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The Plant Cell, Vol. 23: 4334–4347, December 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved. Increased Leaf Angle1, a Raf-Like MAPKKK That Interacts with a Nuclear Protein Family, Regulates Mechanical Tissue Formation 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, China b 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 brassinosteroidrelated 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 signaling mechanisms to initiate cellular responses to external or internal stimuli. One such mechanism is the mitogenactivated protein kinase (MAPK) cascade composed of three levels of Ser/Thr-specific protein kinases (MAPK kinase kinase [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. Compared 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 responses (Asai et al., 2002; Kim et al., 2009). In plants, major 1 These authors contributed equally to this work. correspondence to [email protected]. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Yihua Zhou ([email protected]) and Lizhong Xiong ([email protected]). C Some figures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.111.093419 2 Address challenges still remain in identification of individual kinases, especially due to the existence of more than one hundred components of plant MAPK cascades (Ichimura, 2002). Analysis of the Arabidopsis thaliana genome revealed the presence 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 to C) or two distinct subfamilies based on the sequence of their kinase catalytic domain. Group A comprises the MEKK family, and Groups B and C consist of the Raf-like family (Ichimura, 2002). To date, several MEKK family members have been identified. 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 include Constitutive Triple Response1 (CTR1) and Enhanced Disease 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 ILA1 Regulates Rice Leaf Inclination 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 varieties 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). A more 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, participate in determination of the leaf angle (Cao and Chen, 1995; Shimada et al., 2006). Many of these phytohormones act synergistically with BR (Cohen and Meudt, 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 dwarfism is due to a mutation in TLD1, a gene that encodes an indole3-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 support 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, increased leaf angle1 (ila1), that exhibits abnormal mechanical tissues and cell wall composition in the leaf lamina joint, unrelated to BR responses. ILA1 encodes a Raf-like MAPKKK of 4335 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 proteins [IIPs]). Our findings not only provide unique genetic evidence for the functions of Group C Raf-like MAPKKKs, but also unravel a different mechanism in the regulation of the leaf angle in rice. RESULTS Identification of ila1, a Mutant with a Phenotype Unrelated to BR Responses A T-DNA insertion mutant that shows increased leaf inclination angle (designated ila1) was isolated from the Rice Mutant Database (http://rmd.ncpgr.cn). Identification of the flanking sequence of T-DNA using thermal asymmetric interlaced PCR revealed that the insertion occurs in the fourth exon of an expressed gene (Os06g50920, Rice Genome Annotation Project, http://rice.plantbiology.msu.edu/), 963 nucleotides downstream from the ATG start codon (Figure 1A). The increased leaf inclination cosegregated with the homozygous T-DNA insertion based on PCR analysis (see Supplemental Figure 1A online). RT-PCR assays showed that the intact transcript of Os06g50920 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 elongation 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 4336 The Plant Cell 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%) and Xyl (;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.] 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 ila1 mutant, 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 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 vascular bundles and reduced sclerenchymatous cells in the ila1 lamina joint. Bars = 360 mm. (G) Leaf lamina joint used for measuring mechanical strength. Arrowheads 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 4337 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 ila1 4.4 6 0.2 3.4 6 0.2* 3.1 6 0.2 2.6 6 0.2 99.7 6 6.7 81.3 6 6.6* 212.4 6 18.9 149.4 6 10.9* 11.1 6 0.5 8.6 6 0.8* 44.7 6 3.1 40.2 6 3.1 52.5 6 3.6 34.0 6 3.7* 258.8 6 15.6 175.2 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. in cellulose content, no significant alterations in monosaccharides were found in the ila1 mutant leaf blades (see Supplemental Table 1 online). Deficiencies in mechanical tissues and cellulose content suggested that the mechanical strength of the leaf lamina joint might be affected in the ila1 mutant. We compared the mechanical 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 mechanical 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 from a T-DNA insertion in Os06g50920. 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 Supplemental 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) and BLAST search (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 species revealed that the characteristic features for MAPKKK proteins, 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 tree was 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). The GST protein alone generated undetectable signal and served as a negative control (Figure 4A). Phosphoamino acid analysis was further conducted to determine the phosphorylation ability of ILA1. After separation by two-dimensional thin layer electrophoresis, phosphoserine and phosphothreonine were detected in the phosphorylated ILA1 and MBP, whereas phosphotyrosine was not found (Figures 4B and 4C). These results suggest that ILA1 is a Ser/Thr protein kinase. ILA1 Is Mainly Localized in the Nucleus and Expressed in the Leaf Lamina Joints Next, we fused a green fluorescent protein (GFP) to the N terminus of ILA1 to examine its subcellular localization. Expression 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 localization 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) evaluation 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 the GUS 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 GUSstained 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 4338 The Plant Cell 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. full-length ILA1 protein was used as bait to screen a yeast twohybrid 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 Supplemental 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), respectively. The six IIP proteins range from 628 to 655 amino acids in length and possess a highly conserved domain in the middle. In addition, the IIP proteins have low sequence identity (;30%) with the Arabidopsis WRKY19 transcription factor and were grouped into two subfamilies together with three Arabidopsis 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 in many 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 not with IIPs in subfamily A. We also note that the N-terminal regulatory domain interacts with IIPs. Next, we explored 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/IIP4nYFP) 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 coimmunoprecipitation (Co-IP) assay. We generated a specific polyclonal antibody against IIP4, but we were unable to generate one for IIP2 due to an unknown problem. Using this antibody, we detected IIP4 proteins in the affinity-purified total protein extracts from the transgenic plants harboring a FLAG-tagged ILA1 transgene (see Supplemental Figure 3A online). However, ILA1 Regulates Rice Leaf Inclination 4339 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 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.] 