Fig. 7. Phenotypes of tub2^C239Y. Homozygous seedlings of tub2^C239Y and tub4^P287L (not shown) showed severe defects in anisotropic cell expansion, cytokinesis, and vascular development. (A) Fourteen-day-old seedlings of tub2^C239Y. (B and C) Leaf trichomes. (D and E) Root epidermal cells. Arrows indicate waving root hairs. (F and G) Nuclei visualized by DAPI staining in single root hair cells. Multiple nuclei were also observed in other root cell types. Vascular patterns in cotyledons (H and I) and in true leaves (J and K). (B, D, F, H, and J) Wild type. (A, C, E, G, I, and K) tub2^C239Y.

Fig. 8. Expression of mutant tubulins in wild-type Arabidopsis plants reproduces helical growth phenotypes. Transgenes were tagged with Myc epitopes and expressed under the control of the cauliflower mosaic virus 35S promoter. Root epidermal cells were double stained with antibodies against a-tubulin (red) and the Myc epitope (green). Merged images (yellow) show that expressed tubulin proteins were incorporated into the microtubule polymer, along with wild-type tubulins. This figure illustrates selected examples of mutant tubulins. Other tubulin mutants listed in Table 1 also reproduced the mutant phenotypes and were incorporated into microtubules.

Fig. 9. Comparison of the amino acid sequences of tubulin from pig and Arabidopsis. (A) a-Tubulin. (B) b-Tubulin. Only amino acids that differ from those at the corresponding positions in the pig sequence are shown. Amino acids having identity with those of the pig tubulin at top are indicated by dots. Dashes indicate where gaps were introduced to permit optimal sequence alignment. Mutated amino acids are shown by vertical arrowheads above the pig sequence.

Fig. 10. Quantitative analysis of microtubule orientations. Measurements were taken from immunofluorescence micrographs using anti-a-tubulin antibody in the middle to late elongation zone of 4-day-old seedling roots. Data are displayed as frequency histograms. The average microtubule angles are shown and indicated with arrowheads above the histogram bars. The number of microtubules analyzed (n) is also shown.

Fig. 11. Fluorescence redistribution of tua5^D251N microtubules. The cortical microtubule array in a hypocotyl cell was photobleached by a laser 19-mm in diameter at time 0, and fluorescence images 4 sec before and 12 sec and 60 sec after are shown. (A) Microtubules labeled with GFP-EB1. (B) Microtubules labeled with GFP-TUB6. GFP-EB1 fluorescence on the microtubule lattice recovered completely at 12 sec after photobleaching. We estimate that the half recovery time (T_1/2) is »4 sec. In contrast, microtubule labeling of GFP-TUB6 was not completed even at 60 sec after photobleaching, as reported earlier (1).

1. Shaw SL, Kamyar R, Ehrhardt DW (2003) Science 300: 1715-1718.

Time-lapsed images of Arabidopsis hypocotyl cells expressing GFP-EB1. The images in each sequence were acquired every 4 sec, and each movie consists of 46 frames. S1, wild type; S2, tua5^D251N; and S3, tua4^S178D.

The TUA6 coding region, which contained the single intron and was tagged with a c-Myc epitope at the C terminus (1), and the TUB4 coding region in the Arabidopsis cDNA clone (RZ14g08) were amplified by using gene-specific primers, and cloned into the pDONR221 Gateway entry vector (Invitrogen, Carlsbad, CA). Mutations were introduced by using a KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) and specific primers, and mutated entry vectors were transferred to the pGWB2 (for TUA6) or pGWB18 (for TUB4) Gateway binary vector (obtained from T. Nakagawa, Shimane University). Four copies of the c-Myc epitope were fused to the N terminus of TUB4 in pGWB18. Both vectors expressed transgenes under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The constructs were introduced into the Arabidopsis wild-type plants (Columbia ecotype) using Agrobacterium-mediated infiltration.

Anti-c-myc antibody 9E10 (Roche, Basel, Switzerland) and Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes) were used to detect Myc-tagged tubulins at dilutions of 1:200 and 1:500, respectively. Other immunological procedures were the same as described in the text.

FRAP experiments were conducted with a FV1000 confocal laser scanning microscope system on an IX81 inverted microscope (Olympus, Tokyo Japan). A 488-nm laser line was selected for GFP excitation, and images were collected using 4-sec scan times. Photobleaching was conducted using a SIM scanner with a 405-nm laser line at 100% power for 4 sec.

1 Thitamadee S, Tuchihara K, Hashimoto T (2002) Nature 417:193-196