Phragmoplast Microtubule Arrays Research Articles

Arabidopsis α-Aurora kinase plays a role in cytokinesis through regulating MAP65-3 association with microtubules at phragmoplast midzone

The α-Aurora kinase is a crucial regulator of spindle microtubule organization during mitosis in plants. Here, we report a post-mitotic role for α-Aurora in reorganizing the phragmoplast microtubule array. In Arabidopsis thaliana, α-Aurora relocated from spindle poles to the phragmoplast midzone, where it interacted with the microtubule cross-linker MAP65-3. In a hypomorphic α-Aurora mutant, MAP65-3 was detected on spindle microtubules, followed by a diffuse association pattern across the phragmoplast midzone. Simultaneously, phragmoplast microtubules remained belatedly in a solid disk array before transitioning to a ring shape. Microtubules at the leading edge of the matured phragmoplast were often disengaged, accompanied by conspicuous retentions of MAP65-3 at the phragmoplast interior edge. Specifically, α-Aurora phosphorylated two residues towards the C-terminus of MAP65-3. Mutation of these residues to alanines resulted in an increased association of MAP65-3 with microtubules within the phragmoplast. Consequently, the expansion of the phragmoplast was notably slower compared to wild-type cells or cells expressing a phospho-mimetic variant of MAP65-3. Moreover, mimicking phosphorylation reinstated disrupted MAP65-3 behaviors in plants with compromised α-Aurora function. Overall, our findings reveal a mechanism in which α-Aurora facilitates cytokinesis progression through phosphorylation-dependent restriction of MAP65-3 associating with microtubules at the phragmoplast midzone.

Maize tubulin folding cofactor B is required for cell division and cell growth through modulating microtubule homeostasis.

Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.

MOR1/MAP215 acts synergistically with katanin to control cell division and anisotropic cell elongation in Arabidopsis.

The MAP215 family of microtubule (MT) polymerase/nucleation factors and the MT severing enzyme katanin are widely conserved MT-associated proteins (MAPs) across the plant and animal kingdoms. However, how these two essential MAPs coordinate to regulate plant MT dynamics and development remains unknown. Here, we identified novel hypomorphic alleles of MICROTUBULE ORGANIZATION 1 (MOR1), encoding the Arabidopsis thaliana homolog of MAP215, in genetic screens for mutants oversensitive to the MT-destabilizing drug propyzamide. Live imaging in planta revealed that MOR1-green fluorescent protein predominantly tracks the plus-ends of cortical MTs (cMTs) in interphase cells and labels preprophase band, spindle and phragmoplast MT arrays in dividing cells. Remarkably, MOR1 and KATANIN 1 (KTN1), the p60 subunit of Arabidopsis katanin, act synergistically to control the proper formation of plant-specific MT arrays, and consequently, cell division and anisotropic cell expansion. Moreover, MOR1 physically interacts with KTN1 and promotes KTN1-mediated severing of cMTs. Our work establishes the Arabidopsis MOR1-KTN1 interaction as a central functional node dictating MT dynamics and plant growth and development.

Role of the BUB3 protein in phragmoplast microtubule reorganization during cytokinesis.

The evolutionarily conserved WD40 protein budding uninhibited by benzimidazole 3 (BUB3) is known for its function in spindle assembly checkpoint control. In the model plant Arabidopsis thaliana, nearly identical BUB3;1 and BUB3;2 proteins decorated the phragmoplast midline through interaction with the microtubule-associated protein MAP65-3 during cytokinesis. BUB3;1 and BUB3;2 interacted with the carboxy-terminal segment of MAP65-3 (but not MAP65-1), which harbours its microtubule-binding domain for its post-mitotic localization. Reciprocally, BUB3;1 and BUB3;2 also regulated MAP65-3 localization in the phragmoplast by enhancing its microtubule association. In the bub3;1 bub3;2 double mutant, MAP65-3 localization was often dissipated away from the phragmoplast midline and abolished upon treatment of low doses of the cytokinesis inhibitory drug caffeine that were tolerated by the control plant. The phragmoplast microtubule array exhibited uncoordinated expansion pattern in the double mutant cells as the phragmoplast edge reached the parental plasma membrane at different times in different areas. Upon caffeine treatment, phragmoplast expansion was halted as if the microtubule array was frozen. As a result, cytokinesis was abolished due to failed cell plate assembly. Our findings have uncovered a novel function of the plant BUB3 in MAP65-3-dependent microtubule reorganization during cytokinesis.

