RESUMO
The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.
Assuntos
Parede Celular , Citocinese , Citoesqueleto , Células Vegetais , Fenômenos Fisiológicos Vegetais , Transdução de Sinais , Biologia CelularRESUMO
Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric (14)N/(15)N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.
Assuntos
Arabidopsis/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteômica , Transdução de Sinais , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Proteínas dos Microtúbulos/genética , Microtúbulos/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , Xilema/citologia , Xilema/genética , Xilema/crescimento & desenvolvimento , Xilema/fisiologiaRESUMO
The preprophase band of microtubules performs the crucial function of marking the plane of cell division. Although the preprophase band depolymerises at the onset of mitosis, the division plane is 'memorized' by a cortical division zone to which the phragmoplast is attracted during cytokinesis. Proteins have been discovered that are part of the molecular memory but little is known about how they contribute to phragmoplast guidance. Previously, we found that the microtubule-associated protein AIR9 is found in the cortical division zone at preprophase and returns during cell plate insertion but is absent from the cortex during the intervening mitosis. To identify new components of the preprophase memory, we searched for proteins that interact with AIR9. We detected the kinesin-like calmodulin-binding protein, KCBP, which can be visualized at the predicted cortical site throughout division. A truncation study of KCBP indicates that its MyTH4-FERM domain is required for linking the motor domain to the cortex. These results suggest a mechanism by which minus-end-directed KCBP helps guide the centrifugally expanding phragmoplast to the cortical division site.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Cinesinas/metabolismo , Microtúbulos/metabolismoRESUMO
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.
RESUMO
Light and dark have antagonistic effects on shoot elongation, but little is known about how these effects are translated into changes of shape. Here we provide genetic evidence that the light/gibberellin-signaling pathway affects the properties of microtubules required to reorient growth. To follow microtubule dynamics for hours without triggering photomorphogenic inhibition of growth, we used Arabidopsis thaliana light mutants in the gibberellic acid/DELLA pathway. Particle velocimetry was used to map the mass movement of microtubule plus ends, providing new insight into the way that microtubules switch between orthogonal axes upon the onset of growth. Longitudinal microtubules are known to signal growth cessation, but we observed that cells also self-organize a strikingly bipolarized longitudinal array before bursts of growth. This gives way to a radial microtubule star that, far from being a random array, seems to be a key transitional step to the transverse array, forecasting the faster elongation that follows. Computational modeling provides mechanistic insight into these transitions. In the faster-growing mutants, the microtubules were found to have faster polymerization rates and to undergo faster reorientations. This suggests a mechanism in which the light-signaling pathway modifies the dynamics of microtubules and their ability to switch between orthogonal axes.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Hipocótilo/metabolismo , Hipocótilo/efeitos da radiação , Luz , Microtúbulos/metabolismo , Proteínas de Arabidopsis/metabolismoRESUMO
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.
Assuntos
Arabidopsis/citologia , Hipocótilo/crescimento & desenvolvimento , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Epiderme Vegetal/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Fatores de Transcrição de Zíper de Leucina Básica/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/fisiologia , Proteínas de Fluorescência Verde/química , Hipocótilo/efeitos dos fármacos , Hipocótilo/ultraestrutura , Microtúbulos/efeitos dos fármacos , Nitrilas/farmacologia , Paclitaxel/farmacologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/ultraestrutura , Rotação , Especificidade da Espécie , Tiazolidinas/farmacologia , Fatores de Tempo , Tubulina (Proteína)/químicaRESUMO
