SPIRAL1 encodes a plant-specific microtubule-localized protein required for directional control of rapidly expanding Arabidopsis cells

Highly organized interphase cortical microtubule (MT) arrays are essential for anisotropic growth of plant cells, yet little is known about the molecular mechanisms that establish and maintain the order of these arrays. The Arabidopsis thaliana spiral1 (spr1) mutant shows right-handed helical growth in roots and etiolated hypocotyls. Characterization of the mutant phenotypes suggested that SPR1 may control anisotropic cell expansion through MT-dependent processes. SPR1 was identified by map-based cloning and found to encode a small protein with unknown function. Proteins homologous to SPR1 occur specifically and ubiquitously in plants. Genetic complementation with green fluorescent protein fusion proteins indicated that the SPR1 protein colocalizes with cortical MTs and that both MT localization and cell expansion control are conferred by the conserved N- and C-terminal regions. Strong SPR1 expression was found in tissues undergoing rapid cell elongation. Plants overexpressing SPR1 showed enhanced resistance to an MT drug and increased hypocotyl elongation. These observations suggest that SPR1 is a plant-specific MT-localized protein required for the maintenance of growth anisotropy in rapidly elongating cells.     

Role of the SPIRAL1 gene family in anisotropic growth of Arabidopsis thaliana

Arabidopsis spiral1 (spr1) mutants show a right-handed helical growth phenotype in roots and etiolated hypocotyls due to impaired directional growth of rapidly expanding cells. SPR1 encodes a small protein with as yet unknown biochemical functions, though its localization to cortical microtubules (MTs) suggests that SPR1 maintains directional cell expansion by regulating cortical MT functions. The Arabidopsis genome contains five SPR1-LIKE (SP1L) genes that share high sequence identity in N- and C-terminal regions. Overexpression of SP1Ls rescued the helical growth phenotype of spr1, indicating that SPR1 and SP1L proteins share the same biochemical functions. Expression analyses revealed that SPR1 and SP1L genes are transcribed in partially overlapping tissues. A combination of spr1 and sp1l mutations resulted in randomly oriented cortical MT arrays and isotropic expansion of epidermal cells. These observations suggest that SPR1 and SP1Ls act redundantly in maintaining the cortical MT organization essential for anisotropic cell growth, and that the helical growth phenotype of spr1 results from a partially compromised state of cortical MTs. Additionally, inflorescence stems of spr1 sp1l multiple mutants showed a right-handed tendril-like twining growth, indicating that a directional winding response may be conferred to the non-directional nutational movement by modulating the expression of SPR1 homologs.     

Low concentrations of propyzamide and oryzalin alter microtubule dynamics in Arabidopsis epidermal cells

Previous studies showed that sub-micromolar concentrations of the microtubule-targeting herbicide propyzamide cause a right-handed helical arrangement of cortical microtubule arrays and left-handed twisting in elongating Arabidopsis epidermal cells. When seedlings were grown in the presence of 1-2 microM propyzamide or 50-100 nM oryzalin, we show that microtubules spent more time in a paused state in which they exhibited little net change in length. The drug treatment also resulted in slower growth and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. A reduction in microtubule dynamic turnover may underlie the drug-induced rearrangement of cortical arrays.     

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

Cortical microtubules are responsible for gravity resistance in plants

Mechanical resistance to the gravitational force is a principal gravity response in plants distinct from gravitropism. In the final step of gravity resistance, plants increase the rigidity of their cell walls. Here we discuss the role of cortical microtubules, which sustain the function of the cell wall, in gravity resistance. Hypocotyls of Arabidopsis tubulin mutants were shorter and thicker than the wild-type, and showed either left-handed or right-handed helical growth at 1 g. The degree of twisting phenotype was intensified under hypergravity conditions. Hypergravity also induces reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells. In tubulin mutants, the percentage of cells with longitudinal microtubules was high even at 1 g, and it was further increased by hypergravity. The left-handed helical growth mutants had right-handed microtubule arrays, whereas the right-handed mutant had left-handed arrays. Moreover, blockers of mechanoreceptors suppressed both the twisting phenotype and reorientation of microtubules in tubulin mutants. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in signal perception in gravity resistance. Space experiments will confirm whether this view is applicable to plant resistance to 1 g gravity, as to the resistance to hypergravity.Key words: cortical microtubules, gravity, gravity resistance, hypergravity, mechanoreceptor, microgravity, tubulin mutants     