The Mitotic Function of Augmin Is Dependent on Its Microtubule-Associated Protein Subunit EDE1 in Arabidopsis thaliana

The augmin complex plays an essential role in microtubule (MT)-dependent MT nucleation by recruiting the γ-tubulin complex to MT walls to generate new MTs [1]. The complex contains eight subunits (designated AUG) including AUG8, which is an MT-associated protein (MAP). When this complex is isolated from etiolated seedlings consisting of primarily interphase cells in Arabidopsis thaliana, AUG8 is an integral component [2]. EDE1 (Endosperm DEfective 1) is homologous to AUG8 [3]. Here, we demonstrate that EDE1, but not AUG8, is associated with acentrosomal spindle and phragmoplast MT arrays in patterns indistinguishable from those of the AUG1-7 subunits and the γ-tubulin complex proteins (GCPs) that exhibit biased localization toward MT minus ends. Consistent with this colocalization, EDE1 directly interacts with AUG6 invivo. Moreover, a partial loss-of-function mutation, ede1-1, compromises the localization of augmin and γ-tubulin on the spindle and phragmoplast MT arrays and leads to serious distortions in spindle MT remodeling during mitosis. However, mitosis continues even when kinetochore fibers are not obviously discernable, and cytokinesis takes place following the formation of elongated bipolar phragmoplast MT arrays in the mutant. Hence, we conclude that the mitotic function of augmin is dependent on its MAP subunit EDE1, which cannot be replaced by AUG8, and that the cell-cycle-dependent function of augmin can be differentially regulated by employing distinct MAP subunits. Our results also illustrate that plant cells can respond flexibly to serious challenges of compromised MT-dependent MT nucleation to complete mitosis and cytokinesis.

Mechanism of microtubule array expansion in the cytokinetic phragmoplast

In land plants, the cell plate partitions the daughter cells at cytokinesis. The cell plate initially forms between daughter nuclei and expands centrifugally until reaching the plasma membrane. The centrifugal development of the cell plate is driven by the centrifugal expansion of the phragmoplast microtubule array, but the molecular mechanism underlying this expansion is unknown. Here, we show that the phragmoplast array comprises stable microtubule bundles and dynamic microtubules. We find that the dynamic microtubules are nucleated by γ-tubulin on stable bundles. The dynamic microtubules elongate at the plus ends and form new bundles preferentially at the leading edge of the phragmoplast. At the same time, they are moved away from the cell plate, maintaining a restricted distribution of minus ends. We propose that cycles of attachment of γ-tubulin complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by minus-end-directed motility, drive the centrifugal development of the phragmoplast.

Characterization of the Arabidopsis Augmin Complex Uncovers Its Critical Function in the Assembly of the Acentrosomal Spindle and Phragmoplast Microtubule Arrays

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim γ-tubulin 3, plays a critical role in γ-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of γ-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.

Two Arabidopsis Phragmoplast-Associated Kinesins Play a Critical Role in Cytokinesis during Male Gametogenesis

In plant cells, cytokinesis is brought about by the phragmoplast. The phragmoplast has a dynamic microtubule array of two mirrored sets of microtubules, which are aligned perpendicularly to the division plane with their plus ends located at the division site. It is not well understood how the phragmoplast microtubule array is organized. In Arabidopsis thaliana, two homologous microtubule motor kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, localize exclusively at the juxtaposing plus ends of the antiparallel microtubules in the middle region of the phragmoplast. When either kinesin was knocked out by T-DNA insertions, mutant plants did not show a noticeable defect. However, in the absence of both kinesins, postmeiotic development of the male gametophyte was severely inhibited. In dividing microspores of the double mutant, microtubules often became disorganized following chromatid segregation and failed to form an antiparallel microtubule array between reforming nuclei. Consequently, the first postmeiotic cytokinesis was abolished without the formation of a cell plate, which led to failures in the birth of the generative cell and, subsequently, the sperm. Thus, our results indicate that Kinesin-12A and Kinesin-12B jointly play a critical role in the organization of phragmoplast microtubules during cytokinesis in the microspore that is essential for cell plate formation. Furthermore, we conclude that Kinesin-12 members serve as dynamic linkers of the plus ends of antiparallel microtubules in the phragmoplast.

Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: A quantitative and spatial analysis

Cytokinesis in plants involves both the formation of a new wall and the partitioning of organelles between the daughter cells. To characterize the cellular changes that accompany the latter process, we have quantitatively analyzed the cell cycle-dependent changes in cell architecture of shoot apical meristem cells of Arabidopsis thaliana. For this analysis, the cells were preserved by high-pressure freezing and freeze-substitution techniques, and their Golgi stacks, multivesicular bodies, vacuoles and clathrin-coated vesicles (CCVs) characterized by means of serial thin section reconstructions, stereology and electron tomography techniques. Interphase cells possess approximately 35 Golgi stacks, and this number doubles during G2 immediately prior to mitosis. At the onset of cytokinesis, the stacks concentrate around the periphery of the growing cell plate, but do not orient towards the cell plate. Interphase cells contain approximately 18 multivesicular bodies, most of which are located close to a Golgi stack. During late cytokinesis, the appearance of a second group of cell plate-associated multivesicular bodies coincides with the onset of CCV formation at the cell plate. During this period a 4x increase in CCVs is paralleled by a doubling in number and a 4x increase in multivesicular bodies volume. The vacuole system also undergoes major changes in organization, size, and volume, with the most notable change seen during early telophase cytokinesis. In particular, the vacuoles form sausage-like tubular compartments with a 50% reduced surface area and an 80% reduced volume compared to prometaphase cells. We postulate that this transient reduction in vacuole volume during early telophase provides a means for increasing the volume of the cytosol to accommodate the forming phragmoplast microtubule array and associated cell plate-forming structures.

A novel plant kinesin-related protein specifically associates with the phragmoplast organelles.

In higher plants, the formation of the cell plate during cytokinesis requires coordinated microtubule (MT) reorganization and vesicle transport in the phragmoplast. MT-based kinesin motors are important players in both processes. To understand the mechanisms underlying plant cytokinesis, we have identified AtPAKRP2 (for Arabidopsis thaliana phragmoplast-associated kinesin-related protein 2). AtPAKRP2 is an ungrouped N-terminal motor kinesin. It first appeared in a punctate pattern among interzonal MTs during late anaphase. When the phragmoplast MT array appeared in a mirror pair, AtPAKRP2 became more concentrated near the division site, and additional signal could be detected elsewhere in the phragmoplast. In contrast, the previously identified AtPAKRP1 protein is associated specifically with bundles of MTs in the phragmoplast at or near their plus ends. Localization of the tobacco homolog(s) of AtPAKRP2 was altered by treatment of brefeldin A in BY-2 cells. We discuss the possibility that AtPAKRP1 plays a role in establishing and/or maintaining the phragmoplast MT array, and AtPAKRP2 may contribute to the transport of Golgi-derived vesicles in the phragmoplast.

CDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana.

Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated an Arabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSS (Arabidopsis thaliana katanin-like protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.

Kinesin-Related Proteins in Plant Cytokinesis

Cytokinesis in higher plant cells is mediated by the phragmoplast. The framework of the phragmoplast consists of two anti-parallel sets of microtubules with their plus ends facing each other at or near the division site. During cytokinesis, the phragmoplast microtubule array undergoes dynamic reorganization from a solid cylinder to a hollow array. Concomitant with the microtubule reorganization, Golgi-originated vesicles are rapidly transported along microtubules toward their plus ends to give rise to the centrifugally growing cell plate. Kinesin-related motor proteins play crucial roles in microtubule reorganization and vesicle transport. To date, a number of plant proteins in the kinesin superfamily have been localized to the phragmoplast, and possibly exert roles in cytokinesis. Plus end-directed motors in the BIMC subfamily play a role in sliding anti-parallel microtubules apart to establish the phragmoplast microtubule array, while microtubule minus end-directed Ncd/Kar3-like motors in the C-terminal motor subfamily likely act antagonistically to balance the force generated by the BIMC-like kinesins. The novel C-terminal motor kinesin KCBP probably regulates microtubule organization and cross-linkage of the microtubule minus ends in a Ca ++ /calmodulin-dependent manner. By acting at or near the plus ends of the microtubules, the Arabidopsis phragmoplast-associated kinesin-related protein AtPAKRP1 likely plays a role in establishing and/or maintaining the organization of phragmoplast microtubules. We anticipate more phragmoplast-associated motors to be revealed in the next few years as the completed Arabidopsis genome contains more than 45 genes encoding kinesin-related proteins. Orchestrated forces generated by different motors are required for microtubule-dependent activities to take place in the phragmoplast.