Microtubules are classically described as being transverse, which is perpendicular to the direction of cell elongation. However, fixation studies have indicated that microtubules can be variably aligned across the epidermis of elongating shoots. In addition, microtubules are reported to have different orientations on inner and outer epidermal surfaces, undermining the idea of hoop-reinforcement. Here, long-term movies of Arabidopsis seedlings expressing GFP-TUA6 allowed microtubule alignment to be directly correlated with the rate of elongation within individual growing cells. We also investigated whether microtubule alignment at the inner or the outer epidermal wall better reflected the growth rate. Movies confirmed that transverse microtubules form on the inner wall throughout elongation, but orientation of microtubules is variable at the outer wall, where they tend to become transverse only during episodes of accelerated growth. Because this appears to contradict the concept that circumferential arrays of transverse microtubules or microfibrils are essential for cell elongation, we checked the organisation of cellulose synthase tracks using GFP-CESA3 and found a similar mismatch between trajectories on inner and outer epidermal surfaces. We conclude that microtubule alignment on the inner wall appears to be a more stable predictor of growth anisotropy, whereas outer-wall alignment is more sensitive to the elongation rate.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Glucosiltransferases/metabolismo , Hipocótilo/metabolismo , Microtúbulos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Glucosiltransferases/genética , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Luz , Microtúbulos/genética , Epiderme Vegetal/genética , Epiderme Vegetal/crescimento & desenvolvimento , Epiderme Vegetal/metabolismo , Transporte ProteicoRESUMO
Plant shoots have thick, polylamellate outer epidermal walls based on crossed layers of cellulose microfibrils, but the involvement of microtubules in such wall lamellation is unclear. Recently, using a long-term movie system in which Arabidopsis seedlings were grown in a biochamber, the tracks along which cortical microtubules move were shown to undergo slow rotary movements over the outer surface of hypocotyl epidermal cells. Because microtubules are known to guide cellulose synthases over the short term, we hypothesised that this previously unsuspected microtubule rotation could, over the longer term, help explain the cross-ply structure of the outer epidermal wall. Here, we test that hypothesis using Arabidopsis plants expressing the cellulose synthase GFP-CESA3 and show that cellulose synthase trajectories do rotate over several hours. Neither microtubule-stabilising taxol nor microtubule-depolymerising oryzalin affected the linear rate of GFP-CESA3 movement, but both stopped the rotation of cellulose synthase tracks. Transmission electron microscopy revealed that drug-induced suppression of rotation alters the lamellation pattern, resulting in a thick monotonous wall layer. We conclude that microtubule rotation, rather than any hypothetical mechanism for wall self-assembly, has an essential role in developing cross-ply wall texture.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Hipocótilo/enzimologia , Microtúbulos/metabolismo , Arabidopsis/ultraestrutura , Parede Celular/ultraestrutura , Hipocótilo/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a-GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/citologia , Microtúbulos/ultraestrutura , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , Microscopia Confocal , Proteínas Recombinantes de Fusão/genéticaRESUMO
Translation of most mRNAs is performed in a cap-dependent manner, requiring a protein complex, the cap complex, to regulate the accessibility of the message to the 40S ribosome. The cap complex initiates protein translation by binding to the 5' cap of an mRNA and recruiting ribosomes to begin translation. Compared to animals and yeast, there are significant plant-specific differences in the regulation of cap-dependent mRNA translation, but these are poorly understood. Here, we purified proteins that bind to the 5' cap during the Arabidopsis growth cycle. The protein profile of the cap-binding complexes varies during the various stages of the growth cycle in suspension culture cells. Using Western blotting, the cap complexes of quiescent cells were found to be composed of only three major proteins: eIF4isoE, which is primarily a cytoplasmic protein, and eIF4E and CBP80, which accumulate in the nucleus. However, when cells proliferate, at least 10 major proteins bind directly or indirectly to the 5' cap. Proteomic, Western blotting and immunoprecipitation data establish that the spectrum of RNA helicases in the cap complexes also changes during the growth cycle. Cap complexes from proliferating cultures mainly contain eIF4A, which associates with at least four cap complexes, but eIF4A is replaced by additional helicases in quiescent cells. These findings suggest that the dynamic and selective recruitment of various proteins to mRNA 5' cap complexes could play an important role in the regulation of gene expression.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Biossíntese de Proteínas , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , RNA Helicases/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Before mitosis, a band of microtubules accurately forecasts where the next cross-wall will be inserted but then depolymerizes. How is this division plane memorized until cytokinesis? The molecular memory is being uncovered.