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 the microarray analysis, 820 probes showed more than fourfold alterations, corresponding to 548 downregulated 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 biosynthesis 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 downregulated genes, no significant alterations were observed in the remaining hormone-related genes and transcription factors for 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 investigation. The Gene Ontology database (http://www.geneontology.org/) annotated IIPs as putative transcription factors. Because nuclear localization is a significant feature of tran- 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. 4340 The Plant Cell DISCUSSION ILA1 Is a Functional Kinase of the Group C Raf-Like MAPKKK Family 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. 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 the wild 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. MAPKKKs act at the top level of MAPK cascades and show great sequence diversity (Ichimura, 2002). As determined by the highly conserved kinase domain, plant MAPKKKs are categorized either as MEKKs 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 Raflike 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 traditional MAPK cascades. ILA1 Regulates Mechanical Strength in the Leaf Lamina Joint To our knowledge, functions of Group C Raf-like MAPKKKs 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 regulating mechanical 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 ILA1 Regulates Rice Leaf Inclination 4341 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. 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 glycosyltransferase (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; Peña 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 generally 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 biosynthesis or signaling are not significantly altered in ila1 mutants, except for BZR1, Brassinosteroid Insensitive1 (BRI1), and BRdeficient 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 4342 The Plant Cell be that ILA1 is specifically activated in leaf lamina joints or that leaf lamina joints have a different structure than leaves, especially 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 a Group C Raflike MAPKKK member. Through yeast two-hybrid screening of the cDNA library, six interaction proteins (IIPs) were found; these Figure 8. Characterization of IIPs. (A) ILA1 phosphorylates IIP4. The phosphorylated proteins were separated 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/-LeuTrp-His with 3-AT) and by b-Gal assays. 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 expression levels of ILA1 in both leaves and leaf lamina joints. However, ila1 exhibits a visible phenotype mainly 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 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). ILA1 Regulates Rice Leaf Inclination 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 phosphorylation of a transcription factor by a MAPKKK has been previously reported (Miao et al., 2007). Many transcription factors have been found that are involved in modulating rice leaf inclination. 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 wildtype 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 Developmental 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 measurement. 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 4343 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 ila1 mutant 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 was measured 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 Agrobacterium-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 terminator at the sites of SalI and KpnI in pUC19 to generate a construct for the transformation of rice protoplasts. Next, the CDS of ILA1 was fused to the C terminus of GFP and cloned into the GATEWAY destination binary 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 scanning microscope (Leica TCS SP2). The roots of transgenic plants were 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. GUS activity assays were performed in the T0 and T1 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 mg mL21 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 photographed with a stereomicroscope (Leica MZ FLIII). Phosphorylation Analysis The CDSs of ILA1 and IIP4 were cloned into BamHI and EcoRI sites of pGEX 6p-1 and GATEWAY destination pDEST17 vectors, respectively, for expression of the recombinant proteins in Escherichia coli. The ILA1-GST and 4344 The Plant Cell His-IIP4 recombinant proteins were purified with Glutathione Sepharose 4B 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 30 min, after which it was terminated by the addition of 4 mL of stop buffer (0.35 M Tris-HCl, pH 6.8, 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-PAGE gels, the gels were subjected to autoradiography of 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 transferred 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 plates were stained with 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 ProQuest Two-Hybrid System Manual (Invitrogen). The coding region of ILA1 was cloned into the Gateway vector pDEST32 (Invitrogen). A yeast twohybrid 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 transformants 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. 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, the CDS of ILA1 was amplified from Zhonghua11 and fused with 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 resin was washed three times with 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 transactivation assays. For examination of the expression of the HIS3 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) into KpnI and BamHI 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. Bioinformatics Analyses Microarray Analysis The full-length cDNAs of ILA1, IIP2, and IIP4 were 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. Cotransformation 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 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 Hybridization Oven 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 the wild type and ila1 was 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 To examine ILA1 expression level in wild-type and mutant plants, total RNA was isolated from wild-type and ila1 leaves using TRIzol reagent (Invitrogen). cDNA was synthesized with Superscript 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. ILA1 Regulates Rice Leaf Inclination 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 RNAs were treated with 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), At MEKK2 (AAC28188), Bn MAP3Kb1 (CAA08997), At MEKK4 (CAB40943), At MAP3Kb3 (CAA08996), At MEKK3 (AAC28187), At MAP3Ka (CAA08994), Bn MAP3Ka1 (CAA08995), At MAP3Kg (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), and UBQ5 (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. 4345 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. 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Increased Leaf Angle1, a Raf-Like MAPKKK That Interacts with a Nuclear Protein Family, Regulates Mechanical Tissue Formation in the Lamina Joint of Rice Jing Ning, Baocai Zhang, Nili Wang, Yihua Zhou and Lizhong Xiong Plant Cell 2011;23;4334-4347; originally published online December 29, 2011; DOI 10.1105/tpc.111.093419 This information is current as of June 18, 2017 Supplemental Data /content/suppl/2011/12/29/tpc.111.093419.DC1.html References This article cites 72 articles, 30 of which can be accessed free at: /content/23/12/4334.full.html#ref-list-1 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY