PRS and WOX1 are required for promoting cell pro-liferation in outgrowth of leaf blade, maybe due to activated celldivision via auxin and cytokinin (Nakata et al., 2012). In the nsmutants of maize, microarray data showed that TIR1 and ARF-GAP, auxin signalling- and transport-related genes are downregu-lated (Zhang et al., 2007). In addition, STF of Medicagotruncatula and LAM1 of tobacco, the Arabidopsis WOX1 ortho-logues, also affect auxin concentrations (Tadege et al., 2011),strongly suggesting that the WOX family acts in the regulation ofauxin biosynthesis, signalling or transport.

Our physiological and molecular data suggest that OsWOX3Aregulates the transcription of genes involved in auxin synthesis,signalling and/or polar transport for lateral cell proliferation dur-ing vegetative and reproductive organ development (Table S2,Table S4). Further identification of as-yet-unknown regulatorygenes downstream of OsWOX3A will provide further insightsinto the function of theWOX3 subfamily.

Acknowledgements

This work was supported by a grant from the Next-GenerationBioGreen 21 Program (Plant Molecular Breeding Center No.PJ008128), Rural Development Administration, Republic ofKorea.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Scanning electron microscopy of the adaxial and abaxialsurfaces of nal2/3 leaves.

Fig. S2 The shoot apex region of the nal2/3 mutants displayedreduced size of the shoot apical meristem (SAM) and defectivemarginal structure in leaf primordia.

Fig. S3 nal2/3 displayed normal morphology in pistil and stamendevelopment.

Fig. S4 nal2/3 showed a significantly increased number of tillersat both active and maximum tillering stages.

Fig. S5Characterization of nal2 and nal3 mutant proteins andOsWOX3A homologues.

Fig. S6 Phylogenic tree of the WOX families.

Fig. S7Complementation of nal2/3 by OsWOX3A.

Fig. S8 Endogenous free-IAA concentrations in the whole plantsof 2-wk-old wild-type (WT) and nal2/3 using IAA ELISA analy-sis.

Table S1 Primers used in this study

Table S2Morphological characteristics of nal2/3

Table S3Cell size and cell number in the marginal region oflemma of wild-type and nal2/3 plants

Table S4 Agronomic traits in nal2/3

Table S5 Accession numbers of WOX proteins in the phylogenictree (Fig. S6)

Methods S1Quantification of endogenous IAA.

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