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

Arabidopsis thaliana tortifolía2 carries a point mutation in α-tubulin 4 and shows aberrant cortical microtubule dynamics. The microtubule defect of tortifolia2 leads to overbranching and right-handed helical growth in the single-celled leaf trichomes. Here, we use tortifolia2 to further our understanding of microtubules in plant cell differentiation. Trichomes at the branching stage show an apical ring of cortical microtubules, and our analyses support that this ring is involved in marking the prospective branch site. tortifolia2 showed ectopic microtubule bundles at this stage, consistent with a function for microtubules in selecting new branch sites. Overbranching of tortifolia2 required the C-terminal binding protein/brefeldin A-ADP ribosylated substrate protein ANGUSTIFOLIA1, and our results indicate that the angustifolia1 mutant is hypersensitive to alterations in microtubule dynamics. To analyze whether actin and microtubules cooperate in the trichome cell expansion process, we generated double mutants of tortifolia2 with distorted1, a mutant that is defective in the actin-related ARP2/3 complex. The double mutant trichomes showed a complete loss of growth anisotropy, suggesting a genetic interaction of actin and microtubules. Green fluorescent protein labeling of F-actin or microtubules in tortifolia2 distorted1 double mutants indicated that F-actin enhances microtubule dynamics and enables reorientation. Together, our results suggest actin-dependent and -independent functions of cortical microtubules in trichome differentiation.     

The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the 'distorted' class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.     

Regulation of cell expansion by the <Emphasis Type="Italic"> DISTORTED </Emphasis>genes in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: actin controls the spatial organization of microtubules

The control of the directionality of cell expansion was investigated using a class of eight genes, the so-called DISTORTED (DIS) genes, that are required for proper expansion of leaf trichomes in Arabidopsis thaliana. By tracing the separation of latex beads placed on the trichome surface, we demonstrate that trichomes grow by diffuse rather than tip growth, and that in dis mutants deviations from the normal orientation of growth can occur in all possible directions. We could not detect any differences in intracellular organization between wild-type and dis-group mutants by electron microscopy. The analysis of double mutants showed that although the expression of the dis phenotype is generally independent of branching and endoreduplication, dis mutations act synthetically in combination lesions in the ZWI gene, which encodes a kinesin motor protein. Using a MAP4:GFP marker line, we show that the organization of cortical microtubules is affected in dis-group mutants. The finding that most dis-group mutants have actin defects suggested to us that actin is involved in organizing the orientation of microtubules. By analyzing the microtubule organization in plants treated with drugs that bind to actin, we verified that actin is involved in the positioning of cortical microtubules and thereby in plant cell expansion.     

The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton

The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.     

Trichome morphogenesis: a cell-cycle perspective

Arabidopsis leaf hairs (trichomes) are polyploid epidermal cells with a predictable branching pattern. More than 15 genes have been identified that are involved in the regulation of branching. The cloning of the ZWICHEL, ANGUSTIFOLIA and STICHEL genes points to two mechanistic aspects of branch formation: (i) a role of the microtubule cytoskeleton; and (ii) a link to the regulation of cell divisions. The latter aspect is supported by the recent identification of an Arabidopsis mutant with multicellular trichomes, the siamese mutant, suggesting that Arabidopsis trichomes are evolutionarily derived from multicellular forms. We speculate that the spatial information for branch formation is derived from mechanisms employed in cell divisions.     

Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls

In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis.     

The IRREGULAR TRICHOME BRANCH loci regulate trichome elongation in Arabidopsis

The proper control of cell expansion is vital to plant development. It is responsible for shaping individual cells and, together with cell division, it plays a lead role in shaping plant organs. Much of the underlying mechanism by which plant cells expand anisotropically is not understood. We are taking a genetic approach to cell expansion by isolating mutants that affect the branching pattern of Arabidopsis trichomes. Here we report the identification of four new loci that control trichome morphogenesis. These loci were named the IRREGULAR TRICHOME BRANCH (ITB) loci because of the deleterious effects on branch position and length in the mutants. Our analysis of branch expansion in itb mutants shows that the ITB genes act as positive regulators of branch elongation, and that the branch position defects are caused by altered expansion of the trichome stalk. The itb mutations display synergistic effects in double mutant combinations with certain branch number mutations, suggesting that the ITB genes also play key roles in branch initiation. These results demonstrate that the ITB genes are key regulators of anisotropic cell expansion in trichomes.     

Gravitropic microtubule reorientation can be uncoupled from growth

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90°), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation. Received: 23 November 1999 / Accepted: 10 May 2000     

Actin control over microtubules suggested by DISTORTED2 encoding the Arabidopsis ARPC2 subunit homolog

In Arabidopsis, based on the randomly misshapen phenotype of leaf epidermal trichomes, eight genes have been grouped into a 'DISTORTED' class. Three of the DIS genes, WURM, DISTORTED1 and CROOKED have been cloned recently and encode the ARP2, ARP3 and ARPC5 subunits respectively, of a conserved actin modulating ARP2/3 complex. Here we identify a fourth gene, DISTORTED2 as the Arabidopsis homolog of the ARPC2 subunit of the ARP2/3 complex. Like other mutants in the complex dis2 trichomes also display supernumerary, randomly localized cortical actin patches. In addition dis2 trichomes possess abnormally clustered endoplasmic microtubules near sites of actin aggregation. Since microtubules are strongly implicated in the establishment and maintenance of growth directionality in higher plants our observations of aberrant microtubule clustering in dis2 trichomes suggests a convincing explanation for the randomly distorted trichome phenotype in dis mutants. In addition, the close proximity of microtubule clusters to the arbitrarily dispersed cortical actin patches in the dis mutants provides fresh insights into cytoskeletal interactions leading us to suggest that in higher plants microtubule arrangements directed towards the establishment and maintenance of polar growth-directionality are guided by cortical actin behavior and organization.     