Assuntos
Parede Celular/metabolismo , Citocinese/fisiologia , Mitose/fisiologia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microtúbulos/metabolismoRESUMO
In plants, the preprophase band (PPB) of microtubules marks the cortical site where the cross-wall will fuse with the parental wall during cytokinesis . This band disappears before metaphase, and it is not known how the division plane is "memorized". One idea is that the PPB leaves behind molecules involved in the maturation of the cell plate . Here, we report on the proteomic isolation of a novel 187 kDa microtubule-associated protein, AIR9, conserved in land plants and trypanosomatid parasites. AIR9 decorates cortical microtubules and the PPB but is downregulated during mitosis. AIR9 reappears at the former PPB site precisely when the cortex is contacted by the outwardly growing cytokinetic apparatus. AIR9 then moves inward on the new cross-wall and thus forms a torus. Truncation studies show that formation of the torus requires a repeated domain separate from AIR9's microtubule binding site. Cell plates induced to insert outside the predicted division site do not elicit an AIR9 torus, suggesting that AIR9 recognizes a component of the former PPB. Such misplaced walls remain immature, based on their prolonged staining for the cell-plate polymer callose. We propose that AIR9 may be part of the mechanism ensuring the maturation of those cell plates successfully contacting the "programmed" cortical division site.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Citocinese/fisiologia , Proteínas dos Microtúbulos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Biologia Computacional , Regulação para Baixo , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteômica , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Fenótipo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Primers do DNA , DNA Complementar/genética , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação/genética , Técnicas de Amplificação de Ácido Nucleico , Alinhamento de Sequência , Análise de Sequência de DNA , Transformação GenéticaRESUMO
MAP65 comprises a multigene family specific to plants. To see which isoform is utilised for the unique mechanism of cell expansion, uncomplicated by division structures, carrot cells were deprived of auxin whereupon they stopped dividing and elongated instead. During elongation, a MAP65 protein triplet reduced to a single band. Mass spectrometric analysis demonstrated that this corresponded to a single carrot cDNA; it also corresponded to the major protein previously shown to form filamentous cross-bridges between microtubules in vitro. This MAP65 isoform is concluded to have a major role in establishing the parallel microtubule arrays characteristic of cells undergoing directional expansion.
Assuntos
Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células Vegetais , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Células Cultivadas , DNA Complementar/isolamento & purificação , Daucus carota/citologia , Daucus carota/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Filogenia , Plantas/genética , Isoformas de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In a proteomics-based screen for proteins interacting with cyclin-dependent protein kinase (CDK), we have identified a novel CDK complex containing the eukaryotic translation initiation factor, eIF4A. Reciprocal immunoprecipitations using antibodies against eIF4A indicate that the interaction is specific. The CDKA-eIF4A complex is abundant in actively proliferating and growing cells but is absent from cells that have ceased dividing. The CDKA-eIF4A complex contains kinase activity that is sensitive to the CDK-specific inhibitor roscovitine. This interaction points to a possible molecular mechanism linking cell proliferation with translational control.
Assuntos
Divisão Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Biossíntese de Proteínas/fisiologia , Arabidopsis/citologia , Arabidopsis/enzimologia , Western Blotting , Quinases Ciclina-Dependentes/química , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4A em Eucariotos/química , Testes de Precipitina/métodos , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Purinas/farmacologia , Roscovitina , Nicotiana/citologia , Nicotiana/enzimologiaRESUMO
How do plant actin filaments attach to membranes? Recent studies reveal the presence of plant-specific connectors.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas dos Microfilamentos/fisiologiaRESUMO
The relationship between microtubules and cell-wall texture has had a fitful history in which progress in one area has not been matched by progress in the other. For example, the idea that wall texture arises entirely from self-assembly, independently of microtubules, originated with electron microscopic analyses of fixed cells that gave no clue to the ability of microtubules to reorganize. Since then, live-cell studies have established the surprising dynamicity of plant microtubules involving collisions, changes in angle, parallelization, and rotation of microtubule tracks. Combined with proof that cellulose synthases do track along shifting microtubules, this offers more realistic models for the dynamic influence of microtubules on wall texture than could have been imagined in the electron microscopic era-the era from which most ideas on wall texture originate. This review revisits the classical literature on wall organization from the vantage point of current knowledge of microtubule dynamics.
Assuntos
Parede Celular/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Plantas/ultraestrutura , Celulose/metabolismo , Celulose/ultraestrutura , Desenvolvimento Vegetal , Plantas/metabolismo , Conformação Proteica , Propriedades de SuperfícieRESUMO
The current model of the plant cytokinetic apparatus, describing it as being composed of treadmilling microtubules, is challenged by a new study showing that these microtubules display dynamic instability.
Assuntos
Citocinese , Nicotiana/citologia , Nicotiana/metabolismoRESUMO
Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that-as for the growth of all plant organs-are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls.
Assuntos
Arabidopsis/citologia , Padronização Corporal , Parede Celular/metabolismo , Xilema/citologia , Xilema/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Padronização Corporal/genética , Parede Celular/genética , Parede Celular/ultraestrutura , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Imageamento Tridimensional , Proteínas Associadas aos Microtúbulos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Xilema/genética , Xilema/ultraestruturaRESUMO
This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays.