increased leaf angle1, a raf-like mapkkk that interacts with a

Increased Leaf Angle1, a Raf-Like MAPKKK That Interacts witha Nuclear Protein Family, Regulates Mechanical TissueFormation in the Lamina Joint of Rice C W

Jing Ning,a,1 Baocai Zhang,b,1 Nili Wang,a Yihua Zhou,b,2 and Lizhong Xionga

a National Key Laboratory of Crop Genetic Improvement and National Center for Plant Gene Research (Wuhan), Huazhong

Agricultural University, Wuhan 430070, Chinab State Key Laboratory of Plant Genomics and National Centre for Plant Gene Research (Beijing), Institute of Genetics and

Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Mitogen-activated protein kinase kinase kinases (MAPKKKs), which function at the top level of mitogen-activated protein

kinase cascades, are clustered into three groups. However, no Group C Raf-like MAPKKKs have yet been functionally

identified. We report here the characterization of a rice (Oryza sativa) mutant, increased leaf angle1 (ila1), resulting from a

T-DNA insertion in a Group C MAPKKK gene. The increased leaf angle in ila1 is caused by abnormal vascular bundle

formation and cell wall composition in the leaf lamina joint, as distinct from the mechanism observed in brassinosteroid-

related mutants. Phosphorylation assays revealed that ILA1 is a functional kinase with Ser/Thr kinase activity. ILA1 is

predominantly resident in the nucleus and expressed in the vascular bundles of leaf lamina joints. Yeast two-hybrid

screening identified six closely related ILA1 interacting proteins (IIPs) of unknown function. Using representative IIPs, the

interaction of ILA1 and IIPs was confirmed in vivo. IIPs were localized in the nucleus and showed transactivation activity.

Furthermore, ILA1 could phosphorylate IIP4, indicating that IIPs may be the downstream substrates of ILA1. Microarray

analyses of leaf lamina joints provided additional evidence for alterations in mechanical strength in ila1. ILA1 is thus a key

factor regulating mechanical tissue formation at the leaf lamina joint.

INTRODUCTION

Over the course of evolutionary history, plants have developed

sophisticated signalingmechanisms to initiate cellular responses to

external or internal stimuli. One such mechanism is the mitogen-

activated protein kinase (MAPK) cascade composed of three levels

ofSer/Thr-specificprotein kinases (MAPKkinasekinase [MAPKKK],

MAPK kinase [MAPKK], and MAPK) (Jonak et al., 2002). MAPK

cascades act as important signal modules for diverse cellular

activities, including cell division and differentiation, responses to

abiotic and biotic stresses, and programmed cell death (Tena et al.,

2001; Nakagami et al., 2005). Due to their functional importance,

MAPK cascades are evolutionarily conserved in eukaryotes. Com-

pared with the numerous MAPK cascades documented in yeasts

and animals, the complete MAPK cascades identified in plants are

far fewer. One example is MEKK1–MKK4/MKK5–MPK3/MPK6–

WRKY22/WRKY29, acting downstream of the flagellin receptor

Flagellin Sensitive2; its function is to regulate plant defense re-

sponses (Asai et al., 2002; Kim et al., 2009). In plants, major

challenges still remain in identification of individual kinases, espe-

cially due to the existence of more than one hundred components

of plant MAPK cascades (Ichimura, 2002).

Analysis of the Arabidopsis thaliana genome revealed the pres-

ence of 20 MAPKs, 10 MAPKKs, and 80 MAPKKKs (Colcombet

and Hirt, 2008). As the first level of the phosphorylating cascade,

the MAPKKK family has the most members, the majority of which

have been identified based only on gene sequences (Ichimura,

2002). MAPKKKs have been further divided into three groups

(Groups A toC) or two distinct subfamilies based on the sequence

of their kinase catalytic domain. Group A comprises the MEKK

family, andGroupsBandCconsist of theRaf-like family (Ichimura,

2002). To date, several MEKK family members have been iden-

tified. Tobacco (Nicotiana tabacum) Protein Kinase1 (NPK1) was

the first isolated plant MEKK and is known to regulate cytokinesis

(Banno et al., 1993; Nishihama et al., 2001, 2002). Overexpression

of the kinase domain of NPK1 enhances abiotic stress tolerance

(Soyano et al., 2003; Shou et al., 2004). Compared with the

functions of the MEKK family, the functions of the Raf-like family

are largely unknown. The well-studied Raf-like MAPKKKs in-

clude Constitutive Triple Response1 (CTR1) and Enhanced Dis-

ease Resistance1 (EDR1), which negatively regulate ethylene

and defense responses in Arabidopsis, respectively (Kieber

et al., 1993; Frye et al., 2001). The rice (Oryza sativa) homolog

Accelerated Cell Death and Resistance1/EDR1 was found to

play a positive role in the regulation of disease resistance (Kim

et al., 2009). Drought hypersensitive mutant1 (DSM1), another

rice Raf-like MAPKKK, mediates drought resistance through

1 These authors contributed equally to this work.2 Address correspondence to [email protected] authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Yihua Zhou([email protected]) and Lizhong Xiong ([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.111.093419

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

reduces the time to publication by several weeks.

The Plant Cell Preview, www.aspb.org ã 2011 American Society of Plant Biologists. All rights reserved. 1 of 14

ROS scavenging (Ning et al., 2010). However, all of the reported

Raf-like MAPKKKs are members of Group B. No Group C

members have yet been functionally characterized; they are

known only by sequencing and/or biochemical description

(Tregear et al., 1996; Ichimura et al., 1997).

Rice is one of the world’s most important crops and is also a

model organism. Increasing rice production to support human

population growth is a great challenge, and breeding rice vari-

eties with ideal architecture is an important strategy for the

further improvement of grain yield (Yuan, 1997; Jiao et al., 2010).

Among numerous factors, leaf phenotypes (e.g., leaf angle, leaf

temperature, and leaf senescence) are highly correlated with

yield potential (Murchie et al., 1999; Long et al., 2006; Mitchell

and Sheehy, 2006). Amore erect leaf facilitates the penetration of

sunlight, enhancing photosynthetic efficiency and occupying

less space in dense planting (Duncan, 1971; Sakamoto et al.,

2006; Doust, 2007).

Leaf angle is the inclination between the leaf blade and vertical

culm (Zhao et al., 2010). The leaf lamina joint joins the rice leaf

blade and sheath, contributing significantly to this trait. Any

effects on the development of the leaf lamina joint may thus

regulate leaf angle. Leaf angle is a complex trait; several related

quantitative trait loci have been reported (Li et al., 1998, 1999;

Sakamoto et al., 2006). It has been found that most of the

identified rice mutants with altered leaf inclination have arisen

from abnormal division and expansion of adaxial cells in the

collar (Nakamura et al., 2009; L.Y. Zhang et al., 2009; Zhao et al.,

2010). The corresponding genes are largely involved in the

biosynthesis or signaling of the phytohormone brassinosteroids

(BRs) (Wada et al., 1981; Yamamuro et al., 2000; Wang et al.,

2008; D. Li et al., 2009; Tanaka et al., 2009). Stimulation of leaf

inclination is thus a typical effect of BR in rice (Wada et al., 1981).

Increasing evidence shows that not only BR but also other

phytohormones, including auxin, ethylene, and gibberellin, par-

ticipate in determination of the leaf angle (Cao and Chen, 1995;

Shimada et al., 2006). Many of these phytohormones act syner-

gistically with BR (Cohen andMeudt, 1983; Shimada et al., 2006;

Hardtke et al., 2007; Song et al., 2009). Exceptions have also

been found: For example, a gain-of-function rice mutant that

exhibits increased tiller number, enlarged leaf angle, and dwarf-

ism is due to a mutation in TLD1, a gene that encodes an indole-

3-acetic acid amido synthetase (S.W. Zhang et al., 2009). This

finding highlights the fact that regulation of rice leaf inclination is

complicated and its mechanism is not yet fully understood.

Considering that the mechanical tissues consisting of vascular

bundles and sclerenchymatous cells provide mechanical sup-

port for the plant body, the status of these tissues in the leaf

lamina joint should correlate with altered leaf angle. Several

studies have revealed that abnormalities in culm mechanical

tissues often result in inferior mechanical strength and cause

plant lodging (Li et al., 2003; Tanaka et al., 2003; B. Zhang et al.,

2009; Zhou et al., 2009; Zhang et al., 2010). However, there is

currently no evidence to support this hypothesis regarding

tissues in the leaf lamina joint.

Here, we report the characterization of a rice mutant, in-

creased leaf angle1 (ila1), that exhibits abnormal mechanical

tissues and cell wall composition in the leaf lamina joint, unre-

lated to BR responses. ILA1 encodes a Raf-like MAPKKK of

Group C and is mainly expressed in the leaves and leaf lamina

joint. Our results show that ILA1 is involved in mechanical tissue

formation in the leaf lamina joint and that ILA1 physically interacts

with a family of uncharacterized proteins (ILA1 interacting pro-

teins [IIPs]). Our findings not only provide unique genetic ev-

idence for the functions of Group C Raf-like MAPKKKs, but also

unravel a differentmechanism in the regulation of the leaf angle in

rice.

RESULTS

Identificationof ila1, aMutantwithaPhenotypeUnrelated to

BR Responses

A T-DNA insertion mutant that shows increased leaf inclination

angle (designated ila1) was isolated from the Rice Mutant Data-

base (http://rmd.ncpgr.cn). Identification of the flanking se-

quence of T-DNA using thermal asymmetric interlaced PCR

revealed that the insertion occurs in the fourth exon of an

expressed gene (Os06g50920, RiceGenomeAnnotation Project,

http://rice.plantbiology.msu.edu/), 963 nucleotides downstream

from the ATG start codon (Figure 1A). The increased leaf incli-

nation cosegregated with the homozygous T-DNA insertion

based on PCR analysis (see Supplemental Figure 1A online).

