BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development

Abstract

The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants. However, the molecular mechanisms linking the actin cytoskeleton to these processes remain largely unknown. Here, we report the functional analysis of rice (Oryza sativa) FH5/BENT UPPERMOST INTERNODE1 (BUI1), which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice. The bui1 mutant displayed pleiotropic phenotypes, including bent uppermost internode, dwarfism, wavy panicle rachis, and enhanced gravitropic response. Cytological observation indicated that the growth defects of bui1 were caused mainly by inhibition of cell expansion. Map-based cloning revealed that BUI1 encodes the class II formin FH5. FH5 contains a phosphatase tensin-like domain at its amino terminus and two highly conserved formin-homology domains, FH1 and FH2. In vitro biochemical analyses indicated that FH5 is capable of nucleating actin assembly from free or profilin-bound monomeric actin. FH5 also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end. Interestingly, the FH2 domain of FH5 could bundle actin filaments directly and stabilize actin filaments in vitro. Consistent with these in vitro biochemical activities of FH5/BUI1, the amount of filamentous actin decreased, and the longitudinal actin cables almost disappeared in bui1 cells. The FH2 or FH1FH2 domains of FH5 could also bind to and bundle microtubules in vitro. Thus, our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments, which are important for proper cell expansion and rice morphogenesis.

Figure 1.

Phenotypes of Wild-Type and bui1 Mature Plants. (A) Wild-type (Zhejing 22; left) and bui1 (right) plants. Bar = 2 cm. (B) Panicle exsertion of wild-type (left three) and bui1 (right three) plants. Bar = 2 cm. (C) and (D) Longitudinal sections of the uppermost internodes of the wild type (C) and bui1 (D) at heading stage. The regions used for analysis are indicated with squares in (B). Bars = 100 μm. (E) Panicles of the wild type (left) and bui1 (right) at the mature stage. Bar = 2 cm. (F) to (H) Longitudinal sections of the uppermost internode regions (indicated with squares in [E]) of the wild type (F) and bui1 (G). Severely slanted cells of the same bui1 internode region are shown in (H). Bars = 50 μm. (I) Panicle rachis of the wild type (left) and bui1 (right). Note the wavy rachis in bui1. Bar = 2 cm. (J) Grains of the wild type (top) and bui1 (bottom). Bar = 0.5 cm.

Figure 2.

Comparison of Wild-Type and bui1 Seedlings. (A) Wild-type (WT) and bui1 seedlings (7 d old). Note that the bui1 shoots are bent. Bar = 2 cm. (B) to (D) Longitudinal sections of seedling shoots of the wild type (C) and bui1 (D). (B) shows 9-d-old seedlings of the wild type (left) and bui1 (right). Bar in (B) = 2 cm; bars in (C) and (D) = 100 μm. (E) Roots of the wild type (left) and bui1 (right) grown in half-strength medium. Bar = 1 cm. (F) and (G) Cell morphology of wild-type (F) and bui1 (G) roots by propidium iodide staining. The regions used for analysis are indicated with squares in (E). Bars = 50 μm.

Figure 3.

Map-Based Cloning of BUI1. (A) The BUI1 locus was mapped on chromosome 7 between two SSR markers, RM1132 and RM505. (B) Fine-mapping of BUI1. BUI1 was narrowed to a 60-kb region between two makers, 7WB8 and 7WB16, on a single BAC (AP004275), which contains three predicted genes. (C) Sequence comparison revealed a substitution of A to G in one intron of the gene FH5/Os07g0596300 in bui1. WT, wild type. (D) Detection of altered splicing in bui1 by RT-PCR analysis. Rice UBI1 was used as an internal control. (E) RNA gel blot analysis to confirm the length of the FH5/BUI1 transcript. Total RNA (10 μg) extracted from the wild type and bui1 was used for the analysis (shown below the blot). M, RNA ladder. (F) Confirmation of the full-length FH5/BUI1 transcript by RT-PCR. The primer pairs (P1 and P2) used for RT are indicated in (C). (G) Complementation test of the FH5/BUI1 gene. One representative line (1300-BUI1) of complementation is shown. Bar = 2 cm. (H) Expression pattern of FH5 revealed by RNA gel blot. YS, Young seedling; FL, flower; RA, rachis; LF, leaf; LS, leaf sheath; ND, node; IN, internode; RT, root.

Figure 4.

F-Actin Organization in Wild-Type and bui1 Cells. F-actin organization was visualized by AlexaFluor488–phalloidin staining. Each image is a maximum projection of the fluorescence signals. (A) and (B) F-actin organization in the cortex cells of the root elongation regions of the wild type (A) and bui1 (B). Bars = 20 μm. (C) Quantitative analysis of F-actin levels in wild-type (WT) and bui1 cells as detected in (A) and (B). Data shown are means ± se of fluorescence intensity of 144 cells in the wild type and bui1. P < 0.01, by t test. (D) and (E) F-actin organization in the root elongation region cells of the wild type (D) and bui1 (E). Confocal settings for bui1 were increased to give clear signals. Bars = 20 μm. (G) and (H) F-actin organization in the root transition region cells of the wild type (G) and bui1 (H). Confocal settings for bui1 were increased to give clear signals. Bars = 20 μm. (F) and (I) Fluorescence intensities corresponding to the regions marked in (D)/(E) and (G)/(H), respectively.

Figure 5.

FH5 Nucleates Actin Assembly from G-Actin and the Profilin/Actin Complex. (A) and (B) Time course of actin polymerization in the presence of FH5 FH2 (A) or FH5 FH1FH2 (B) monitored by pyrene fluorescence. Various concentrations of FH5 FH2 or FH5 FH1FH2 were added to 2 μM 5% pyrene-labeled actin before the initiation of actin polymerization. (C) Nucleation efficiency of FH5 FH2 and FH5 FH1FH2. The efficiency was calculated at half-maximal actin polymerization according to Blanchoin et al. (2000). (D) FH5 FH2 was not able to nucleate actin from the profilin/actin complex. The reactions were conducted with different combinations as indicated at right. (E) FH5 FH1FH2 was able to nucleate actin assembly from the profilin/actin complex. The reactions were conducted with different combinations as indicated at right.

Figure 6.

Visualization of the Effect of FH5 on Profilin/Actin Polymerization by TIRFM. Time-lapse evanescent wave microscopy was conducted with 1.5 μM ATP-Oregon-green-actin (100% labeled) and 5 μM human profilin I in the absence or presence of FH5. Images were acquired at various times as indicated below the panels. The green arrows indicate the ends of typical actin filaments during elongation. Conditions were as follows: 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 50 mM DTT, 0.2 mM ATP, 50 mM CaCl₂, 100 μg/mL Glc oxidase, 15 mM Glc, 20 μg/mL catalase, and 1.0% methylcellulose. Bar = 10 μm. (A) to (E) Time-lapse micrographs of profilin/Oregon-green-actin polymerization. The profilin/Oregon-green-actin complex was perfused into the flow cell coated with NEM–myosin. See also Supplemental Movie 4 online. (F) to (J) Time-lapse micrographs of profilin/Oregon-green-actin polymerization in the presence of FH5 FH2. The profilin/Oregon-green-actin complex with 10 nM FH5 FH2 was perfused into the flow cell coated with NEM–myosin. See also Supplemental Movie 5 online. (K) to (O) Time-lapse micrographs of profilin/Oregon-green-actin polymerization in the presence of FH5 FH1FH2. The profilin/Oregon-green-actin complex with 5 nM FH5 FH1FH2 was perfused into the flow cell coated with NEM–myosin. See also Supplemental Movie 6 online. (P) The mean elongation rate (±se) of actin filaments. The elongation rates were measured with profilin/actin alone (10.8 ± 0.3; n = 17), profilin/actin plus 5 nM FH5 FH1FH2 (4.2 ± 0.3; n = 11), and profilin/actin plus 10 nM FH5 FH2 (7.7 ± 0.1; n = 17). P < 0.01, by ANOVA test.

Figure 7.

