Quantitative changes in microtubule distribution correlate with guard cell function in Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.     

The function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.     

Guard cells of Commelina communis L. do not respond metabolically to osmotic stress in isolated epidermis: Implications for stomatal responses to drought and humidity

We investigated the hypothesis that stomatal aperture is regulated by epidermal water status. Detached epidermal peels of Commelina communis L. or leaf disks with epidermis attached were incubated in graded solutions of mannitol (0–1.2 M) containing KCl. In isolated epidermis, guard-cell solute content of open stomata did not decrease in response to desiccation. Guard cells of closed stomata accumulated solutes to the same extent in all levels of mannitol tested. There was no evidence of stress-induced hydroactive closure nor of inhibition of hydroactive opening, even when guard cells of closed stomata were initially plasmolyzed. Hydropassive, osmometer-like, changes in stomatal aperture in the isolated epidermis were induced by addition or removal of mannitol, but these did not involve changes in guard-cell solute content. In leaf disks, stomata exhibited clear hydroactive stomatal responses. Steady-state guard-cell solute content of initially open and initially closed stomata decreased substantially with increasing mannitol. Stomata were completely closed above approx. 0.4 M mannitol, near the turgor-loss point for the bulk leaf tissue. Stomata of Commelina did not exhibit direct hydroactive responses to environmental or epidermal water status. Stomatal responses to water deficit and low humidity may be indirect, mediated by abscisic acid or other signal metabolite(s) from the mesophyll.Abbreviations ABA abscisic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Regulation of stomatal movement by cortical microtubule organization in response to darkness and ABA signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Microtubule dynamics are essential for plant cell development and in producing responses to external stimuli. However, little is known about the regulation of microtubule dynamics or crosstalk between microtubule and stomatal movement. Here we identified microtubule reorganization as a crucial factor determining guard cell responses to dark and abscisic acid (ABA) signaling. As stomata opened, guard cells exhibited radially arranged cortical microtubules, which depolymerized into the cytosol when exposed to darkness and ABA. Suppression of microtubule disassembly by paclitaxel, a microtubule-stabilizing drug, significantly enhanced stomatal aperture under light, and partially blocked ABA- or darkness-induced stomatal closure. However, treatment with only the anti-microtubule drug, oryzalin, did not affect stomatal movement with or without external stimuli. Phosphatidic acid (PA) bound to a clade A type 2C protein phosphatase (PP2C), PP2CA, and deletion of PP2CA partially inhibited PA-induced microtubule depolymerization and stomatal closure. Moreover, microtubule reorganization was altered in the ABA-insensitive mutant pldα1, but not in the ABA-hypersensitive mutant pp2ca. We propose that a faithfully balanced reorganization of microtubules fulfills fundamental functions to enable the fast change of stomata in plant adaptive responses to developmental and environmental cues.     

The stomata of the cork-oak, Quercus suber. An ultrastructural approach

In the evergreen leaves of Quercus suber, stomata play a major role in adaptation to drought and temperature stress. The leaf is of zygostomic type and has about 430 stomata per square milimeter of abaxial leaf surface. The stomatal complex is of the anomocytic type. The guard cells protrude from the epidermal plane. The guard cell nucleus contains heterochromatin in small granules. The guard cell cytoplasm is characterised by a large number of well developed mitochondria, amyloplasts with stroma and grana, and a well developed cytoskeleton with a cortical array of microtubules oriented pa railed to the slit axis that persist even in mature cells. Guard cell walls are asymmetrically thickened and devoid of plasmodesmata. No area of cell walls was free of cuticle or covered by a thin cuticular layer and apparently no area of limited cuticular development provides evaporation when the stomata are closed.     

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 ULTRASTRUCTURAL CYTOLOGY OF THE DIFFERENTIATING GUARD CELLS OF VIGNA SINENSIS