RT-PCR assays showed that the intact transcript ofOs06g50920

was undetectable in the ila1 mutant (Figure 1B), indicating that

the T-DNA insertion nearly represses expression of the targeted

gene.

The mutant plants show normal growth with the exception of

increased leaf angle and slight dwarfism. In ila1, the bending of

the leaf blade occurs at the lamina joint. As shown in Figures 1C

to 1E, the leaf angle of ila1 was significantly greater (76%) than

that of wild-type plants. This phenotype was not observed in the

newly developing leaves of ila1. We therefore examined the

dynamic alterations of leaf angle during leaf development at the

tillering stage. Three days after the appearance of a new leaf in

ila1, the angle of that leaf gradually increased and reached a

maximum of ;678 on the eighth day, whereas the leaf angle of

wild-type plants reached a plateau at 188 (see Supplemental

Figures 1B and 1C online). Therefore, alteration of leaf angle in

ila1 mutants is developmentally modulated.

Altered leaf inclination is a typical response to BR in rice (Wada

et al., 1981). To test whether the ila1 phenotype is related to BR

responses, we treated the wild-type and mutant seedlings with

different concentrations of BR and compared the length of

coleoptiles and roots because BR can promote coleoptile elon-

gation and decrease root length in rice (Yamamuro et al., 2000).

The wild-type plants and the ila1 mutants showed no significant

differences in coleoptile and root length, suggesting that the ila1

mutant has the same BR response as the wild-type plants (see

Supplemental Figures 2A and 2B online). BR-induced rice leaf

inclination often results from rapid expansion and propagation of

collar adaxial cells. We used scanning electron microscopy to

observe the adaxial surface and longitudinal sections of ial1 and

wild-type leaf lamina joints. No significant alterations in cell

expansion and propagation were found in the adaxial region (see

Supplemental Figures 2C to 2H online). Therefore, we conclude

2 of 14 The Plant Cell

that the increased leaf angle of ila1 is not due to altered BR

responses.

Increased Leaf Angle in ila1 Results from Abnormal

Mechanical Tissues at the Leaf Lamina Joint

To investigate the cellular mechanism of increased leaf angle in

the ila1mutant, we examined the anatomical structure of the leaf

lamina joint in the wild-type and ila1 plants. Fresh hand-cut cross

sections of leaf lamina joints showed that the vascular bundles in

ila1 plants were smaller than those in the wild-type plants

(Figures 2A to 2F). The smaller vascular bundles in the ila1 leaf

lamina joint were characterized by reduced sclerenchymatous

cells (Figures 2C to 2F). The abnormal appearance of the

mechanical tissues at the leaf lamina joint suggested an altered

cell wall composition; we therefore compared the cell wall

compositions of the wild-type and ila1 leaf lamina joint. As

predicted, the cellulose content was reduced by ;32% in the

mutant (Table 1). The levels of noncellulosic neutral sugars were

also generally reduced, with the largest reductions in Glc (;35%)

andXyl (;30%) (Table 1). However, except for an;8%reduction

Figure 1. Characterization of ila1, a T-DNA Insertion Mutant of Rice.

(A) Insertion position of T-DNA in the ILA1 gene. Exons and introns are

indicated in closed and open boxes, respectively. LB, left border; RB,

right border.

(B) RT-PCR analysis of ILA1 in the wild-type (WT) and ila1 plants. Actin1

was used as an internal control.

(C)Comparison of leaf angle in the wild-type and ila1 plants at the tillering

stage. Values are the means 6 SD (n = 30). **P <0.01 (t-test).

(D) and (E) A wild-type (D) and ila1 plant (E). The leaf angle in the mutant

plants is increased, as shown in the figures embedded at the right

corners. Arrows indicate the leaf lamina joint.

[See online article for color version of this figure.]

Figure 2. The ila1 Leaf Lamina Joint Has Smaller Vascular Bundles and

Reduced Mechanical Strength.

(A) and (B) Cross sections of a wild-type (A) and ila1 (B) leaf lamina joint.

The red broken lines indicate the abaxial vascular bundles, and the black

broken lines indicate the adaxial vascular bundles. Bars = 1 mm.

(C) and (D) Enlargements of the areas denoted by the red and black

rectangles, respectively, in (A). Bars = 360 mm.

(E) and (F) Enlargements of the areas denoted by the red and black

rectangles, respectively, in (B), showing the significantly smaller vas-

cular bundles and reduced sclerenchymatous cells in the ila1 lamina

joint. Bars = 360 mm.

(G) Leaf lamina joint used for measuring mechanical strength. Arrow-

heads indicate the breakage point induced by the measurements. WT,

wild type.

(H) Triple measurements of the breaking force and extension length in

wild-type and ila1 leaf lamina joints.

Ab, abaxial; Ad, adaxial; Sc, sclerenchymatous cells; V, vascular bundles.

ILA1 Regulates Rice Leaf Inclination 3 of 14

in cellulosecontent, no significant alterations inmonosaccharides

were found in the ila1mutant leaf blades (see Supplemental Table

1 online). Deficiencies inmechanical tissues and cellulose content

suggested that the mechanical strength of the leaf lamina joint

might be affected in the ila1 mutant. We compared the mechan-

ical properties of the leaf lamina joint in the wild-type and ila1

plants. The force required to break the mutant leaf lamina joint

was only two-thirds of the force required for the wild-type leaf

lamina joint (Figures 2G and 2H), and the extension length of the

ial1 leaf lamina joint was;47% greater than that of the wild-type

leaf lamina joint (Figure 2H). Therefore, abnormalities in mechan-

ical tissue formation cause altered cell wall composition and

inferior mechanical strength at the mutant leaf lamina joint,

consequently leading to the increased leaf angle in ila1 plants.

ILA1 Encodes a Group C Raf-Like MAPKKK with Ser/Thr

Kinase Activity

Thermal asymmetric interlaced PCR showed that the increased

leaf angle of ila1 arises fromaT-DNA insertion inOs06g50920. To

confirm the sequence identity of ILA1, a complementation test

was performed by transforming the ila1 mutant with an 8.8-kb

genomic region that included the complete open reading frame

(ORF) and putative promoter region for ILA1 (pILA1) (see Sup-

plemental Figure 3A online). All of the complementation lines

showed the wild-type leaf inclination (see Supplemental Figures

3B and 3C online), suggesting that Os06g50920 is ILA1.

According to the rice genome annotation database (http://rice.

plantbiology.msu.edu) andBLASTsearch (www.ncbi.nlm.nih.gov/

BLAST/), ILA1 encodes a putative MAPKKK protein with a length

of 564 amino acids and a molecular mass of 63 kD. According to

the Pfam database, the deduced ILA1 has an N-terminal ACT

domain (PF01842) and a C-terminal kinase domain (PF07714)

(Figure 3A), which likely confer a protein regulatory feature and

kinase activity, respectively. Sequence alignment of ILA1 with

several identified MAPKKKs from three representative plant spe-

cies revealed that the characteristic features for MAPKKK pro-

teins, including all of the 11 subdomains and a Lys in the ATP

binding site, are highly conserved in ILA1 (see Supplemental

Figure 4 online). To assign ILA1 to a specific MAPKKK family, a

phylogenetic treewas built using ILA1 and its homologs. ILA1 was

clustered into Group C of the Raf-like family (Figure 3B; see

Supplemental Data Set 1 online).

As a putative Raf-like MAPKKK, ILA1 may possess kinase

activity. To test this possibility, the coding sequence (CDS) of

ILA1 was fused with glutathione S-transferase (GST) for protein

purification; the resulting protein was subjected to a kinase

activity assay. Upon incubation with 32P-labeled ATP, GST-ILA1

phosphorylated itself and myelin basic protein (MBP), a widely

used universal substrate for kinase activity assays (Eichberg and

Iyer, 1996). TheGSTprotein alone generated undetectable signal

and served as a negative control (Figure 4A). Phosphoamino acid

analysis was further conducted to determine the phosphoryla-

tion ability of ILA1. After separation by two-dimensional thin layer

electrophoresis, phosphoserine and phosphothreonine were

detected in the phosphorylated ILA1 and MBP, whereas phos-

photyrosine was not found (Figures 4B and 4C). These results

suggest that ILA1 is a Ser/Thr protein kinase.

ILA1 IsMainly Localized in theNucleus andExpressed in the

Leaf Lamina Joints

Next, we fused a green fluorescent protein (GFP) to the N

terminus of ILA1 to examine its subcellular localization. Expres-

sion of this fusion protein in rice protoplasts revealed that the

GFP signals were found in both the cytoplasm and the nucleus,

with a majority in the nucleus (Figure 5A). Transgenic rice plants

harboring the GFP-ILA1 transgene showed a consistent locali-

zation pattern: The fluorescence signals of the fusion protein

were mainly observed in the nuclei (Figure 5B).

