Helical microtubule arrays in a collection of twisting tubulin mutants of Arabidopsis thaliana

Abstract

Anisotropic expansion of plant cells requires organized arrays of cortical microtubules. Mutations in microtubule-associated proteins and a particular mutation in ␣-tubulins were reported to cause abnormal microtubule arrays and result in helical growth in Arabidopsis thaliana. However, the way in which these mutations affect the organization of microtubules remains unknown. We here identified 32 Arabidopsis twisting mutants that have either missense or amino acid deletion mutations in ␣-or ␤-tubulins. Mutations were mapped to the GTPase-activating region in ␣-tubulin, intra-and interdimer interfaces of tubulin heterodimers, and lateral contact regions among adjacent protofilaments. These dominant-negative tubulin mutants were incorporated into the microtubule polymer and formed shallow helical arrays of distinct handedness along the long axis of the root epidermal cells. A striking correlation exists between the direction in which cortical helical arrays are skewed and the growth direction of elongating roots. The GTPase-activating-region mutant had left-handed helical arrays composed of highly stabilized microtubules, which could be decorated along the entire microtubule lattices with the otherwise tip-localized End Binding 1 protein. A mutation at the intradimer interface, on the other hand, generated highly dynamic microtubules and right-handed helical arrays. Cortical microtubules in wild type and these two tubulin mutants were composed mainly of 13 protofilaments. This comprehensive analysis of tubulin mutations provides insights into the mechanism by which tubulin structures influence microtubule dynamics and organization.dynamic instability ͉ GTPase-activating region ͉ helix D ifferentiated plant cells show a striking diversity of shapes that are important to their functions. As a plant cell expands under the force of turgor pressure, cellulose microfibrils in the wall function as the primary load-bearing structure, and their spatial arrangement generally determines the direction of cell expansion and final cell shape (1). A long-standing paradigm proposes that plasma membrane-associated microtubules direct or constrain the movement of the cellulose-synthesizing complex and thus dictate the deposition patterns of cellulose microfibrils, although the relationship between the cortical microtubules and cell morphogenesis may be more complicated (1, 2).Rapidly expanding cells in the roots and the etiolated hypocotyls grow longitudinally to form highly elongated cylindrical cells and possess arrays of cortical microtubules that are arranged largely perpendicular to the cell's long axis (3). The importance of transverse arrays for straight growth has been indicated by pharmacological studies and experiments with helical growth mutants of Arabidopsis thaliana. The rapidly elongating cells of these mutants skew consistently either to the right or to the left and exhibit cortical microtubule arrays that form shallow helices with fixed handedness (4). Plant-specific microtubule-associated proteins are mutated in t...

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus

“…Similar phenotypes were reported for tortifolia and spiral mutants (Furutani et al, 2000;Buschmann et al, 2004;Shoji et al, 2004). Helical growth defects commonly correlate with an increased transverse alignment of MTs (Ishida et al, 2007), which are visible upon the overexpression of YFP-IQD11, GFP-IQD14, and GFP-IQD16 (Figs. 6I and 7C).…”

“…6, A-D), indicating the functionality of the fusion protein. Helical growth aberrations and altered leaf shape were reported for tomato sun lines (Wu et al, 2011) and often are associated with defects in MT functions (Buschmann et al, 2004;Ishida et al, 2007). Moreover, the growth defects are reminiscent of plants overexpressing LONGIFOLIA1/TON1 RECRUITING MOTIF2 (TRM2), which encodes a plant-specific MAP (Lee et al, 2006;Drevensek et al, 2012).…”

“…Additional support for IQD functions at MTs is indirectly provided by studies in tomato, which showed that duplication of the IQD12/SUN locus causes elongated fruits, altered cell shape, and twisted stems (Xiao et al, 2008;Wu et al, 2011). These phenotypes are reminiscent of plants with mutations in tubulin subunits (Ishida et al, 2007) and several MAPs (Buschmann et al, 2004;Sedbrook et al, 2004). Collectively, the limited evidence points to regulatory roles of IQD proteins in Ca 2+ -CaM signaling and plant development.…”

Computer simulation and mathematical models of the noncentrosomal plant cortical microtubule cytoskeleton

“…The simulations of Eren et al [2010] showed that changing the microtubule polymer dynamics as experimentally observed in Arabidopsis tua4 S178D and tua5 D251N twisted growth mutants [Ishida et al, 2007] was not sufficient to induce skewed arrays. Inspired by the conceptual framework for the role of CMT nucleation, and branch-form nucleation in particular, in CMT array orientation [Wasteneys and Ambrose, 2009], Eren et al [2010] used their 3D simulations with nonperiodic boundaries to test several scenarios with branch-form nucleation and boundary conditions such as increasing or decreasing the mean branch angle, introducing a bias for branching from one side of the mother CMT, and assigning only one of the end walls of the cylinder as a nonperiodic boundary.…”

Actin-Dependent and -Independent Functions of Cortical Microtubules in the Differentiation ofArabidopsisLeaf Trichomes

“…The tor2 mutant shows several distinct defects in the dynamics of cortical microtubules. Many of the observed aberrations argue for microtubules being hyperstabilized in the tor2 background, which is probably caused by a decreased GTP hydrolysis rate in tor2 microtubules (Ishida et al, 2007;Buschmann et al, 2009). Interestingly, the tor2 mutant shows right-handed cell expansion even in the single-celled leaf trichomes.…”