Salt stress affects cortical microtubule organization and helical growth in Arabidopsis

Cortical microtubule arrays are critical in determining the growth axis of diffusely growing plant cells, and various environmental and physiological factors are known to affect the array organization. Microtubule organization is partly disrupted in the spiral1 mutant of Arabidopsis thaliana, which displays a right-handed helical growth phenotype in rapidly elongating epidermal cells. We show here that mutations in the plasma membrane Na(+)/H(+) antiporter SOS1 and its regulatory kinase SOS2 efficiently suppressed both microtubule disruption and helical growth phenotypes of spiral1, and that sos1 and sos2 roots in the absence of salt stress exhibited altered helical growth response to microtubule-interacting drugs at low doses. Salt stress also altered root growth response to the drugs in wild-type roots. Suppression of helical growth appeared to be specific to spiral1 since other helical growth mutants were not rescued. The effects of sos1 in suppressing spiral1 defects and in causing abnormal drug responses were nullified in the presence of the hkt1 Na(+) influx carrier mutation in roots but not in hypocotyls. These results suggest that cytoplasmic salt imbalance caused by insufficient SOS1 activity compromises cortical microtubule functions in which microtubule-localized SPIRAL1 is specifically involved.     

Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants

The proper organization of cortical microtubule arrays is essential for anisotropic growth in plants but how distinct array patterns are formed is not understood. Here, we report a relationship between microtubule dynamics and array organization using transgenic plants expressing modified tubulins. When green fluorescent protein (GFP) or a hemaglutinin epitope tag was fused to the N-terminus of tubulins and expressed in Arabidopsis plants, these tubulins were incorporated into microtubules along with endogenous tubulins. Plants expressing the modified beta-tubulins were phenotypically normal and possessed transversely oriented cortical arrays in the epidermal cells of the root elongation zone; however, the expression of modified alpha-tubulins caused right-handed helical growth, increased trichome branching, and a shallow left-handed (S-form) helical array organization. In cells expressing the modified alpha-tubulins, microtubule dynamicity was suppressed and polymerization was promoted, and GFP-EB1 (End Binding 1) labeled larger regions of the microtubule end more frequently, when compared with control cells. We propose that the N-terminal appendage introduced into alpha-tubulin inhibits GTP hydrolysis, thus producing polymerization-prone microtubules with an extended GTP cap. Consistent with this interpretation, plants expressing an alpha-tubulin mutated in the GTPase-activating domain exhibited similar microtubule properties, with regard to dynamics and the localization of GFP-EB1, and showed right-handed helical growth.     

Helical Growth of the Arabidopsis Mutant tortifolia2 Does Not Depend on Cell Division Patterns but Involves Handed Twisting of Isolated Cells

Several factors regulate plant organ growth polarity. tortifolia2 (tor2), a right-handed helical growth mutant, has a conservative replacement of Arg-2 with Lys in the α-tubulin 4 protein. Based on a published high-resolution (2.89 Å) tubulin structure, we predict that Arg-2 of α-tubulin forms hydrogen bonds with the GTPase domain of β-tubulin, and structural modeling suggests that these contacts are interrupted in tor2. Consistent with this, we found that microtubule dynamicity is reduced in the tor2 background. We investigated the developmental origin of the helical growth phenotype using tor2. One hypothesis predicts that cell division patterns cause helical organ growth in Arabidopsis thaliana mutants. However, cell division patterns of tor2 root tips appear normal. Experimental uncoupling of cell division and expansion suggests that helical organ growth is based on cell elongation defects only. Another hypothesis is that twisting is due to inequalities in expansion of epidermal and cortical tissues. However, freely growing leaf trichomes of tor2 mutants show right-handed twisting and cortical microtubules form left-handed helices as early as the unbranched stage of trichome development. Trichome twisting is inverted in double mutants with tor3, a left-handed mutant. Single tor2 suspension cells also exhibit handed twisting. Thus, twisting of tor2 mutant organs appears to be a higher-order expression of the helical expansion of individual cells.     