The expression pattern of ILA1 was examined using two

methods: (1) quantitative real-time PCR (qRT-PCR) and (2) eval-

uation of the expression of the b-glucuronidase (GUS) reporter,

driven by the ILA1 putative promoter in rice plants. qRT-PCR

analysis showed that the expression level of ILA1 is quite low in the

examined organs, with relatively higher levels in leaves and leaf

lamina joints (Figure 6A). Examination of GUS activity in three

independent transgenic lines further revealed that theGUS signals

were strongly observed in the leaf lamina joints and were also

detected in vascular bundles of leaves and coleoptiles (Figures 6B

to 6D). In addition, appearance of GUS activity in the leaf lamina

joint is developmentally regulated. GUS activity was absent in the

first leaves (the youngest one from the top), increased from the

second to the third leaves, and remained in the mature leaves

(Figures 6E to 6H). Fresh hand-cut cross sectioning of the GUS-

stained leaf lamina joint further showed that the GUS signals were

restricted to mechanical tissues, including vascular bundles and

sclerenchymatous cells (Figures 6I and 6J). The tissue-specific

and developmental expression pattern of ILA1 matches well with

the phenotypes of ila1 described above.

ILA1 Interacts with a Functionally Unidentified

Protein Family

MAPKKKs generally phosphorylate target proteins to mediate

signal transduction. To investigate the target protein of ILA1, the

Table 1. Compositional Analysis of Wall Residues from Wild-Type and ila1 Leaf Lamina Joints

Samples Rha Fuc Ara Xyl Man Gal Glc Cellulose

Wild type 4.4 6 0.2 3.1 6 0.2 99.7 6 6.7 212.4 6 18.9 11.1 6 0.5 44.7 6 3.1 52.5 6 3.6 258.8 6 15.6

ila1 3.4 6 0.2* 2.6 6 0.2 81.3 6 6.6* 149.4 6 10.9* 8.6 6 0.8* 40.2 6 3.1 34.0 6 3.7* 175.2 6 12.6*

AIRs were prepared from the leaf lamina joint of ila1 and wild-type plants. The alditol acetate derivatives were analyzed by gas chromatography–mass

spectrometry. The results are given as the means (mg/g of AIR) of three replicates 6 SD. The asterisk indicates a significant difference between the

wild type and mutant determined by the least significant difference t test at P < 0.01.


full-length ILA1 protein was used as bait to screen a yeast two-

hybrid library of rice. After screening for approximate one million

transformants, 74 interaction clones were identified. Notably, 69

of them were reproducibly derived from six ORFs (see Supple-

mental Figure 5 online). Bioinformatic analysis showed that these

six genes represent an uncharacterized small family. These IIPs

were designated as IIP1 through IIP6 (Os01g43370,Os02g15880,

Os02g36590, Os04g38520, Os04g54830, and Os06g33180), re-

spectively. The six IIP proteins range from 628 to 655 amino acids

in length and possess a highly conserved domain in themiddle. In

addition, the IIP proteins have low sequence identity (;30%)with

the Arabidopsis WRKY19 transcription factor and were grouped

into two subfamilies togetherwith threeArabidopsis IIP homologs

(see Supplemental Data Set 2 and Supplemental Figure 6 online).

qRT-PCR and microarray data (L. Wang et al., 2010) further

showed that these genes are ubiquitously expressed inmany rice

organs, although the expression levels vary (see Supplemental

Figure 7 online).

To confirm the interaction of ILA1 with IIPs, the ILA1 protein

was split into kinase (ILA1K) and regulatory (ILA1R) domains and

subjected to interaction analysis. As shown in Figure 7A, the

kinase domain of ILA1 is involved in the interaction with IIPs in

subfamily B, but notwith IIPs in subfamily A.We also note that the

N-terminal regulatory domain interacts with IIPs. Next, we ex-

plored the interaction of ILA1 and IIPs in vivo using a bimolecular

fluorescence complementation (BiFC) approach. Two IIPs (IIP2

and IIP4) were chosen as representatives for subfamilies A and B

in the following examinations. Coexpression of the ILA1-fused

C-terminal part of yellow fluorescent protein (ILA1-cYFP) and the

IIP-fused (IIP2/IIP4) N-terminal part of YFP (IIP2-nYFP/IIP4-

nYFP) in rice protoplasts produced obvious fluorescent signals

in the nuclei (Figure 7B). However, no signal was observed by

coexpressing each of these constructs with an empty vector

(Figure 7B). This interaction was further confirmed by a coimmu-

noprecipitation (Co-IP) assay. We generated a specific polyclo-

nal antibody against IIP4, but wewere unable to generate one for

IIP2 due to an unknown problem. Using this antibody, we

detected IIP4 proteins in the affinity-purified total protein ex-

tracts from the transgenic plants harboring a FLAG-tagged

ILA1 transgene (see Supplemental Figure 3A online). However,

Figure 3. ILA1 Is a RAF-Like MAPKKK of Group C.

(A) Domain structure of ILA1.

(B) Phylogenetic tree of ILA1 and other MAPKKKs in plants. Prefixes on protein names indicate species of origin. At, Arabidopsis thaliana; Bn, Brassica

napus; Nt, Nicotiana tabacum; Os, Oryza sativa; Le, Solanum lycopersicum var lycopersicum; Cm, Cucumis melo; Fs, Fagus sylvatica; Ah, Arachis

hypogaea.


IIP4 was not detected in the extracts from plants expressing the

FLAG-tagged DSM1, another Raf-like MAPKKK (Ning et al.,

2010) (Figure 7C). These results suggest that ILA1 interacts with

IIP proteins.

IIPs, the Likely Targets of ILA1, Are Nuclear Proteins with

Transactivation Activity

In light of the evidence for interaction between ILA1 and IIPs, it

seemed that IIPs may be the phosphorylated substrates of ILA1.

We selected IIP4 as a representative to test this hypothesis. IIP4

fused to a polyhistidine tag (His-IIP4) was purified and incubated

with GST-ILA1 for an in vitro kinase activity assay. As shown in

Figure 8A, radioactively labeled IIP4 was detected. These results

reveal that IIP4 is a phosphorylated substrate of ILA1, as is likely

the case for the other IIPs.

The function of the IIPs was the focus of our next investi-

gation. The Gene Ontology database (http://www.geneontol-

ogy.org/) annotated IIPs as putative transcription factors.

Because nuclear localization is a significant feature of tran-

scription factors, we fused GFP to the N terminus of IIP2

and IIP4 and transformed the rice protoplasts. Detection of the

fusion proteins in the nuclei of the transformed cells suggested

that IIP2 and IIP4 are nuclear-localized proteins (Figures 8B and

8C). We further examined the transactivation activity of IIP2 and

IIP4 in yeast by fusing each of them with the GAL4 DNA binding

domain. As shown by the yeast growth status on the selective

medium and the b-Gal assays, both IIP2 and IIP4 exhibited

transcription activity in yeast (Figure 8D).

Expression Levels of Genes Involved in Cell Wall Synthesis

Are Significantly Downregulated in ila1

To investigate the molecular basis of ILA1’s effects on rice leaf

angle, we examined the expression profiles of ila1 and wild-type

leaf lamina joints using an Affymetrix Rice GeneChip. Of the

57,381 probe sets in themicroarray analysis, 820 probes showed

more than fourfold alterations, corresponding to 548 downregu-

lated and 38 upregulated annotated genes (see Supplemental

Tables 2 and 3 online). Previous studies have revealed that some

transcription factors and genes involved in hormone biosynthe-

sis or signaling regulate leaf angle in rice (Hong et al., 2002; Bai

et al., 2007; Zhao et al., 2010). We therefore compared the

expression levels of these genes using the wild-type and mutant

microarray data. Except for two upregulated and three down-

regulated genes, no significant alterations were observed in the

remaining hormone-related genes and transcription factors for

Figure 4. ILA1 Has Ser/Thr Kinase Activity.

(A) Phosphorylation analysis of ILA1, showing that ILA1 phosphorylates

itself and the general substrate MBP. GST proteins added instead of

GST-ILA1 were used as a negative control. The phosphorylated proteins

were separated in SDS-PAGE gels and subjected to autoradiography

(left panel) or stained with Coomassie blue (right panel).

(B) and (C) Phosphoamino acid analysis of the GST-ILA1–phosphorylated

ILA1 (B) and MBP (C). The positions of phosphoamino acids (pSer, pThr,

and pTyr) were revealed by autoradiography (left panel) or by spraying

with ninhydrin (right panel).

[See online article for color version of this figure.]

Figure 5. Subcellular Localization of GFP-ILA1.

(A) Transient expression of GFP-ILA1 in rice protoplasts. DIC, differential

interference contrast. Bar = 5 mm.

(B) Fluorescent signals in transgenic rice plants expressing GFP-ILA1.

The nuclei were counterstained with 4’,6-diamidino-2-phenylindole

(DAPI). Bar = 30 mm.


either data set (Figures 9A and 9B). However, many genes

possibly involved in cell wall formation were downregulated in

ila1 (see Supplemental Table 3 online). As verified by qRT-PCR,

eight cell wall synthesis–related genes that we examined showed

reduced transcriptional levels (ranging from 83 to 38%of thewild

type) in the mutant plants (Figure 9C), providing direct support

for the effects of ILA1 in sclerenchymatous cell formation and

cell wall biosynthesis. We conclude that the ILA1 mutation

significantly represses the expression of genes involved in

cell wall synthesis and consequently causes the increased leaf

angle.

