Elucidating the Mechanism of Cortical Microtubule Reorientation in Plant Cells

Reorientation of the cortical microtubule array is an essential component of cellular development in plants. However, mechanistic details of this process are unknown. The cortical microtubule array of freshly isolated protoplasts (obtained from Nicotiana tabacum BY-2 suspension culture) is relatively random, but upon culturing the cell wall regenerates and the microtubules begin to reorganize. Because cortical microtubules are highly dynamic, we postulated that their reorganization is accomplished solely by the depolymerization of disordered microtubules, followed by repolymerization into an ordered array. This hypothesis was tested on freshly isolated protoplasts using drugs that alter the dynamic status of microtubules by either hyperstabilizing the polymer (taxol); or preventing the addition of subunits to the microtubules (amiprophosmethyl; APM). Microtubule arrays that were hyperstabilized with 10 μM taxol not only reordered, but did so more quickly than untreated cells. Moreover, protoplasts treated with taxol and 20 μM APM also showed accelerated reorganization. Control experiments, performed in vivo and in vitro, confirmed that subunit addition was hindered by APM. Thus, microtubules appear capable of reorienting as relatively intact units. Sodium azide (1 mM) and sodium cyanide (1 mM) can prevent reorientation, indicating that cellular energy is required for this event but this energy is not used by the actin-myosin system because the microfilament-disrupting drug cytochalasin D (50 μM) did not affect reorientation. These results indicate that cortical microtubule array reorganization is a complex process that can involve polymer movement.