Identification of a phragmoplast-associated kinesin-related protein in higher plants.

The phragmoplast executes cytokinesis in higher plants. The major components of the phragmoplast are microtubules, which are arranged in two mirror-image arrays perpendicular to the division plane [1]. The plus ends of these microtubules are located near the site of the future cell plate. Golgi-derived vesicles are transported along microtubules towards the plus ends to deliver materials bound for the cell plate [2] [3]. During cell division, rapid microtubule reorganization in the phragmoplast requires the orchestrated activities of microtubule motor proteins such as kinesins. We isolated an Arabidopsis cDNA clone of a gene encoding an amino-terminal motor kinesin, AtPAKRP1, and have determined the partial sequence of its rice homolog. Immunofluorescence experiments with two sets of specific antibodies revealed consistent localization of AtPAKRP1 and its homolog in Arabidopsis and rice cells undergoing anaphase, telophase and cytokinesis. AtPAKRP1 started to accumulate along microtubules towards the spindle midzone during late anaphase. Once the phragmoplast microtubule array was established, AtPAKRP1 conspicuously localized to microtubules near the future cell plate. Our results provide evidence that AtPAKRP1 is a hitherto unknown motor that may take part in the establishment and/or maintenance of the phragmoplast microtubule array.

Suppression of endogenous alpha and beta tubulin synthesis in transgenic maize calli overexpressing alpha and beta tubulins.

Maize Black Mexican Sweetcorn cells have been transformed with constructs containing alpha and beta tubulin coding sequences either singly or together. It is shown that recovery of stable maize transformants is dependent on the co-expression of transfected alpha and beta tubulin in the same lines, indicating that plant cells cannot tolerate an imbalance in the ratio of alpha tubulin to beta tubulin within the cytoplasm. The co-expression of transfected alpha and beta tubulin in maize cells results in an increase in the overall tubulin content (approximately threefold). The transfected alpha and beta tubulins are incorporated into cortical, spindle and phragmoplast microtubule arrays indicating that they are functional. Furthermore, the co-expression of the transfected alpha and beta tubulins results in the suppression of endogenous alpha and beta tubulin synthesis. This suppression increases both with the strength of the promoter in the constructs and with the number of copies of the transgenes inserted into the maize genome. The implications for the post-transcriptional and post-translational regulation of tubulin synthesis in plant cells are discussed.

Reorganization of the actin and microtubule cytoskeleton throughout blue-light-induced differentiation of characean protonemata into multicellular thalli