Structural differentiation of the guard cells of Vigna sinensis results from the integration of the following interrelated processes: a) intense activity of ribosomes, dictyosomes, endoplasmic reticulum (ER) membranes and mitochondria and patterned organization of microtubules; b) unequal thickening and ordered micellation of their walls and opening of the stomatal pore; and c) the divergent differentiation of the plastids. In differentiating guard cells, microtubules appear anticlinally oriented and more or less evenly distributed along the unthickened part of the dorsal wall and in the middle part of the ventral wall where thickening of the future pore occurs. In periclinal walls, microtubules fan away from the margins of the increasing thickening of the ventral wall and, later, from the rims of the stomatal pore towards the dorsal walls, parallel to the depositing radial microfibrils. Microtubules may be the cytoplasmic elements underlying guard-cell morphogenesis. Although cell-plate organization in guard-cell mother cells does not seem to differ from that of other protodermal cells, the middle lamella of the ventral wall becomes electron-translucent. The stomatal pore develops schizogenously from the internal and/or external ends of the ventral wall and proceeds inwards, remaining incomplete in most of the stomata of plants grown for 30 days in darkness and in some malformed ones which were developed after a prolonged action of colchicine. The guard cell, when approaching maturity, loses its organelle complexity and plasmodesmata, but it keeps a significant portion of its cytoplasm and organelles. Perigenous stomata generally exceed the size of mesoperigenous and mesogenous ones, develop large vacuoles and appear able to induce oriented divisions in their vicinity.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.     

Dissecting the Nanoscale Distributions and Functions of Microtubule-End-Binding Proteins EB1 and ch-TOG in Interphase HeLa Cells

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other’s localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.     

Touch, Grasp, Deliver and Control: Functional Cross-Talk Between Microtubules and Cell Adhesions

Cross-talk between microtubule networks and sites of cell–matrix and cell–cell adhesion has profound impact on these structures and is essential for proper cell organization, polarization and motility. Components of adhesion sites can interact directly with microtubules or with proteins that specifically associate with microtubule plus ends and minus ends and in this way capture, stabilize or destabilize microtubules. In their turn, microtubules can serve as routes for delivery of structural and regulatory factors that control adhesion site turnover. In addition, the microtubule lattice or growing microtubule plus ends can serve as diffusional sinks that accumulate and scaffold regulatory molecules, thereby affecting their activity in the vicinity of adhesions. Combination of these mechanisms underlies the functional co-operation between microtubules and adhesion sites and defines their dynamic behavior.     

Organisation of microtubules and actin filaments in the cortex of differentiating Selaginella guard cells

Summary Using fluorescent probes and confocal laser scanning microscopy we have examined the organisation of the microtubule and actin components of the cytoskeleton in kidney-shaped guard cells of six species of Selaginella. The stomata of Selaginella exhibit novel cytoskeletal arrangements, and at different developmental stages, display similarities in microtubule organisation to the two major types of stomata: grass (dumbbell-shaped) and non-grass (kidney-shaped). Initially, cortical microtubules and F-actin radiate from the stomatal pore and extend across the external and internal periclinal cell surfaces of the guard cells. As the stomata differentiate, the cytoskeleton reorients only along the internal periclinal walls. Reorganisation is synchronous in guard cells of the same stoma. Microtubules on the inner periclinal walls of the guard cells now emanate from areas of the ventral wall on either side of the pore and form concentric circles around the pore. The rearrangement of F-actin is similar to that of microtubules although F-actin is less well organised. Radial arrays of both microtubules and F-actin are maintained adjacent to the external surfaces. Subsequently, in two of the six species of Selaginella examined, microtubules on both the internal and external walls become oriented longitudinally and exhibit no association with the ventral wall. In the other four species, microtubules adjacent to the internal walls revert to the initial radial alignment. These findings may have implications in the development and evolution of the stomatal complex.Abbreviations GC guard cell - MT microtubule     

EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells

Highly conserved EB1 family proteins bind to the growing ends of microtubules, recruit multiple cargo proteins, and are critical for making dynamic microtubules in vivo. However, it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. In this paper, we identify a novel EB1 cargo protein, Sentin. Sentin depletion in Drosophila melanogaster S2 cells, similar to EB1 depletion, resulted in an increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. We demonstrate that Sentin's association with EB1 was critical for its plus end localization and function. Furthermore, the EB1 phenotype was rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Knockdown of Sentin attenuated plus end accumulation of Msps (mini spindles), the orthologue of XMAP215 microtubule polymerase. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip.     

Array and distribution of actin filaments in guard cells contribute to the determination of stomatal aperture

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.     