Consistent handedness of microtubule helical arrays in maize and Arabidopsis primary roots

Summary The orientation of cortical microtubules in plant cells has been extensively studied, in part because of their influence on the expansion of most plant cell types. Cortical microtubules are often arranged in helical arrays, which are well known to occur with a specific pitch as a function of development or experimental treatment; however, it is not known if the handedness of helical arrays can also be specified. We have studied the handedness of helical arrays by using Vibratome sectioning of maize primary roots and confocal microscopy of Arabidopsis primary roots. In cortical cells of maize roots, the helical array was found to have the same handedness at a given position, not only for the cells of a single root, but also for the cells of more than one hundred roots examined. Quantification of angular distribution of apparent individual microtubules showed that defined regions of the root were composed of cells with highly uniform microtubule orientation. In the region between transverse and longitudinal microtubules (5–10.5 mm from the tip), the array formed a right-handed helix, and basal of cells with longitudinal microtubules (11.5–15 mm from the tip), the array formed a left-handed helix. Similarly, in epidermal cells of Arabidopsis roots right-handed helical arrays were found in the region between transverse and longitudinal microtubules. These results suggest that, in addition to the orientation of microtubules, the handedness of helical microtubule arrays is under cellular control.Abbreviations Cy3 indocarbocyanine - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis-[2-ethanesulfonic acid]     

An <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> tubulin mutant with conditional root-skewing phenotype

Low doses of microtubule-interacting drugs cause wild-type Arabidopsis thaliana seedling roots to twist in a left-handed helical direction. We here report molecular characterization of an A. thaliana tubulin mutant whose roots twist in a right-handed direction and have shallow left-handed cortical microtubule arrays when challenged with low doses of microtubule drugs. In the absence of the drug, growth and development of the mutant was apparently normal. In this conditional twisting mutant, Cys213 of α-tubulin6 was exchanged with Tyr. The mutant tubulin was incorporated into the microtubule polymer with wild-type tubulins, and thus acted as a dominant-negative mutation. These results suggest that compromised microtubules in wild-type and mutant roots are qualitatively distinct and affect skewing direction differently.     

Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants

We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by 1g gravity and hypergravity in tubulin mutants.The direction of cell expansion is important for determining the shape of whole plant body. Cortical microtubules are assumed to be responsible for anisotropic expansion of plant cells (Wasteneys and Galway, 2003; Lloyd and Chan, 2004; Mathur, 2004; Baskin, 2005; Paredez et al., 2008). The prevailing view is that cortical microtubule arrays direct or constrain the movement of the cellulose synthase complexes and thus align nascent cellulose microfibrils in the same direction in the innermost layer of the cell wall (Baskin, 2001), although some other mechanisms may also be involved (Baskin, 2001; Sugimoto et al., 2003; Wasteneys, 2004).It is evident that orientation of cortical microtubules plays an essential role in creating the distinct shape of higher plant organs, even if there is uncertainty over the mechanism by which microtubules influence morphogenesis. The importance of cortical microtubule arrays for anisotropic growth has been documented by pharmacological studies and experiments with helical growth mutants of Arabidopsis (Arabidopsis thaliana). Mutants on α- and β-tubulins as well as microtubule-associated proteins show either left-handed or right-handed helical growth (Thitamadee et al., 2002; Nakajima et al., 2004; Sedbrook et al., 2004; Shoji et al., 2004). 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 (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). Cortical microtubule arrays in the left-handed helical growth mutants form right-handed helix, whereas those in right-handed helical growth mutants form left-handed helix (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). These results indicate that dysfunctional cortical microtubules are arranged in helical arrays and affect the direction of cell expansion.The gravitational force is one of the environmental factors that determine the plant body shape. Under hypergravity conditions produced by centrifugation, plants generally have a shorter and thicker body (Soga et al., 2006). Namely, hypergravity modifies growth anisotropy. In Arabidopsis hypocotyls, the expression of most α- and β-tubulin genes was up-regulated by hypergravity (Yoshioka et al., 2003; Matsumoto et al., 2007). In protoplasts of Brassica hypocotyls, hypergravity stimulated the regeneration of cortical microtubules into parallel arrays (Skagen and Iversen, 1999), and in azuki bean (Vigna angularis) epicotyls it increased the percentage of cells with longitudinal cortical microtubules (Soga et al., 2006). The reorientation of cortical microtubules from transverse to longitudinal directions may be involved in modifications by hypergravity to growth anisotropy.The aim of this study was to clarify the roles of cortical microtubules in gravity-induced modifications to development of stem organs. For this purpose, we examined the changes in growth, morphology, and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis amino acid substitution mutants in α-tubulin structure, tua3, tua4, and tua6, grown under 1g and 300g conditions. We have reported the possible involvement of mechanosensitive ion channels (mechanoreceptors) in hypergravity-induced modifications to growth and cell wall properties (Soga et al., 2004, 2005, 2006). Thus, we also examined the effect of blockers of mechanoreceptors on helical growth and orientation of cortical microtubule arrays in the tubulin mutants.     

Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein

Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.     

Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis

Profilin (PFN) is an ubiquitous, low-M(r), actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.     

WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana

Arabidopsis WAVE-DAMPENED 2 (WVD2) was identified by forward genetics as an activation-tagged allele that causes plant and organ stockiness and inversion of helical root growth handedness on agar surfaces. Plants with high constitutive expression of WVD2 or other members of the WVD2-LIKE (WDL) gene family have stems and roots that are short and thick, have reduced anisotropic cell elongation, are suppressed in a root-waving phenotype, and have inverted handedness of twisting in hypocotyls and roots compared with wild-type. The wvd2-1 mutant shows aberrantly organized cortical microtubules in peripheral root cap cells as well as reduced branching of trichomes, unicellular leaf structures whose development is regulated by microtubule stability. Orthologs of the WVD2/WDL family are found widely throughout the plant kingdom, but are not similar to non-plant proteins with the exception of a C-terminal domain distantly related to the vertebrate microtubule-associated protein TPX2. in vivo, WVD2 and its closest paralog WDL1 are localized to interphase cortical microtubules in leaves, hypocotyls and roots. Recombinant glutathione-S-transferase:WVD2 or maltose binding protein:WVD2 protein bind to and bundle microtubules in vitro. We speculate that a C-terminal domain of TPX2 has been utilised by the WVD2 family for functions critical to the organization of plant microtubules.     

Molecular genetic analysis of left-right handedness in plants

Handedness in plant growth may be most familiar to us when we think of tendrils or twining plants, which generally form consistent right- or left-handed helices as they climb. The petals of several species are sometimes arranged like fan blades that twist in the same direction. Another less conspicuous example is 'circumnutation', the oscillating growth of axial organs, which alternates between a clockwise and an anti-clockwise direction. To unravel molecular components and cellular determinants of handedness, we screened Arabidopsis thaliana seedlings for helical growth mutants with fixed handedness. Recessive spiral1 and spiral2 mutants show right-handed helical growth in roots, hypocotyls, petioles and petals; semi-dominant lefty1 and lefty2 mutants show opposite left-handed growth in these organs. lefty mutations are epistatic to spiral mutations. Arabidopsis helical growth mutants with fixed handedness may be impaired in certain aspects of cortical microtubule functions, and characterization of the mutated genes should lead us to a better understanding of how microtubules function in left-right handedness in plants.     

Spiral cell wall growth in zygnemataceae

Using two species ofSpirogyra and one species ofZygnema, it was demonstrated on a quantitative basis that these algal filaments grow while twisting around their own axis. The sense of spiral growth of the cell wall inSpirogyra-1 was always left-handed being coincident with the sense of chloroplast helix. InSpirogyra-2, the growth vector of the cell wall was likewise left-handed in most cases, but there occurred right-handed growth also. InZygnema both left-handed and right-handed senses of spiral growth were found in nearly equal frequencies. Besides the natural cell wall growth, the effects of longitudinal tension and turgor pressure on elongation and twisting of the filaments were briefly studied. It was shown that the cell wall of Zygnemataceae exhibited mechanical anisotropy in helical direction.     

NIMA-related kinases regulate directional cell growth and organ development through microtubule function in Arabidopsis thaliana

NIMA-related kinase 6 (NEK6) regulates cellular expansion and morphogenesis through microtubule organizaiton in Arabidopsis thaliana. Loss-of-function mutations in NEK6 (nek6/ibo1) cause ectopic outgrowth and microtubule disorganization in epidermal cells. We recently found that NEK6 forms homodimers and heterodimers with NEK4 and NEK5 to destabilize cortical microtubules possibly by direct binding to microtubules and the β-tubulin phosphorylation. Here, we identified a new allele of NEK6 and further analyzed the morphological phenotypes of nek6/ibo1 mutants, along with alleles of nek4 and nek5 mutants. Phenotypic analysis demonstrated that NEK6 is required for the directional growth of roots and hypocotyls, petiole elongation, cell file formation, and trichome morphogenesis. In addition, nek4, nek5, and nek6/ibo1 mutants were hypersensitive to microtubule inhibitors such as propyzamide and taxol. These results suggest that plant NEKs function in directional cell growth and organ development through the regulation of microtubule organization.