Cortical microtubule arrays undergo rotary movements in Arabidopsis hypocotyl epidermal cells

Plant-cell expansion is controlled by cellulose microfibrils in the wall with microtubules providing tracks for cellulose synthesizing enzymes. Microtubules can be reoriented experimentally and are hypothesized to reorient cyclically in aerial organs, but the mechanism is unclear. Here, Arabidopsis hypocotyl microtubules were labelled with AtEB1a-GFP (Arabidopsis microtubule end-binding protein 1a) or GFP-TUA6 (Arabidopsis alpha-tubulin 6) to record long cycles of reorientation. This revealed microtubules undergoing previously unseen clockwise or counter-clockwise rotations. Existing models emphasize selective shrinkage and regrowth or the outcome of individual microtubule encounters to explain realignment. Our higher-order view emphasizes microtubule group behaviour over time. Successive microtubules move in the same direction along self-sustaining tracks. Significantly, the tracks themselves migrate, always in the direction of the individual fast-growing ends, but twentyfold slower. Spontaneous sorting of tracks into groups with common polarities generates a mosaic of domains. Domains slowly migrate around the cell in skewed paths, generating rotations whose progressive nature is interrupted when one domain is displaced by collision with another. Rotary movements could explain how the angle of cellulose microfibrils can change from layer to layer in the polylamellate cell wall.     

Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis

Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern.     

IRREGULAR TRICHOME BRANCH1 in Arabidopsis encodes a plant homolog of the actin-related protein2/3 complex activator Scar/WAVE that regulates actin and microtubule organization

The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.     

Extragenic suppressors of the arabidopsis zwi-3 mutation identify new genes that function in trichome branch formation and pollen tube growth.

The plant cytoskeleton plays a pivotal role in determining the direction of cell wall expansion, and ultimately the cell's final shape. However, the mechanisms by which localized expansion events are initiated remain obscure. Mutational analysis of the trichome (plant hair) morphogenic pathway in Arabidopsis has identified at least eight genes that determine trichome branch number. One of these genes, ZWICHEL (ZWI), encodes a novel member of the kinesin superfamily of motor proteins. Mutations in the ZWI gene cause a reduction in the number of trichome branches. To identify additional genes involved in trichome branch initiation, we screened for extragenic suppressors of the zwi-3 mutation and isolated three suppressors that rescued the branch number defect of zwi-3. These suppressors define three genes, named suz, for suppressor of zwichel-3. All of the suppressors were shown to be allele specific. One of the suppressors, suz2, also rescued the trichome branch number defect of another branch mutant, furca1-2. Plants homozygous for suz2 have more than the wild-type number of trichome branches. This suggests that SUZ2 is a negative regulator of trichome branching and may interact with ZWI and FURCA1. The suz1 and suz3 mutants display no obvious phenotype in the absence of the zwi-3 mutation. The suz1 zwi-3 double mutants also exhibited a male-sterile phenotype due to a defect in pollen tube germination and growth, whereas both the suz1 and the zwi-3 single mutants are fertile. The synthetic male sterility of the suz1 zwi-3 double mutants suggests a role for SUZ1 and ZWI in pollen germination and pollen tube growth. DNA sequence analysis of the zwi-3 mutation indicated that only the tail domain of the zwi-3 protein would be expressed. Thus, the suz mutations show allele-specific suppression of a kinesin mutant that lacks the motor domain.     

Trichome cell growth in Arabidopsis thaliana can be derepressed by mutations in at least five genes

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.     

The effect of okadaic acid on Arabidopsis thaliana root morphology and microtubule organization in its cells

To investigate the role of protein hyperphosphorylation in plant cells, the effect of okadaic acid, a specific inhibitor of protein phosphatases PPI and PP2A, on the general morphology of Arabidopsis thaliana primary roots and the structural-functional characteristics of cortical microtubules in different cell types in all primary root growth zones was studied. It was found that okadaic acid affects microtubule organization in a different manner depending on the type of cells and functional zones of the primary root. Cortical microtubules in the epidermis and cortex cells of the elongation zone proved to be most sensitive to 0.1, 1, and 10 nM okadaic acid which completely depolymerized after inhibitor treatment. In trichoblasts, atrichoblasts of differentiation zone treatment with okadaic acid caused the microtubules stabilization. The treatment with okadaic acid significantly affected the morphology of root hairs, causing their swelling and branching as a result of abnormal microtubule orientation. The results of this study suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays a crucial key in microtubule organization in plant cells.