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. 2011 Dec;23(12):4334-47.
doi: 10.1105/tpc.111.093419. Epub 2011 Dec 29.

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  1 , Baocai Zhang, Nili Wang, Yihua Zhou, Lizhong Xiong
Affiliations

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 et al. Plant Cell. 2011 Dec.

Abstract

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.

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Figures

Figure 1.
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 ± 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.
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 μm. (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 μm. (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.
Figure 3.
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.
Figure 4.
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.
Figure 5.
Subcellular Localization of GFP-ILA1. (A) Transient expression of GFP-ILA1 in rice protoplasts. DIC, differential interference contrast. Bar = 5 μm. (B) Fluorescent signals in transgenic rice plants expressing GFP-ILA1. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Bar = 30 μm.
Figure 6.
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.
Figure 7.
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 β-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.
Figure 8.
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 μm. (C) Expression of GFP-tagged IIP4 in rice protoplasts. Bar = 5 μm. (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 β-Gal assays.
Figure 9.
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).
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