FH5 Binds to the Barbed End of Actin Filaments and Inhibits Dilution-Mediated Actin Depolymerization. (A) Kinetics of actin filament barbed end elongation. Preformed actin filaments (1.0 μM) were incubated with various concentrations of FH5 FH2 before the addition of 0.4 μM pyrene-labeled actin monomers. a.u., absorbance units. (B) Plot of the initial rate of actin elongation versus the concentrations of FH5 FH2 and FH5 FH1FH2. The equilibrium dissociation constant was calculated by fitting the data with Equation 1 (see Methods). The representative K_d is 1.2 nM for FH5 FH2 and 14.6 nM for FH5 FH1FH2. (C) and (D) Kinetics of actin depolymerization in the presence of various concentrations of FH5 FH2 (C) and FH5 FH1FH2 (D) monitored by the decrease in pyrene fluorescence. F-actin (5 μM) was incubated with various concentrations of FH5 FH2 or FH5 FH1FH2 for 5 min at room temperature, and actin depolymerization was initiated by diluting the mixtures 25-fold into buffer G.

Figure 8.

FH5 Binds and Bundles F-Actin in Vitro. (A) A high-speed cosedimentation assay was performed to determine the binding between FH5 and F-actin. A mixture of 3 μM F-actin and 3 μM FH5 FH2 or 3 μM AFH1 FH1FH2 was centrifuged at 150,000g for 30 min at 4°C. Equal amounts of supernatant and pellet were separated by 10% SDS-PAGE and stained with Coomassie blue. S, Supernatant; P, pellet. (B) Increasing concentrations of FH5 FH2 (0.2–3.5 μM) were cosedimented with 3 μM F-actin. The concentration of bound FH5 FH2 was plotted against the concentration of free FH5 FH2 and fitted with a hyperbolic function. The representative K_d value was calculated to be 0.30 μM for FH5 FH2. (C) Identical experiments were performed with AFH1 FH1FH2 (0.33–3.3 μM). The representative K_d value was calculated to be 0.32 μM for AFH1 FH1FH2. (D) Bundling activity of FH5 6×His-FH2-Cter-6×His was tested by low-speed cosedimentation assay. Lanes 1 and 2, Actin filaments (3 μM) alone; lanes 3 to 8, actin filaments with various concentrations of FH5 6×His-FH2-Cter-6×His; lanes 9 and 10, FH5 6×His-FH2-Cter-6×His alone. (E) Light-scattering assays were performed to confirm the bundling activity of FH5 6×His-FH2-Cter-6×His. Lane 1, Actin filaments alone; lanes 2 to 4, actin filaments plus various concentrations of FH5 6×His-FH2-Cter-6×His (lane 2, 170 nM; lane 3, 340 nM; lane 4, 500 nM); lane 5, actin filaments plus 150 nM FH1FH2 of AFH1. (F) to (J) Micrographs of actin bundles: actin filaments alone (F); actin filaments plus 500 nM FH5 FH2 (G); actin filaments plus 500 nM FH5 6×His-FH2-Cter-6×His (H); actin filaments plus 500 nM FH5 FH1FH2 (I); actin filaments plus 500 nM AFH1 FH1FH2 (J). Bar in (J) = 20 μm.

Figure 9.

FH5 Binds and Bundles Microtubules in Vitro. (A) FH5 FH2 binds to taxol-stabilized microtubules. Taxol-stabilized microtubules (5 μM) were incubated with various concentrations of FH5 proteins for 30 min at room temperature, and the mixtures were then subjected to centrifugation at 25,000g for 30 min at 25°C. The supernatants (S) and pellet (P) were analyzed by SDS-PAGE. Lanes 1 and 2, Tubulin alone; lanes 3 to 8, tubulin with various concentrations of FH5 6×His-FH2-6×His; lanes 9 and 10, FH5 6×His-FH2-6×His alone; lanes 11 and 12, tubulin with 1 μM FH5 GST-FH2; lanes 13 and 14, 1 μM FH5 GST-FH2 alone. (B) The amounts of FH5 6×His-FH2-6×His bound were plotted versus the total concentration of FH5. Bound represents the FH5 6×His-FH2-6×His protein in the pellet. (C) to (G) Micrographs of microtubules in the absence or presence of FH5 proteins or Nt MAP65 as indicated: microtubules alone (C); microtubules plus 500 nM FH5 FH2 (D); microtubules plus 500 nM FH5 6×His-FH2-6×His (E); microtubules plus 500 nM FH5 FH1FH2 (F); microtubules plus 500 nM Nt MAP65 (G). Bar in (G) = 20 μm.