The {gamma}-tubulin complex protein GCP4 is required for organizing functional microtubule arrays in Arabidopsis thaliana

Abstract

Microtubule (MT) nucleation and organization depend on the evolutionarily conserved protein gamma -tubulin, which forms a complex with GCP2-GCP6 (GCP for gamma -Tubulin Complex Protein). To date, it is still unclear how GCP4-GCP6 (the non-core GCPs) may be involved in acentrosomal MT nucleation in plant cells. We found that GCP4 was associated with gamma -tubulin in vivo in Arabidopsis thaliana. When GCP4 expression was repressed by an artificial microRNA, transgenic plants exhibited phenotypes of dwarfism and reduced organ size. In mitotic cells, it was observed that the gamma -tubulin signal associated with the mitotic spindle, and the phragmoplast was depleted when GCP4 was downregulated. Consequently, MTs failed to converge at unified spindle poles, and the bipolar phragmoplast MT array frequently had discrete bundles with extended minus ends, resulting in failed cytokinesis as reflected by cell wall stubs in leaf epidermal cells. In addition, cortical MTs in swollen guard cells and pavement cells of the leaf epidermis became hyperparallel and bundled, which was likely caused by frequent MT nucleation with shallow angles on the wall of extant MTs. Therefore, our results support the notion that GCP4 is an indispensable component for the function of gamma -tubulin in MT nucleation and organization in plant cells.

Figure 1.

Interaction between At GCP4 and γ -Tubulin in Vivo. Proteins affinity purified from transgenic Arabidopsis lines expressing GCP4-FLAG were probed with anti-AtGCP4 (lane 1) and anti-FLAG (lane 2) to detect isolated GCP4 and with the antibodies GTA (lane 3) and R-70 (lane 4) to detect γ -tubulin. The molecular mass markers (in kD) are shown at the left.

Figure 2.

Knocking Down the Expression of Arabidopsis GCP4 by amiR-GCP4. (A) Predicted folding of miR164b (top) and replacement of the miRNA164b and miRNA164b* sequences with the amiR-GCP4 and amiR-GCP4* sequences, respectively (bottom). (B) Growth phenotypes of 7-week-old plants of amiR-GCP4 lines (#1, #4, #17, #9, and #24) compared with the wild-type control. (C) to (E) Close-up views of amiR-GCP4 lines 1, 4, and 17, respectively. (F) In wild-type leaves, trichomes predominantly bear three branches. (G) In amiR-GCP4 leaves, trichomes often bear four or five branches (arrows). (H) Assessment of the expression levels of At GCP4 in the representative amiR-GCP4 lines (#4, 0.20 ± 0.057; #17, 0.24 ± 0.028; #9, 0.43 ± 0.056; and #24, 0.50 ± 0.065) compared with the wild-type control (1.00 ± 0.19) by quantitative RT-PCR. Bars = 0.5 cm in (C) to (E) and 500 μ m (F) and (G).

Figure 3.

Outlines of Leaf Epidermal Cells in Wild-Type and amiR-GCP4 Plants. (A) Propidium iodide staining highlights the outlines of lobed pavement cells and pairs of guard cells (arrow) in a wild-type leaf. (B) In the leaf of amiR-GCP4 plants, some differentiated guard cells are abnormally swollen (arrow). Frequently, no paired guard cells were formed between pavement cells due to failed cell division (asterisks). Cell wall stubs were found in both pavement cells and aborted guard cells (arrowheads). Bar = 20 μ m.

Figure 4.

Abnormal MT Organization in the Mitotic Spindle and the Phragmoplast of amiR-GCP4 Cells. (A) Wild-type spindles have MTs converging toward spindle poles (asterisks). In the mutant spindles, MTs often converge in several discrete sites (arrows) and have prominent nonspindle MTs (arrowheads). (B) MTs in wild-type phragmoplasts are organized in a bipolar fashion. In amiR-GCP4 cells, some early phragmoplasts have disorganized MTs that extend beyond the region normally occupied by the phragmoplast. In mature phragmoplasts of wild-type cells, MTs have the most prominent signals toward the periphery. In amiR-GCP4 cells, MTs often appear in discrete, thin bundles or extend beyond normal positions (arrowheads). (C) Quantitative assessment of abnormal spindles and phragmoplasts in amiR-GCP4 plants compared with the control ones. Bars = 5 μ m.

Figure 5.

Reduction of the γ -Tubulin Signal on Spindle and Phragmoplast MTs in amiR-GCP4 Cells. (A) In wild-type cells, γ -tubulin appears prominently on spindle MTs. Greatly reduced signals are associated with abnormal amiR-GCP4 spindle MTs. (B) The wild-type phragmoplast also contains a prominent γ -tubulin signal. In amiR-GCP4 mutant cells, the γ -tubulin signal is greatly diminished in phragmoplast MTs. (C) Quantitative assessment of relative signal intensity in the control cells and amiR-GCP4 cells. Bars = 5 μ m.

Figure 6.

MT Organization in Epidermal Cells of the Wild-Type Control and amiR-GCP4 Leaves Expressing GFP-TUB6. (A) In the control leaf epidermal pavement cells, cortical MTs exhibit a complex fine network. (B) In the amiR-GCP4 mutant, cortical MTs exhibit highly ordered parallel bundles in most pavement cells and enlarged guard cells. (C) In a control leaf, all pavement cells exhibit fine cortical MTs with nonuniform orientations within individual cells. (D) In an amiR-GCP4 mutant leaf, highly bundled MTs exhibit parallel configurations. Bars = 20 μ m in (A) and (B) and 10 μ m in (C) and (D).

Figure 7.

Abnormal MT Organization in Swollen Guard Cells. (A) In the control guard cells, radial MTs decorated by GFP-TUB6 are nucleated from the central region facing the ventral cell wall surrounding the stomatal pore (asterisk). (B) to (D) In the amiR-GCP4 line expressing GFP-TUB6, abnormally swollen guard cells contain highly bundled parallel cortical MTs (B). Other cells derived from failed division of the guard mother cell. Cortical MTs are aligned uniformly in all directions, resulting in enlarged spherical cells ([C] and [D]). Bar = 10 μ m.

Figure 8.

MT Nucleation on Extant MTs in Control and amiR-GCP4 Cells. (A) Two representative nucleation events are shown in both control cells and amiR-GCP4 cells expressing GFP-TUB6. In the control panels, two nucleation sites are indicated by an arrowhead and an arrow with respective MT-branching angles of 39.7 ° and 41.6 °. In the amiR-GCP4 panels, the two MT-branching angles are 14.0 ° (arrowhead) and 30.6 ° (arrow). Bar = 2.5 μ m. (B) Quantitative analysis of MT branching angles in both the control (n = 80) and amiR-GCP4 cells expressing GFP-TUB6 (n = 71). Statistically significant difference was shown between amiR-GCP4 cells and the control cells (Student's t test; P < 0.01).