DISCUSSION

ILA1 Is a Functional Kinase of the Group C Raf-Like

MAPKKK Family

MAPKKKs act at the top level ofMAPK cascades and show great

sequence diversity (Ichimura, 2002). As determined by the highly

conserved kinase domain, plant MAPKKKs are categorized

either asMEKKs and Raf-like families or as three groups (Groups

A to C). Among the eighty MAPKKKs in Arabidopsis, only a

limited number of MEKKs (Group A) and Group B Raf-like

MAPKKKs have been identified (Kieber et al., 1993; Frye et al.,

2001; Nishihama et al., 2001, 2002; Soyano et al., 2003; Shou

et al., 2004). In this study, ILA1 was identified as a putative Raf-

like MAPKKK of Group C, based on its deduced amino acid

sequence. In vitro biochemical assays further verified that ILA1

can phosphorylate itself and the general substrate MBP at Ser

and Thr residues. ILA1 is therefore a functional Ser/Thr kinase. In

classic MAPK cascades, the downstream targets of MAPKKKs

are MAPKKs, followed by MAPKs. In plants, the identified

complete MAPK cascades are limited to the MEKK members,

including the pathways MEKK1–MKK4/MKK5–MPK3/MPK6–

WRKY22/WRKY29 (Asai et al., 2002), YDA–MKK4/MKK5–

MPK3/MPK6 (Bergmann et al., 2004; Wang et al., 2007), and

NPK1–NQK1–NRK1 (Soyano et al., 2003). Such cascades have

not been identified for Raf-like family members. The substrates

of EDR1 and CTR1, two well-characterized Raf-like MAPKKKs,

are still unclear (Frye et al., 2001). CTR1 was reportedly able to

inhibit MKK9–MPK3/MPK6 activation and probably acts as an

unconventional MAPKKK (Yoo et al., 2008). As a previously

undescribed Raf-like MAPKKK of Group C, ILA1 may or may not

follow the traditional MAPK cascades. A pairwise interaction test

with all rice MAPKKs did not reveal any positive interactions

(J. Ning and L. Xiong, unpublished data). IIPs, the identified

interacting proteins of ILA1, are not MAPKKs. Therefore, ILA1

probably mediates a signaling pathway distinct from the tradi-

tional MAPK cascades.

ILA1 Regulates Mechanical Strength in the Leaf

Lamina Joint

To our knowledge, functions of Group C Raf-likeMAPKKKs have

not been described previously. The T-DNA insertion mutation in

ILA1 provides a valuable opportunity to evaluate their functions.

Here, a series of evidence has revealed that ILA1 affects leaf

angle by regulatingmechanical tissue formation at the leaf lamina

joint. First, the ila1 mutant displays increased leaf angle. This

phenotype gradually manifests as leaf development progresses.

Second, anatomic analysis revealed smaller vascular bundles

and a reduced number of sclerenchymatous cells in the ila1 leaf

lamina joint. The abnormal mechanical tissues in the ila1 leaf

lamina joint result in low levels of cellulose and other cell wall

monosaccharides, leading to inferior mechanical support for

mutant leaves. Third, ILA1 is developmentally expressed in the

leaf lamina joint. Strong GUS signals were observed in vascular

bundles and sclerenchymatous cells in the leaf lamina joint.

Fourth, genome-wide exploration of the expression profiles of

ila1 and wild-type leaf lamina joints showed that the expression

Figure 6. Expression Patterns of ILA1.

(A) qRT-PCR analysis of ILA1 expression in different rice organs. The

error bars represent the SE of the mean values of two biological

replicates.

(B) to (H) Examination of GUS activity in the transgenic plants expressing

ILA1pro:GUS. The GUS activity is shown by arrows in the leaf lamina

joints (B) and vascular bundles of leaves (C) and coleoptiles (D). GUS

activity (indicated by arrows) is further examined in the first (E), second

(F), third (G), and developed (H) leaves of transgenic plants at the tillering

stage. Bars = 4 mm.

(I) and (J) Fresh hand-cut cross sections of leaf lamina joints, showing

the GUS activity in vascular bundles and sclerenchymatous cells. Bars =

1 mm.


of many genes involved in cell wall formation is downregulated in

the mutants. Cellulose synthase (CESA) and COBRA-like genes

are known to participate in cellulose biosynthesis in rice (Li et al.,

2003; Tanaka et al., 2003; B. Zhang et al., 2009). Although the

functions of many cellulose synthase-like (CSL) and glycosyl-

transferase (GT) genes have not been verified in rice, their

homologous genes in Arabidopsis were reported to function in

noncellulosic polysaccharide synthesis (Liepman et al., 2005;

Lerouxel et al., 2006; Pena et al., 2007; Persson et al., 2007).

Rice leaf angle is one of the important agronomic traits

affecting plant architecture and yield. Most of the previously

documented rice leaf inclination mutants arise from BR-induced

cell division and/or elongation at the adaxial surface of the leaf

lamina joint. Suppression of BR biosynthesis or signaling gener-

ally blocks cell propagation/expansion and results in an erect

leaf; in the presence of BR, cell division/expansion occurs and

induces an increased leaf angle. For example, RNA interference

suppression of Brassinazole Resistant1 (BZR1), a transcription

factor involved in the BR signaling pathway, causes erect leaves

(Bai et al., 2007). Increased Lamina Inclination1 (ILI1) functions

downstream of BZR1. Overexpression of ILI1 stimulates the BR

signaling pathway and increases rice leaf angle (L.Y. Zhang et al.,

2009). However, the ila1 mutants show similar BR responses

to the wild-type plants. Microscopy analysis did not reveal

abnormal cell expansion and propagation at the adaxial surface

of the ila1 leaf lamina joint. In addition, based on the microarray

data, the expression levels of many components in BR biosyn-

thesis or signaling are not significantly altered in ila1 mutants,

except for BZR1, Brassinosteroid Insensitive1 (BRI1), and BR-

deficient dwarf1 (BRD1). BZR1 and BRI1 are downregulated in

ila1, which contradicts the previous finding that blocking their

expression generally causes erect leaves. BRD1, a gene involved

Figure 7. ILA1 Interacts with IIPs.

(A) Examination of the interaction between IIPs and the regulatory and kinase domains of ILA1 in yeast. The interactions were verified by growing the

yeast on selective medium (SC/-Leu-Trp-His with 3-AT) and conducting b-Gal assays. The regulatory domain (ILA1R) and the kinase domain (ILA1K) are

indicated in white and gray, respectively.

(B) BiFC assay to verify the interaction of ILA1 and IIPs in rice protoplasts. Transformants expressing ILA1-cYFP/IIP2-nYFP/IIP4-nYFP and the empty

vector were used as negative controls, and those expressing ZIP63 were used as a positive control.

(C) Co-IP assay to show the interaction between ILA1 and IIP4 in transgenic rice plants expressing ILA1-FLAG (pILA1cF) or DSM1KD-FLAG

(pDSM1KDF). Plant proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose

membranes, and analyzed by protein gel blotting with antibodies as indicated. The asterisk indicates a nonspecific product. WT, wild type.


in BR biosynthesis (Hong et al., 2002), is significantly upregulated

in ila1, which might be a feedback effect because ila1 does not

exhibit responses characteristic of BR overdose.

As a whole, our evidence suggests that ILA1 represents a

potentially unique paradigm in the regulation of leaf angle in rice.

It should be noted that qRT-PCR analysis revealed high expres-

sion levels of ILA1 in both leaves and leaf lamina joints. However,

ila1 exhibits a visible phenotypemainly in its leaf lamina joints but

not in its leaves. There are many possible causes for this type of

inconsistency, which has also been found in previous studies of

other genes (M. Wang et al., 2010). In this case, the reason might

be that ILA1 is specifically activated in leaf lamina joints or that

leaf lamina joints have a different structure than leaves, espe-

cially in the mechanical tissues where ILA1 is expressed.

Possible Mechanism of ILA1 Action

Identification of ILA1 phosphorylation substrates is critical for

understanding the signal transduction pathway of aGroupCRaf-

like MAPKKK member. Through yeast two-hybrid screening of

the cDNA library, six interaction proteins (IIPs) were found; these

Figure 9. Comparison of Gene Expression Levels between ila1 and Wild

Type in Determining the Leaf Angle Inclination.

(A) mRNA chip data showing the expression levels of hormone-related

genes reported to affect leaf inclination. WT, wild type.

(B) mRNA chip data showing the expression levels of transcription

factors reported to affect leaf inclination.

(C) qRT-PCR analysis of the genes involved in cell wall synthesis. The

rice UBQ5 gene was amplified as the internal control.

The error bars in (A) and (B) represent the SE, whereas those in (C)

represent the SD of the mean values of two biological replicates.

Asterisks indicate a significant difference with respect to the wild type

(t test at *P < 0.05 and **P < 0.01).

Figure 8. Characterization of IIPs.

(A) ILA1 phosphorylates IIP4. The phosphorylated proteins were sepa-

rated in SDS-PAGE gels and subjected to autoradiography (left panel) or

stained with Coomassie blue (right panel).

(B) Expression of GFP-tagged IIP2 in rice protoplasts. Bar = 5 mm.

(C) Expression of GFP-tagged IIP4 in rice protoplasts. Bar = 5 mm.

(D) Transactivation activity assays of IIP2 and IIP4 in yeast. The activity is

indicated by the growth status of yeast on selective medium (SC/-Leu-

Trp-His with 3-AT) and by b-Gal assays.


IIPs consist of a small family with unknown functions. The

interactions were further confirmed by the BiFC and Co-IP

analyses of a representative IIP. More importantly, we showed

that ILA1 could phosphorylate an IIP in vitro. IIPs are thus likely to

be the authentic downstream targets of ILA1. We also found that

IIPs may act as transcription factors because (1) they were

suggested as putative transcription factors by bioinformatic

analysis; (2) they are nuclear localized in rice; and (3) they

showed transactivation activity in yeast. In fact, direct phosphor-

ylation of a transcription factor by a MAPKKK has been previ-

ously reported (Miao et al., 2007). Many transcription factors

have been found that are involved in modulating rice leaf incli-

nation. Most of them act through BR signaling pathways (Lee

et al., 2008; Wang et al., 2008; Tanaka et al., 2009; L.Y. Zhang

et al., 2009). Some, not related to BR responses, also function in

cell division or expansion (Zhao et al., 2010). Here, as the

interacting proteins of ILA1, IIPs appear to regulate directly or

indirectly multiple aspects of cell wall–related gene expression in

the leaf lamina joint.