The reorganization of the actin and microtubule (MT) cytoskeleton was immunocytochemically visualized by confocal laser scanning microscopy throughout the photomorphogenetic differentiation of tip-growing characean protonemata into multicellular green thalli. After irradiating dark-grown protonemata with blue or white light, decreasing rates of gravitropic tip-growth were accompanied by a series of events leading to the first cell division: the nucleus migrated towards the tip; MTs and plastids invaded the apical cytoplasm; the polar zonation of cytoplasmic organelles and the prominent actin patch at the cell tip disappeared and the tip-focused actin microfilaments (MFs) were reorganized into a homogeneous network. During prometaphase and metaphase, extranuclear spindle microtubules formed between the two spindle poles. Cytoplasmic MTs associated with the apical spindle pole decreased in number but did not disappear completely during mitosis. The basal cortical MTs represent a discrete MT population that is independent from the basal spindle poles and did not redistribute during mitosis and cytokinesis. Preprophase MT bands were never detected but cytokinesis was characterized by higher-plant-like phragmoplast MT arrays. Cytoplasmic actin MFs persisted as a dense network in the apical cytoplasm throughout the first cell division. They were not found in close contact with spindle MTs, but actin MFs were clearly coaligned along the MTs of the early phragmoplast. The later belt-like phragmoplast was completely depleted of MFs close to the time of cell plate fusion except for a few actin MF bundles that extended to the margin of the growing cell plate. The cell plate itself and young anticlinal cell walls showed strong actin immunofluorescence. After several anticlinal cell divisions, basal cells of the multicellular protonema produced nodal cell complexes by multiple periclinal divisions. The apical-dome cell of the new shoot which originated from a nodal cell becomes the meristem initial that regularly divides to produce a segment cell. The segment cell subsequently divides to produce a single file of alternating internodal cells and multicellular nodes which together form the complexly organized characean thallus. The actin and MT distribution of nodal cells resembles that of higherplant meristem cells, whereas the internodal cells exhibit a highly specialized cortical system of MTs and streaming-generating actin bundles, typical of highly vacuolated plant cells. The transformation from the asymmetric mitotic spindle of the polarized tip-growing protonema cell to the symmetric, higher-plant-like spindle of nodal thallus cells recapitulates the evolutionary steps from the more primitive organisms to higher plants.

The herbicide sindone B disrupts spindle microtubule organizing centers

Sindone B is classified as a “mitotic disrupter herbicide”, but little information is published on the physiological effects of this structurally unique compound. Onion root tips treated with various concentrations of sindone B were examined by light microscopy following Feulgen staining, immunofluorescence of tubulin, and electron microscopy. Numerous multipolar mitotic figures are noted in Feulgen-stained specimens. Spindle microtubule arrays are focused toward many poles instead of the bipolar arrangement of the spindle in control roots. Cortical and preprophase microtubule arrays appear unaffected. Phragmoplast arrays are also multiple and are curved and branched around the multiple nuclei that result from the multipolar spindle arrays. The cell plates established by these abnormal phragmoplast microtubule arrays are branched and reticulate. These observations indicate that sindone B interacts with the spindle microtubule organizing center so that multipolar divisions result. Phragmoplast disruption may be a secondary consequence of the multipolar division. These symptoms are also characteristic of the structurally unrelated carbamate herbicides.

A novel pattern of herbicide cross-resistance in a trifluralin-resistant biotype of green foxtail [ Setaria viridis (L.) beauv.

A trifluralin-resistant (R) biotype of Setaria viridis is found in areas of Manitoba, Canada where trifluralin is utilized as the principal or sole herbicide. In this study, we examine the crossresistance pattern of this biotype to other mitotic disrupter herbicides utilizing growth measurements and electron microscopy to monitor the resistance. Compared to a trifluralin-susceptible (S) biotype, the R biotype is cross-resistant to all dinitroaniline herbicides tested, with I 50 R S ratios [the concentration of herbicide required to inhibit root growth of the R biotype by 50% divided by the concentration which induces the same effect for the S biotype] ranging from 1.6 to 14.8. This R biotype is also cross-resistant at about the same level to amiprophosmethyl and dithiopyr, but exhibits no resistance to pronamide, sindone B, barban, or the microtubule stabilizer taxol. The highest level of resistance is to the structurally unrelated herbicides DCPA (I 50 R S > 50 ) and terbutol (I 50 R S = 13.4 ). This is the first reported incidence of resistance to these two herbicides. Ultrastructural observations show herbicide-induced abnormalities are either reduced or absent in the R biotype. The S biotype is actually less susceptible to chlorpropham and propham ( R S I 50 = 0.7) than the R biotype. The high level of resistance to the phragmoplast-disrupting microtubule herbicide DCPA, as well as terbutol, and a lower level of resistance to a number of other tubulin-interacting microtubule disrupters, indicate that the R biotype may contain an alteration in a cytoskeletal protein that stabilizes phragmoplast microtubule arrays.

