Genetic evidence that cellulose synthase activity influences microtubule cortical array organization

Abstract

To identify factors that influence cytoskeletal organization we screened for Arabidopsis (Arabidopsis thaliana) mutants that show hypersensitivity to the microtubule destabilizing drug oryzalin. We cloned the genes corresponding to two of the 131 mutant lines obtained. The genes encoded mutant alleles of PROCUSTE1 and KORRIGAN, which both encode proteins that have previously been implicated in cellulose synthesis. Analysis of microtubules in the mutants revealed that both mutants have altered orientation of root cortical microtubules. Similarly, isoxaben, an inhibitor of cellulose synthesis, also altered the orientation of cortical microtubules while exogenous cellulose degradation did not. Thus, our results substantiate that proteins involved in cell wall biosynthesis influence cytoskeletal organization and indicate that this influence on cortical microtubule stability and orientation is correlated with cellulose synthesis rather than the integrity of the cell wall.

Figure 1.

Root phenotypes of oryzalin-hypersensitive mutants 16-2 and 52-isx. A to I, Ecoptype Columbia of Arabidopsis (Col-0; A, D, and G); 16-2 (B, E, and H); and 52-isx (C, F, and I). A to C, Roots of 5-d-old seedlings grown on MS media containing 0.017% methanol. D to F, Roots of 5-d-old seedlings grown on MS containing 175 nm oryzalin. Col-0 (A and D) does not exhibit apparent cell swelling when grown on 175 nm oryzalin while line 16-2 (B and E) and 52-isx (C and F) exhibited swollen cells when grown in the presence of oryzalin. G to I, Roots of 7-d-old seedlings first grown on MS media for 4 d and then transferred to 175 nm oryzalin plates for three additional days exhibit more severe swelling. Caliper bar in D to F is set to the width of Col-0 root in D. Scale bar = 100 μm.

Figure 2.

Seedling phenotypes of 16-2 and 52-isx mutants. A, Five-day-old etiolated Col-0, 16-2, and 52-isx seedlings. B to D, Five-day-old Col-0, 16-2, and 52-isx seedlings grown under 24-h light on MS media (B), 1.5 nm isoxaben (C), and 175 nm oryzalin (D). E, Col-0, 16-2, and 52-isx seedlings were grown on MS media for 4 d and then transferred to 175 nm oryzalin for another 3 d. Scale bar = 5 mm.

Figure 3.

Measurement of root swelling in oryzalin-treated seedlings. Control seedlings were germinated on media containing 0.017% methanol and grown for 5 d. Oryzalin seedlings were germinated on 175 nm oryzalin and grown for 5 d. Transferred seedlings were germinated on 0.017% methanol and transferred to plates containing 175 nm oryzalin for another 3 d. Error bars indicate se.

Figure 4.

Effect of oryzalin on microtubule organization in the 16-2 and 52-isx mutants visualized by confocal microscopy of GFP:MAP4. Five-day-old Col-0 (A and D), 16-2 (B and C), and 52-isx (E and F) seedlings were grown under 24-h light on vertical plates containing the indicated inhibitors. Cortical microtubules of the GFP:MAP4 marker line (A and D) were unaffected by 150 nm oryzalin. The 16-2 mutant exhibits disorganized cortical microtubules in the absence of oryzalin (B), and treatment with 150 nm oryzalin causes lateral expansion of elongating root cells (C). E, Microtubule array organization appears normal in 52-isx without oryzalin treatment. F, Treatment with 150 nm oryzalin results in microtubule array reorganization from transverse to longitudinal near the end of the root elongating zone in the 52-isx mutant. Arrowheads mark the end of the root cap and the beginning of the elongation zone (A to C) and root hair emergence marking the end of the elongation zone (D to E). Scale bar =10 μm.

Figure 5.

Abrupt oryzalin exposure causes randomized cortical microtubule organization in the 52-isx mutant. GFP:MAP4 control and 52-isx seedlings were grown on vertical plates with 24-h light directly on coverglass within a thin film of growth medium. Five-day-old seedlings were treated with 175 nm oryzalin for 4.5 h and then imaged by confocal microscopy. Cortical microtubules in the GFP:MAP4 (A) control line remain transverse after oryzalin exposure. Similar treatment of 52-isx mutant seedlings (B) resulted in randomized microtubules throughout the root elongation zone. Scale bar = 10 μm.

Figure 6.

Cortical microtubules of 52-isx are sensitive to 1-butanol. A, Five-day-old GFP-MAP4 untreated. B and C, Five-day-old seedlings carrying GFP:MAP4 were transferred to media containing 0.2% 1-butanol for 22 to 24 h. Some loss of order occurs in MAP4 control (B) but 52-isx (C) is further disorganized by the same treatment, suggesting less stable cortical arrays. (D) Treatment of 52-isx with 0.2% 2-butanol does not affect MT organization demonstrating the specificity of the 1-butanol affects. Scale bar = 10 μm.

Figure 7.

Disruption of CESA activity affects microtubule organization. All seedlings were grown on 0.5× MS under 24-h light for 5 d and carried GFP:MAP4 to facilitate visualization of microtubules. A, Control seedlings mounted in 0.01% dimethyl sulfoxide for 2.5 h. B, MAP4-GFP seedlings mounted in 100 nm isoxaben for 2.5 h, resulting in oblique and longitudinally organized microtubules. C, MAP4-GFP seedlings treated with .1% cellulase R10. D, MAP4-GFP seedlings treated with 150 nm oryzalin for 3.5 h. E, Five-day-old prc1-20 seedlings mounted in 150 nm oryzalin for 3.5 h. The oryzalin treatment results in randomized microtubules. F, Five-day-old control seedlings treated with 0.1% cellulase R10 and 150 nm oryzalin. Caliper bars are set to the width of the cell at the top of the file. Scale bar = 10 μm.

Figure 8.

CESA6:YFP dynamics are altered in kor1-3 and kor1-1. A and B, Average of 60 frames acquired over 10 min in wild type (A) and kor1-3 (B). Red circle in B marks a slow moving particle (78 nm/min) that is seen as a bright spot in the average projection. Scale bar = 10 μm. C, Histogram of measured particle velocities; the mean is 353 nm/min in wild type and 280 nm/min in kor1-3. D to F, Kymographs from 10-min observations of wild type (D) and kor1-3 (E and F). Particle velocities are constant in the wild type (D); note the linear streaks. Kymograph (E) corresponds to the slow moving particle marked by the red circle in B; note the shallow slope indicating slow movement. Particle velocities can be seen to slow down in kor1-3 during the period of imaging (F; arcing streaks indicated by red arrowheads), while the particle marked with a green arrowhead remained at a constant velocity. G, Average projected image of kor1-1 as in A and B. H, Kymograph of YFP:CESA6 in kor1-1 showing consistently slow velocities measured to be 143 ± 43 nm/min (n = 50). Scale bar = 10 μm.