However, the above hypothesis needs additional support. It is

still unknown whether the kinase activity and/or the interaction

with IIPs are required for mechanical strength at the leaf lamina

joint, although we demonstrated that ILA1 interacts with IIPs and

phosphorylates IIP4. In addition, genetic evidence for IIP function

is unavailable at this time. We attempted to generate transgenic

plants with RNA interference to suppress the expression of IIP5.

These transgenic plants showed a wild-type appearance (data

not shown), which is very likely due to the redundancy of IIP

genes.We also surveyed the expression profiles of IIP genes. The

universal expression patterns indicate that IIPs are important for

plant growth. Their function in specific tissue may be determined

by the interaction proteins, such as ILA1. Therefore, further

studies are required to reveal the complete pathway mediated

by ILA1 in the regulation of leaf angle in rice, which is a different

pathway than the known BR response-related mechanisms.

METHODS

Plant Material and Growth Conditions

The seeds of ila1 (Zhonghua11 background) were obtained from the Rice

Mutant Database (http://rmd.ncpgr.cn) (Wu et al., 2003; Zhang et al.,

2006). The japonica (Oryza sativa) cv Zhonghua11 was used as the wild-

type control. Homozygous mutants were identified and used for further

analyses. All plants used in this study were grown in the fields of

experimental station or in the greenhouse at Huazhong Agricultural

University (Wuhan, China) and at the Institute of Genetics and Develop-

mental Biology, Chinese Academy of Sciences (Beijing, China).

Breaking Force Test and Microscopy

To examine the mechanical strength in leaf lamina joint, the leaf lamina

joints of third leaves (below flag leaves) from developmentally matched ial1

and wild-type plants were collected and immediately used for measure-

ment. The forces that break the samples at leaf lamina joints were

measured with a digital force/length tester (5848 Microtester; Instron).

For microscopy of the anatomical features of the wild-type and mutant

leaf lamina joint, the fresh hand-cut sections (;20 mm thickness) were

prepared and stained with 0.01% (v/v) toluidine blue in PBS buffer (137

mM NaCl, 10 mM sodium phosphate, and 2.7 mM KCl, pH 7.4). The

pictures were taken under a light microscope (Leica). For scanning

electron microscopy, the critical-point-drying samples of wild-type and

mutant leaf lamina joints were coated with gold at 20 mA for 120 s and

observed with an S-3000N scanning electron microscope (Hitachi).

Cell Wall Composition Assay

The leaf lamina joints from age-matched wild-type and ila1mutant plants

were collected and used to prepare alcohol-insoluble residues (AIRs).

Derived alditol acetates were generated from destarched AIRs and

analyzed by an Agilent 7890 Series GC system as previously described

(M. Li et al., 2009). The crystalline cellulose content wasmeasured using a

modified anthrone assay as described (Updegraff, 1969). Briefly, the

residues remained after 2 M trifluoroacetic acid hydrolysis were treated

with Updegraff reagent (acetic acid:nitric acid:water, 8:1:2, v/v) at 1008C

for 30 min. The cooled pellets were then washed with acetone and

hydrolyzed with 72% sulfuric acid. The cellulose content was quantified

via an anthrone assay.

Complementation Test

The 8.8-kb genomic region, including the complete ORF of ILA1 and its

putative promoter, were obtained from the BAC clone a0069I24, digested

with SalI, and inserted into pCAMBIA2301. The resulting construct

(pILA1) was introduced into Agrobacterium tumefaciens strain EHA105

by electroporation and transformed the ila1 mutant plants by the Agro-

bacterium-mediated transformation procedure (Hiei et al., 1994).

Subcellular Localization and GUS Activity Measurements

GFP was fused to the N terminus of ILA1 and inserted between the

cauliflower mosaic virus 35S promoter and the nopaline synthase termi-

nator at the sites of SalI and KpnI in pUC19 to generate a construct for the

transformation of rice protoplasts. Next, the CDS of ILA1was fused to the

C terminusofGFPandcloned into theGATEWAYdestinationbinary vector

pH7WGF2.0 (Karimi et al., 2005). The resulting construct was introduced

into the rice variety Zhonghua11. GFP fluorescence was examined in the

young roots of 2-week-old T1 transgenic plants under a confocal laser

scanningmicroscope (Leica TCSSP2). The roots of transgenic plantswere

incubated with 2 mg/mL of 4’,6-diamidino-2-phenylindole (Sigma-Aldrich)

to counterstain the nuclei. For subcellular localization of IIPs, the cDNA of

IIP2 and IIP4 was fused with GFP at the N terminus and cloned into the

above pUC19 vector, respectively. Transformation of rice protoplast cells

was performed as described by Zhou et al. (2009). After overnight

incubation at 258C, GFP fluorescence was observed with a confocal laser

scanning microscope (Leica TCS SP2).

The promoter of ILA1 (1433 bp upstream of ATG) was amplified from the

rice genomic DNA of Zhonghua11 and inserted into the pDX2181 binary

vector containing the GUS reporter gene. The construct was introduced

into Zhonghua11 plants by the Agrobacterium-mediated transformation

procedure.GUSactivity assayswere performed in theT0andT1 transgenic

plants using a previously described histochemical staining method (Wu

et al., 2003). Briefly, the tissues were incubated in a staining buffer (50 mM

sodium phosphate at pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 1 mgmL21

of X-Gluc, 100 mg mL21 of chloramphenicol, 1 mM potassium ferricyanide,

1 mM potassium ferrocyanide, and 20% methanol) at 378C and then

cleaned in 70% ethanol. The stained tissues were observed and photo-

graphed with a stereomicroscope (Leica MZ FLIII).

Phosphorylation Analysis

TheCDSsof ILA1and IIP4were cloned intoBamHIandEcoRI sitesofpGEX

6p-1 and GATEWAY destination pDEST17 vectors, respectively, for ex-

pression of the recombinant proteins inEscherichia coli. The ILA1-GST and


His-IIP4 recombinant proteinswerepurifiedwithGlutathioneSepharose4B

and nickel-nitrilotriacetic acid agarose resin, respectively. A kinase assay

was performed in 20 mL of kinase buffer (50 mM Tris-HCl, 10 mM MgCl2,

1 mM DTT, 0.1 mM ATP, and 5 mCi [g-32P]ATP) containing 1 mg of purified

GST-ILA1 protein with or without 1 mg of MBP. The GST protein added

instead of GST-ILA1 was used as a negative control. To examine whether

ILA1 phosphorylates IIP4,;1 mg of GST-ILA1 and;5 mg of His-IIP4 were

incubated in the same reaction buffer. The reaction was incubated at room

temperature for 30min, after which itwas terminatedby the addition of 4mL

of stopbuffer (0.35MTris-HCl, pH6.8, 10.3% [w/v] SDS, 36% [v/v] glycerol,

0.6 M DTT, and 0.012% [w/v] bromophenol blue). After separation in 12%

SDS-PAGEgels, thegelswere subjected to autoradiographyof 32P-labeled

signals and staining with Coomassie Brilliant Blue in the absence of [g-32P]

ATP.

Phosphoamino acid analysis was performed as described previously

(Hunter and Sefton, 1980; Kamps and Sefton, 1989). Briefly, the purified

proteins were labeled with [g-32P]-ATP via phosphorylation assays as

described above. They were then separated by SDS-PAGE and trans-

ferred onto polyvinylidene difluoride membranes. The radioactive protein

bands of interest were excised and hydrolyzed in 5.7 N HCl at 1108C for

1 h. The hydrolyzed samples were concentrated and applied to cellulose

thin layer plates for electrophoresis with three phosphoamino acid

standards (0.3 mg each of PSer, PThr, and PTyr; Sigma-Aldrich). The

plateswere stainedwith ninhydrin solution (0.2% [v/v] in ethanol) and then

subjected to autoradiography.

Yeast Two-Hybrid Assay

Yeast two-hybrid assays were performed in accordance with the Pro-

Quest Two-Hybrid SystemManual (Invitrogen). The coding region of ILA1

was cloned into the Gateway vector pDEST32 (Invitrogen). A yeast two-

hybrid library was constructed from the mRNA of rice roots at the tillering

stage by following the manufacturer’s manual of the SuperScript plasmid

system and Gateway technology for cDNA synthesis and cloning kit

(Invitrogen). The cDNA library was screened using a modified method

described by Hou et al. (2009). Approximately 1.7 3 106 yeast trans-

formants were screened on the selective medium (SC/-Leu-Trp-His) with

15 mM 3-amino-1,2,4-triazole (3-AT). The b-galactosidase activity was

determined in yeast strain MaV203, and the positive candidates were

subjected to sequencing.

Bioinformatics Analyses

The full-length cDNAs of ILA1, IIP2, and IIP4were obtained from the Rice

Genome Resource Center (http://www.rgrc.dna.affrc.go.jp/). Domain

prediction for ILA1 and IIPs was performed using the Pfam database

(http://pfam.sanger.ac.uk/) and the National Center for Biotechnology

Information (NCBI) Conserved Domains database (http://www.ncbi.nlm.

nih.gov/Structure/cdd/cdd.shtml). A search for ILA1 homologs in plants

was performed using the NCBI BLAST server (http://blast.ncbi.nlm.nih.

gov/Blast.cgi). Unrooted neighbor-joining trees of ILA1 homologs and

IIPs were generated using MEGA4 with 1000 bootstrap replicates.