Mitotic Disrupter Herbicides

Approximately one-quarter of all herbicides that have been marketed affect mitosis as a primary mechanism of action. All of these herbicides appear to interact directly or indirectly with the microtubule. Dinitroaniline and phosphoric amide herbicides inhibit microtubule polymerization from free tubulin subunits. Because of the loss of spindle and kinetochore microtubules, chromosomes cannot move to the poles during mitosis, resulting in cells exhibiting an arrested prometaphase configuration. Nuclear membranes re-form around the chromosomal masses to form lobed nuclei. Cortical microtubules, which influence cell shape, are also absent, and, as a result, the cell expands isodiametrically. In root tips and other structures that are normally elongated, these herbicides induce a characteristic club-shaped swelling. Pronamide and MON 7200 induce similar effects, except that tufts of microtubules remain at the kinetochore region of the chromosomes. The carbamate herbicides barban, propham, and chlorpropham alter the organization of the spindle microtubules so that multiple spindles are formed. Chromosomes move to many poles and multiple nuclei result. Abnormal branched cell walls partly separate the nuclei. Terbutol induces “star anaphase” chromosome configurations in which the chromosomes are drawn into an area at the poles in a star-like aggregation. DCPA's most dramatic effect is on phragmoplast microtubule arrays. Multiple, branched, and curved phragmoplasts are found after herbicide treatment. These disrupters should prove to be useful tools in investigations of the proteins and structures required for a successful cell division.

Immunofluorescence and electron microscopic investigations of DCPA-treated oat roots

Immunofluorescence and electron microscopy were used to investigate the effects of DCPA (dimethyl tetrachloroterephthalate) on oat roots. Phragmoplast microtubule arrays were the most consistently affected. Multiple phragmoplasts, undulated phragmoplasts, branched phragmoplasts, and phragmoplasts arising at places other than that predicted by the preprophase band were all observed. Cell wall formations resulting from these abnormal phragmoplast configurations are also abnormal and consist of reticulate cell plates, walls formed at abnormal angles, and wall material in net-like arrays containing pockets of cytoplasm. Cortical and preprophase band microtubule arrays were unaffected, and were similar in both treated and control cells. Minispindle and multipolar spindles were observed in some meristematic cells, although many spindle formations were normal or nearly so. Some multiple and highly lobed nuclei were also found as a consequence of these abnormal divisions. Cell plates were abnormal even when there were no nuclear abnormalities that would cause physical disruption of the division plane and even though preprophase bands (which are predictive of the subsequent plane of division) were normal. These data indicate that DCPA has a direct effect on phragmoplast microtubule organizing centers.

Immunofluorescence and electron microscopic investigations of the effects of dithiopyr on onion root tips

Light, immunofluorescence, and electron microscopy are used to investigate the effects of dithiopyr [ S,S-dimethyl 2-(difluoromethyl)-4-(2-methylpropyl)-6-(trifluoromethyl)-3,5-pyridine-dicarbothioate] on onion root meristems. At high herbicide concentrations (10 −4, 10 −5 M) all of the dividing cells are in an arrested prometaphase configuration although, at lower concentrations (10 −6, 10 −7 M), multipolar mitosis, along with some apparently normal mitotic figures, also are noted. Immunofluorescence microscopy reveals that nearly all of the microtubules are absent after treatment with high concentrations of herbicide, although small tufts of microtubules remain at the kinetochore. These kinetochore tufts are confirmed to be microtubules (and not just aggregates of tubulin protein) by electron microscopic analysis. Phragmoplast arrays are absent at these higher concentrations and no cell plates are formed. Nuclei that reform after the arrested prometaphase or multipolar division are irregularly formed, either highly lobed or fragmented into multiple nuclei. Branched and undulating phragmoplast microtubule arrays oriented in abnormal directions are observed frequently after 10 −6 M treatment. The walls that result from these abnormal phragmoplasts are also unusual. Branched walls, multiple walls/cell, walls with electron opaque depositions, and incomplete reticulate walls are also noted. Because dithiopyr does not bind tubulin, we propose that this herbicide destabilizes microtubules as they are formed so that only short microtubules result. These shortened, destabilized microtubules are ineffective in chromosome movement during mitosis or other cellular functions involving microtubules. Thus, the net effects of dithiopyr are similar to those compounds that cause complete microtubule loss, such as trifluralin or amiprophosmethyl, even though the mechanism of disruption is different.