Generation and regulation of microtubule network asymmetry to drive cell polarity

Microtubules control cell architecture by serving as a scaffold for intracellular transport, signaling, and organelle positioning. Microtubules are intrinsically polarized, and their orientation, density, and post-translational modifications both respond and contribute to cell polarity. Animal cells that can rapidly reorient their polarity axis, such as fibroblasts, immune cells, and cancer cells, contain radially organized microtubule arrays anchored at the centrosome and the Golgi apparatus, whereas stably polarized cells often acquire non-centrosomal microtubule networks attached to the cell cortex, nucleus, or other structures. Microtubule density, longevity, and post-translational modifications strongly depend on the dynamics of their plus ends. Factors controlling microtubule plus-end dynamics are often part of cortical assemblies that integrate cytoskeletal organization, cell adhesion, and secretion and are subject to microtubule-dependent feedback regulation. Finally, microtubules can mechanically contribute to cell asymmetry by promoting cell elongation, a property that might be important for cells with dense microtubule arrays growing in soft environments.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.     

Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules

Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.     

外源喜树碱对植物叶片气孔导度的影响

前人研究表明,保卫细胞微管系统在气孔运动中起到重要作用:保卫细胞质膜上内向K⁺通道的正常活性有赖于微管的正常解聚/聚合的动态变化,微管系统可能通过调节保卫细胞K⁺通道而控制气孔运动,即微管解聚导致内向K⁺通道关闭,保卫细胞无法因膨压调节吸收水分而抑制气孔开放。有学者认为,喜树碱或其类似物能够与微管蛋白结合,并降低微管结构稳定性,其机制则可能是抑制细胞高分子量微管结合蛋白对微管组装的辅助作用,但这方面的实验证据相对匮乏。因此,为了进一步研究喜树碱的生态生物化学功能,我们采取叶面喷施喜树碱和PEG模拟干旱诱导气孔关闭的方法,研究了外源喜树碱对喜树幼苗气孔导度的影响,同时以没有内源喜树碱的烟草为实验材料进行了对照研究。研究结果表明:0.0115mmol·L^-1的喜树碱水饱和溶液对喜树和烟草幼苗叶片气孔导度的影响规律一致,均表现出明显的抑制气孔开放的效果,这为喜树碱与微管蛋白结合提供了部分证据。     

Variation in stomatal characteristics over the lower surface of Commelina communis leaves

Abstract. The silicone rubber impression technique was used to measure stomatal apertures in 9 mm² sampling areas covering the entire lower surface of leaves of Commelina communis L. The data were analysed using a computer program which produced 'iso-aperture' contours illustrating local differences in mean stomatal aperture. Little consistency was seen in the iso-aperture patterns among sampling times, although the stomata were always relatively closed at the leaf tip and base. When stomata in the middle of the lamina were open, those near to the leaf margin tended to be relatively closed. In places, gradients of mean stomatal aperture were as high as 1 μm mm⁻¹. Measurements along a transect across the lower epidermis revealed no correlation of stomatal aperture with the presence of major veins in the mesophyll tissue. Variation in guard-cell size and stomatal frequency on the lower leaf surface was also analysed. The guard cells were smallest and the stomata more frequent near to the leaf margins. The significance of the results is discussed in relation to measurements of leaf conductance and models of stomatal function.     

The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.     

Microtubule dynamics are involved in stomatal movement ofVicia faba L.

Summary To obtain a full picture of microtubule (MT) behavior during the opening and closure of guard cells we have microinjected living guard cells ofVicia faba with fluorescent tubulin, examined fine detail by freeze shattering fixed cells, and used drug treatments to confirm aspects of MT dynamics. Cortical MTs in fully opened guard cells are transversely oriented from the ventral wall to the dorsal wall. When the stomatal aperture was decreased by darkness, these MTs became twisted and patched and broken down into diffuse fragments when stomata were closed. When the closed stomata were opened in response to light, the MTs in guard cells changed from the diffused, transitional pattern back to one in which MTs are transversely oriented from stomatal pore to dorsal wall. This observation indicates a linkage between these MT changes and stomatal movement. To confirm this, we used the MT-stabilizing agent taxol and the MT-depolymerizing herbicide oryzalin and observed their effects on the stomatal aperture and MT dynamics. Both drugs suppressed light-induced stomatal opening and dark-induced closure. MTs are known to be necessary for maintaining the static kidney shape of guard cells; the present data now show that the dynamic properties of polymeric tubulin accompany changes in shape with stomatal movement and may be functionally involved in stomatal movement.