Bootstrap values more than 50% are shown. The alignments used for

these analyses are available as Supplemental Data Sets 1 and 2 online.

BiFC and Co-IP Assays

BiFC assays were performed according to Waadt et al. (2008). ILA1 was

cloned into KpnI- and BamHI-digested pVYCE to generate an ILA1-cYFP

construct. IIP2 and IIP4 were cloned into KpnI and BamHI sites of pVYNE

to generate IIP2-nYFP and IIP4-nYFP constructs, respectively. Cotrans-

formation of ILA1-cYFP and IIP2-nYFP/IIP4-nYFP constructs in rice

protoplasts was performed according to the method described above.

Transformants expressing ILA1-cYFP/IIP2-nYFP /IIP4-nYFP and the

empty vector were used as negative controls, and those expressing

bZIP63-cYFP and bZIP63-nYFP were used as a positive control. After

incubation at 258C overnight, the YFP signal was observed using a

confocal laser scanning microscope (Leica TCS SP2).

For Co-IP assays, theCDSof ILA1was amplified fromZhonghua11 and

fusedwith a 33Flag tag in the p1301U-FLAG vector (Sun and Zhou, 2008)

to transform wild-type rice plants. Total protein extracts were prepared

from the leaves of transgenic plants with 2 volumes of immunoprecipitation

buffer (100 mM HEPES, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM

Na3VO4, 10 mM NaF, 50 mM a-glycerophosphate, 0.1% Triton X-100,

1 mM phenylmethylsulfonyl fluoride, 5 pg/mL aprotinin, 5 pg/mL leupeptin,

and 10% [v/v] glycerol). The extracts were centrifuged at 16,000 rcf for 20

min at 48C, and the supernatant was incubated with anti-FLAG M2 affinity

gel (Sigma-Aldrich) overnight at 48C. The resinwaswashed three timeswith

immunoprecipitation buffer and then eluted in the SDS sample buffer. After

separation on 12% SDS-PAGE gels, the proteins were transferred onto

nitrocellulose membranes and probed with anti-FLAG (Proteintech) and

anti-IIP4 antibodies (Beijing Protein Innovation) with a dilution of 1:1000.

Anti-IIP4 polyclonal antibodies were produced by synthesizing a peptide of

18 amino acid residues (VTATTTSEQRNHPRHPKK) and coupling it into

keyhole limpet hemocyanin for immunization of rabbits. This experiment

was independently repeated three times, using proteins isolated from

plants expressing DSM1-FLAG as a negative control (Ning et al., 2010).

Analysis of the IIPs’ Functions

For transactivation activity assay, the CDS of IIP2 and IIP4 were cloned

into the Gateway destination pDEST32 vector and introduced into yeast

strain MaV203. The transformed yeast strains were then used for trans-

activation assays. For examination of the expression of theHIS3 gene, the

yeast was plated on selective medium (SC/-Leu-Trp-His) with 20 mM

3-AT to observe the growth status. For the b-galactosidase assays, the

yeast was grown on YPAD plates for 2 d, and the b-galactosidase activity

on the filter papers was analyzed as described by the manufacturer’s

instructions for the ProQuest two-hybrid system (Invitrogen).

For generation of IIP5 RNA interference transgenic plants, an RNA

interference construct was prepared by inserting a 530-bp fragment of

IIP5 cDNA (1280 to 1811 bp) intoKpnI andBamHI sites of pDS1301 vector

(Chu et al., 2006). The resulting construct was introduced into a rice

variety Zhonghua11. The phenotype observation was performed on T0

and T1 transgenic plants.

Microarray Analysis

Two biological replicate samples of the leaf lamina joints from the ila1 and

wild-type plants were collected at the tillering stage for RNA extraction.

Total RNAs isolated from each replicate via the TRIzol method (Invitrogen)

were used for target synthesis. The microarray analyses were performed

according to the standard protocols (Affymetrix) with Affymetrix Hybridiza-

tionOven 640, Affymetrix Fluidics Station 450, and by Affymetrix GeneChip

service (CapitalBio). The data collection and analysis were performed with

Affymetrix GeneChip Scanner 3000 and Affymetrix GeneChip Operating

Software (version 1.4). To compare the expression level of individual gene,

the signal ratio of each gene between thewild type and ila1was calculated.

The genes showing more than fourfold alterations in transcript levels are

listed in Supplemental Tables 2 and 3 online.

RT-PCR and qRT-PCR Assays

Toexamine ILA1expression level inwild-typeandmutantplants, totalRNAwas

isolated from wild-type and ila1 leaves using TRIzol reagent (Invitrogen). cDNA

was synthesizedwithSuperscript II reverse transcriptase (Invitrogen) according

to the manufacturer’s instructions. RT-PCR amplification was performed to

examine expression of ILA1 using Actin1 as an endogenous control.


For qRT-PCR analysis of ILA1, IIPs, and the cell wall–related genes,

different organs, including leaf lamina joints, were collected for RNA

extraction using TRIzol reagent. Total RNAswere treatedwith RNase-free

DNase I (Invitrogen) and used to synthesize cDNA. qRT-PCR was

performed with a cycler apparatus (Bio-Rad) using FastStart Universal

SYBR Green Master (Roche). Amplification was conducted in 96-well

optical reaction plates with the following protocol: 948C for 4 min, 40

cycles of 948C for 30 s, 568C for 30 s, and 728C for 30 s. The housekeeping

gene UBQ5 was used as an internal control for normalization of RNA

samples. Expression levels of examined genes were quantified by a

relative quantitation method (DDCT). The statistical significance was

evaluated by Student’s t test. Data are presented as mean values of at

least two biological repeats with SE.

Gene-specific primers are shown in Supplemental Table 4 online.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL

databases under the following accession numbers: ILA1 (AK073747), At

CTR1 (AAA32779), Le CTR1 (CAA73722), At EDR1 (NP563824), Le CTR2

(CAA06334), Os EDR1 (AAN61142), Ah STYPK (AAK11734), At ATN1

(CAA63387), At MRK1 (BAA22079), At ANP1 (BAA21854), At ANP2

(BAA21856), At ANP3 (BAA21857), At ARAKIN (AAA99196), At MEKK1

(BAA09057), AtMEKK2 (AAC28188), BnMAP3Kb1 (CAA08997), AtMEKK4

(CAB40943), At MAP3Kb3 (CAA08996), At MEKK3 (AAC28187), At

MAP3Ka (CAA08994), BnMAP3Ka1 (CAA08995), AtMAP3Kg (CAA74696),

At MAP3K«1 (CAA12272), Bn MAP3K«1 (CAB54520), At MAP3K«2

(AAF21208), Cm CTR1 (AAK67354), At MAP3Kd1 (CAA74591), Hv EDR1

(AAG31142), Fs PK1 (CAC09580), Fs PKF1 (CAA66149), At WRKY19

(NP192939), IIP1 (AK101548), IIP2 (AK100409), IIP3 (AK105763), IIP4

(AK101398), IIP5 (AK062582), IIP6 (AK062190), DSM1 (AK102767), Actin1

(X15865), CESA6 (AK100914), CSLC7 (AK243206), IRX10L (Os01g70200),

GUX1L (AK100345),CSLF6 (AK065259),GT8 (AK070652),GT43 (AK062726),

UGA4E (AK100965), andUBQ5 (AK061988).Microarray data from this article

have been deposited in the NCBI Gene Expression Omnibus data repository

(http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE33361.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. ila1 Shows Increased Leaf Angle.

Supplemental Figure 2. ila1 Shows Indistinguishable BR Responses

Compared with the Wild Type.

Supplemental Figure 3. Complementation Test of ila1.

Supplemental Figure 4. Sequence Alignment of ILA1 with Identified

Plant MAPKKKs.

Supplemental Figure 5. Identification of IIP-ILA1 Interactions by

Yeast Two-Hybrid Assay.

Supplemental Figure 6. Sequence Alignment and Phylogenetic Tree

of the IIP Members.

Supplemental Figure 7. Expression Profiles of IIPs.

Supplemental Table 1. Compositional Analysis of Wall Residues

from Wild-Type and ila1 Leaf Blades.

Supplemental Table 2. Genes Upregulated Fourfold in Microarray

Analysis of the ila1 Leaf Lamina Joints Compared with That of the

Wild Type.

Supplemental Table 3. Genes Downregulated Fourfold in Microarray

Analysis of the ila1 Leaf Lamina Joints Compared with That of the

Wild Type.

Supplemental Table 4. Primer Sequences Used in This Article.

Supplemental Data Set 1. Text File of the Alignment Used to

Generate the Phylogenetic Tree of ILA1 and Other MAPKKKs Shown

in Figure 3B.

Supplemental Data Set 2. Text File of the Alignment Used to Generate

the Phylogenetic Tree of IIPs Shown in Supplemental Figure 6B.

ACKNOWLEDGMENTS

We thank Rongjian Ye (Huazhong Agricultural University) for providing

vector pDX2181, Yunhai Li (Institute of Genetics and Developmental

Biology, Chinese Academy of Sciences) for help with light microscopy

observation, Taihua Zhang (Institute of Mechanics, Chinese Academy of

Sciences) for assistance with breaking force measurements, Rod Wing

(University of Arizona) for the gift of the rice BAC clone a0069I24,

Xianghua Li and Jinghua Xiao (Huazhong Agricultural University) for the

support on facility maintenance, and Dongmei Zhang (Institute of

Genetics and Developmental Biology, Chinese Academy of Sciences)

for assistance in recombinant protein purification. This study was

supported by grants from the National Natural Science Foundation of

China (30725021, 31125019, and 30871326) and the National Program

on High Technology Development (2012AA100103).

AUTHOR CONTRIBUTIONS

J.N., B.Z., Y.Z., and L.X. designed the research. J.N., B.Z., N.W., and Y.Z.

performed the research and analyzed the data. J.N. and Y.Z. wrote the

article. Y.Z. and L.X. contributed equally to this work.

Received November 1, 2011; revised December 5, 2011; accepted

December 14, 2011; published December 29, 2011.

REFERENCES

Asai, T., Tena, G., Plotnikova, J., Willmann, M.R., Chiu, W.L.,

Gomez-Gomez, L., Boller, T., Ausubel, F.M., and Sheen, J.

(2002). MAP kinase signalling cascade in Arabidopsis innate immu-

nity. Nature 415: 977–983.

Bai, M.Y., Zhang, L.Y., Gampala, S.S., Zhu, S.W., Song, W.Y., Chong,

K., and Wang, Z.Y. (2007). Functions of OsBZR1 and 14-3-3 proteins

in brassinosteroid signaling in rice. Proc. Natl. Acad. Sci. USA 104:

13839–13844.

Banno, H., Hirano, K., Nakamura, T., Irie, K., Nomoto, S., Matsumoto,

K., and Machida, Y. (1993). NPK1, a tobacco gene that encodes a

protein with a domain homologous to yeast BCK1, STE11, and Byr2

protein kinases. Mol. Cell. Biol. 13: 4745–4752.

Bergmann, D.C., Lukowitz, W., and Somerville, C.R. (2004). Stomatal

development and pattern controlled by a MAPKK kinase. Science

304: 1494–1497.

Cao, H., and Chen, S. (1995). Brassinosteroid-induced rice lamina joint

inclination and its relation to indole-3-acetic acid and ethylene. Plant

Growth Regul. 16: 189–196.

Cohen, J.D., and Meudt, W.J. (1983). Investigations on the mechanism

of the brassinosteroid response: I. indole-3-acetic acid metabolism

and transport. Plant Physiol. 72: 691–694.

Colcombet, J., and Hirt, H. (2008). Arabidopsis MAPKs: A complex

signalling network involved in multiple biological processes. Biochem.

J. 413: 217–226.

Chu, Z., Yuan, M., Yao, J., Ge, X., Yuan, B., Xu, C., Li, X., Fu, B., Li, Z.,

Bennetzen, J.L., Zhang, Q., and Wang, S. (2006). Promoter mutations


of an essential gene for pollen development result in disease resistance

in rice. Genes Dev. 20: 1250–1255.

Doust, A. (2007). Architectural evolution and its implications for do-

mestication in grasses. Ann. Bot. (Lond.) 100: 941–950.

Duncan, W.G. (1971). Leaf angle, leaf area, and canopy photosynthesis.

Crop Sci. 11: 482–485.

Eichberg, J., and Iyer, S. (1996). Phosphorylation of myelin protein:

Recent advances. Neurochem. Res. 21: 527–535.

Frye, C.A., Tang, D., and Innes, R.W. (2001). Negative regulation of

defense responses in plants by a conserved MAPKK kinase. Proc.

Natl. Acad. Sci. USA 98: 373–378.

Hardtke, C.S., Dorcey, E., Osmont, K.S., and Sibout, R. (2007).

Phytohormone collaboration: Zooming in on auxin-brassinosteroid

interactions. Trends Cell Biol. 17: 485–492.

Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient

transformation of rice (Oryza sativa L.) mediated by Agrobacterium

and sequence analysis of the boundaries of the T-DNA. Plant J. 6:

271–282.

Hong, Z., et al. (2002). Loss-of-function of a rice brassinosteroid

biosynthetic enzyme, C-6 oxidase, prevents the organized arrange-

ment and polar elongation of cells in the leaves and stem. Plant J. 32:

495–508.

Hou, X., Xie, K., Yao, J., Qi, Z., and Xiong, L. (2009). A homolog of

human ski-interacting protein in rice positively regulates cell viability

and stress tolerance. Proc. Natl. Acad. Sci. USA 106: 6410–6415.

Hunter, T., and Sefton, B.M. (1980). Transforming gene product of

Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci.

USA 77: 1311–1315.

Ichimura, K.; MAPK Group (2002). Mitogen-activated protein kinase

cascades in plants: A new nomenclature. Trends Plant Sci. 7:

301–308.

Ichimura, K., Mizoguchi, T., and Shinozaki, K. (1997). ATMRK1, an

Arabidopsis protein kinase related to mammal mixed-lineage kinases

and Raf protein kinases. Plant Sci. 130: 171–179.

Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G.,

Zeng, D., Lu, Z., Zhu, X., Qian, Q., and Li, J. (2010). Regulation of

OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat.

Genet. 42: 541–544.

Jonak, C., Okresz, L., Bogre, L., and Hirt, H. (2002). Complexity, cross

talk and integration of plant MAP kinase signalling. Curr. Opin. Plant

Biol. 5: 415–424.

Kamps, M.P., and Sefton, B.M. (1989). Acid and base hydrolysis of

phosphoproteins bound to immobilon facilitates analysis of phospho-

amino acids in gel-fractionated proteins. Anal. Biochem. 176: 22–27.

Karimi, M., De Meyer, B., and Hilson, P. (2005). Modular cloning in

plant cells. Trends Plant Sci. 10: 103–105.

Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker,

J.R. (1993). CTR1, a negative regulator of the ethylene response

pathway in Arabidopsis, encodes a member of the raf family of protein

kinases. Cell 72: 427–441.

Kim, J.A., et al. (2009). Rice OsACDR1 (Oryza sativa accelerated cell

death and resistance 1) is a potential positive regulator of fungal

disease resistance. Mol. Cells 28: 431–439.

Lee, S., Choi, S.C., and An, G. (2008). Rice SVP-group MADS-box

proteins, OsMADS22 and OsMADS55, are negative regulators of

brassinosteroid responses. Plant J. 54: 93–105.

Lerouxel, O., Cavalier, D.M., Liepman, A.H., and Keegstra, K. (2006).

Biosynthesis of plant cell wall polysaccharides - A complex process.

Curr. Opin. Plant Biol. 9: 621–630.

Li, D., Wang, L., Wang, M., Xu, Y.Y., Luo, W., Liu, Y.J., Xu, Z.H., Li, J.,

and Chong, K. (2009). Engineering OsBAK1 gene as a molecular tool

to improve rice architecture for high yield. Plant Biotechnol. J. 7:

791–806.

Li, M., Xiong, G., Li, R., Cui, J., Tang, D., Zhang, B., Pauly, M., Cheng,

Z., and Zhou, Y. (2009). Rice cellulose synthase-like D4 is essential

for normal cell-wall biosynthesis and plant growth. Plant J. 60: 1055–

1069.

Li, Y., Qian, Q., Zhou, Y., Yan, M., Sun, L., Zhang, M., Fu, Z., Wang,

Y., Han, B., Pang, X., Chen, M., and Li, J. (2003). BRITTLE CULM1,

which encodes a COBRA-like protein, affects the mechanical prop-

erties of rice plants. Plant Cell 15: 2020–2031.

Li, Z.K., Paterson, A.H., Pinson, S.R.M., and Khush, G.S. (1998). A

major gene, Ta1 and QTLs affecting tiller and leaf angles in rice. Rice

Genet. Newsl. 15: 154–156.

Li, Z.K., Paterson, A.H., Pinson, S.R.M., and Stansel, J.W. (1999).

RFLP facilitated analysis of tiller and leaf angles in rice (Oryza sativa L).

Euphytica 9: 79–84.

Liepman, A.H., Wilkerson, C.G., and Keegstra, K. (2005). Expression

of cellulose synthase-like (Csl) genes in insect cells reveals that CslA

family members encode mannan synthases. Proc. Natl. Acad. Sci.

USA 102: 2221–2226.

Long, S.P., Zhu, X.G., Naidu, S.L., and Ort, D.R. (2006). Can improve-

ment in photosynthesis increase crop yields? Plant Cell Environ. 29:

315–330.

Miao, Y., Laun, T.M., Smykowski, A., and Zentgraf, U. (2007).

Arabidopsis MEKK1 can take a short cut: It can directly interact

with senescence-related WRKY53 transcription factor on the protein

level and can bind to its promoter. Plant Mol. Biol. 65: 63–76.

Mitchell, P.L., and Sheehy, J.E. (2006). Supercharging rice photosyn-

thesis to increase yield. New Phytol. 171: 688–693.

Murchie, E.H., Chen, Yz., Hubbart, S., Peng, S., and Horton, P.

(1999). Interactions between senescence and leaf orientation deter-

mine in situ patterns of photosynthesis and photoinhibition in field-

grown rice. Plant Physiol. 119: 553–564.

Nakagami, H., Pitzschke, A., and Hirt, H. (2005). Emerging MAP

kinase pathways in plant stress signalling. Trends Plant Sci. 10:

339–346.

Nakamura, A., Fujioka, S., Takatsuto, S., Tsujimoto, M., Kitano, H.,

Yoshida, S., Asami, T., and Nakano, T. (2009). Involvement of C-22-

hydroxylated brassinosteroids in auxin-induced lamina joint bending

in rice. Plant Cell Physiol. 50: 1627–1635.

Ning, J., Li, X., Hicks, L.M., and Xiong, L. (2010). A Raf-like MAPKKK

gene DSM1 mediates drought resistance through reactive oxygen

species scavenging in rice. Plant Physiol. 152: 876–890.

Nishihama, R., Ishikawa, M., Araki, S., Soyano, T., Asada, T., and

Machida, Y. (2001). The NPK1 mitogen-activated protein kinase

kinase kinase is a regulator of cell-plate formation in plant cytokinesis.

Genes Dev. 15: 352–363.

Nishihama, R., Soyano, T., Ishikawa, M., Araki, S., Tanaka, H.,

Asada, T., Irie, K., Ito, M., Terada, M., Banno, H., Yamazaki, Y.,

and Machida, Y. (2002). Expansion of the cell plate in plant cytoki-

nesis requires a kinesin-like protein/MAPKKK complex. Cell 109:

87–99.

Pena, M.J., Zhong, R., Zhou, G.K., Richardson, E.A., O’Neill, M.A.,

Darvill, A.G., York, W.S., and Ye, Z.H. (2007). Arabidopsis irregular

xylem8 and irregular xylem9: Implications for the complexity of

glucuronoxylan biosynthesis. Plant Cell 19: 549–563.

Persson, S., Caffall, K.H., Freshour, G., Hilley, M.T., Bauer, S.,

Poindexter, P., Hahn, M.G., Mohnen, D., and Somerville, C.

(2007). The Arabidopsis irregular xylem8 mutant is deficient in glucur-

onoxylan and homogalacturonan, which are essential for secondary

cell wall integrity. Plant Cell 19: 237–255.

Sakamoto, T., et al. (2006). Erect leaves caused by brassinosteroid

deficiency increase biomass production and grain yield in rice. Nat.

Biotechnol. 24: 105–109.

Shimada, A., Ueguchi-Tanaka, M., Sakamoto, T., Fujioka, S., Takatsuto,


S., Yoshida, S., Sazuka, T., Ashikari, M., and Matsuoka, M. (2006). The

rice SPINDLY gene functions as a negative regulator of gibberellin signaling

by controlling the suppressive function of the DELLA protein, SLR1, and

modulating brassinosteroid synthesis. Plant J. 48: 390–402.

Shou, H., Bordallo, P., Fan, J.B., Yeakley, J.M., Bibikova, M., Sheen,

J., and Wang, K. (2004). Expression of an active tobacco mitogen-

activated protein kinase kinase kinase enhances freezing tolerance in

transgenic maize. Proc. Natl. Acad. Sci. USA 101: 3298–3303.

Song, Y., You, J., and Xiong, L. (2009). Characterization of OsIAA1

gene, a member of rice Aux/IAA family involved in auxin and

brassinosteroid hormone responses and plant morphogenesis. Plant

Mol. Biol. 70: 297–309.

Soyano, T., Nishihama, R., Morikiyo, K., Ishikawa, M., and Machida,

Y. (2003). NQK1/NtMEK1 is a MAPKK that acts in the NPK1 MAPKKK-

mediated MAPK cascade and is required for plant cytokinesis. Genes

Dev. 17: 1055–1067.

Sun, Q., and Zhou, D.X. (2008). Rice jmjC domain-containing gene

JMJ706 encodes H3K9 demethylase required for floral organ devel-

opment. Proc. Natl. Acad. Sci. USA 105: 13679–13684.

Tanaka, A., et al. (2009). BRASSINOSTEROID UPREGULATED1, en-

coding a helix-loop-helix protein, is a novel gene involved in brassi-

nosteroid signaling and controls bending of the lamina joint in rice.

Plant Physiol. 151: 669–680.

Tanaka, K., Murata, K., Yamazaki, M., Onosato, K., Miyao, A., and

Hirochika, H. (2003). Three distinct rice cellulose synthase catalytic

subunit genes required for cellulose synthesis in the secondary wall.

Plant Physiol. 133: 73–83.

Tena, G., Asai, T., Chiu, W.L., and Sheen, J. (2001). Plant mitogen-

activated protein kinase signaling cascades. Curr. Opin. Plant Biol. 4:

392–400.

Tregear, J.W., Jouannic, S., Schwebel-Dugue, N., and Kreis, M.

(1996). An unusual protein kinase displaying characteristics of both

the serine/threonine and tyrosine families is encoded by the Arabi-

dopsis thaliana gene ATN1. Plant Sci. 117: 107–119.

Updegraff, D.M. (1969). Semimicro determination of cellulose in bio-

logical materials. Anal. Biochem. 32: 420–424.

Waadt, R., Schmidt, L.K., Lohse, M., Hashimoto, K., Bock, R., and

Kudla, J. (2008). Multicolor bimolecular fluorescence complementa-

tion reveals simultaneous formation of alternative CBL/CIPK com-

plexes in planta. Plant J. 56: 505–516.

Wada, K., Marumo, S., Ikekawa, N., Morisaki, M., and Mori, K. (1981).

Brassinolide and homobrassinolide promotion of lamina inclination of

rice seedlings. Plant Cell Physiol. 22: 323–325.

Wang, H., Ngwenyama, N., Liu, Y., Walker, J.C., and Zhang, S.

(2007). Stomatal development and patterning are regulated by envi-

ronmentally responsive mitogen-activated protein kinases in Arabi-

dopsis. Plant Cell 19: 63–73.

Wang, L., Xie, W., Chen, Y., Tang, W., Yang, J., Ye, R., Liu, L., Lin, Y.,

Xu, C., Xiao, J., and Zhang, Q. (2010). A dynamic gene expression

atlas covering the entire life cycle of rice. Plant J. 61: 752–766.

Wang, L., Xu, Y., Zhang, C., Ma, Q., Joo, S.H., Kim, S.K., Xu, Z., and

Chong, K. (2008). OsLIC, a novel CCCH-type zinc finger protein with

transcription activation, mediates rice architecture via brassinoste-

roids signaling. PLoS ONE 3: e3521.

Wang, M., Wang, K., Tang, D., Wei, C., Li, M., Shen, Y., Chi, Z., Gu,

M., and Cheng, Z. (2010). The central element protein ZEP1 of the

synaptonemal complex regulates the number of crossovers during

meiosis in rice. Plant Cell 22: 417–430.

Wu, C., Li, X., Yuan, W., Chen, G., Kilian, A., Li, J., Xu, C., Li, X.,

Zhou, D.X., Wang, S., and Zhang, Q. (2003). Development of

enhancer trap lines for functional analysis of the rice genome. Plant

J. 35: 418–427.

Yamamuro, C., Ihara, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuto,

S., Ashikari, M., Kitano, H., and Matsuoka, M. (2000). Loss of function

of a rice brassinosteroid insensitive1 homolog prevents internode

elongation and bending of the lamina joint. Plant Cell 12: 1591–1606.

Yoo, S.D., Cho, Y.H., Tena, G., Xiong, Y., and Sheen, J. (2008). Dual

control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signal-

ling. Nature 451: 789–795.

Yuan, L.P. (1997). Hybird rice breeding for super yield. Hybrid Rice

12: 1–7.

Zhang, B., Deng, L., Qian, Q., Xiong, G., Zeng, D., Li, R., Guo, L., Li,

J., and Zhou, Y. (2009). A missense mutation in the transmembrane

domain of CESA4 affects protein abundance in the plasma membrane

and results in abnormal cell wall biosynthesis in rice. Plant Mol. Biol.

71: 509–524.

Zhang, J., Li, C., Wu, C., Xiong, L., Chen, G., Zhang, Q., and Wang, S.

(2006). RMD: A rice mutant database for functional analysis of the rice

genome. Nucleic Acids Res. 34(Database issue): D745–D748.

Zhang, L.Y., et al. (2009). Antagonistic HLH/bHLH transcription factors

mediate brassinosteroid regulation of cell elongation and plant devel-

opment in rice and Arabidopsis. Plant Cell 21: 3767–3780.

Zhang, M., Zhang, B., Qian, Q., Yu, Y., Li, R., Zhang, J., Liu, X., Zeng,

D., Li, J., and Zhou, Y. (2010). Brittle Culm 12, a dual-targeting

kinesin-4 protein, controls cell-cycle progression and wall properties

in rice. Plant J. 63: 312–328.

Zhang, S.W., Li, C.H., Cao, J., Zhang, Y.C., Zhang, S.Q., Xia, Y.F.,

Sun, D.Y., and Sun, Y. (2009). Altered architecture and enhanced

drought tolerance in rice via the down-regulation of indole-3-acetic

acid by TLD1/OsGH3.13 activation. Plant Physiol. 151: 1889–1901.

Zhao, S.Q., Hu, J., Guo, L.B., Qian, Q., and Xue, H.W. (2010). Rice leaf

inclination2, a VIN3-like protein, regulates leaf angle through modu-

lating cell division of the collar. Cell Res. 20: 935–947.

Zhou, Y., et al. (2009). BC10, a DUF266-containing and Golgi-located

type II membrane protein, is required for cell-wall biosynthesis in rice

(Oryza sativa L.). Plant J. 57: 446–462.


DOI 10.1105/tpc.111.093419; originally published online December 29, 2011;Plant Cell

Jing Ning, Baocai Zhang, Nili Wang, Yihua Zhou and Lizhong XiongRegulates Mechanical Tissue Formation in the Lamina Joint of Rice

Increased Leaf Angle1, a Raf-Like MAPKKK That Interacts with a Nuclear Protein Family,

This information is current as of April 13, 2018

Supplemental Data /content/suppl/2011/12/29/tpc.111.093419.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

increased leaf angle1, a raf-like mapkkk that interacts with a

Documents