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     

Ultrastructure of stomatal development in Arabidopsis (Brassicaceae) leaves

Stomatal development was studied in wild-type Arabidopsis leaves using light and electron microscopy. Development involves three successive types of stomatal precursor cells: meristemoid mother cells, meristemoids, and guard mother cells (GMCs). The first two types divide asymmetrically, whereas GMCs divide symmetrically. Analysis of cell wall patterns indicates that meristemoids can divide asymmetrically a variable number of times. Before meristemoid division, the nucleus and a preprophase band of microtubules become located on one side of the cell, and the vacuole on the other. Meristemoids are often triangular in shape and have evenly thickened walls. GMCs can be detected by their roughly oval shape, increased starch accumulation, and wall thickenings on opposite ends of the cells. Because these features are also found in developing stomata, stomatal differentiation begins in GMCs. The wall thickenings mark the division site in the GMC since they overlie a preprophase band of microtubules and occur where the cell plate fuses with the parent cell wall. Stomatal differentiation in Arabidopsis resembles that of other genera with kidney-shaped guard cells. This identification of stages in stomatal development in wild-type Arabidopsis provides a foundation for the analysis of relevant genes and of mutants defective in stomatal patterning, cell specification, and differentiation.     

STRUCTURE AND DEVELOPMENT OF WALLS IN FUNARIA STOMATA

The development and structure of the guard cell walls of Funaria hygrometrica Hedw. (Musci) were studied with the light and electron microscopes. The stoma consists of only one, binucleate guard cell as the pore wall does not extend to the ends of the cell. The guard cell wall is thinnest in the dorsal wall near the outer wall but during movement is most likely to flex at thin areas of the outer and ventral walls. The mature wall contains a mottled layer sandwiched between two, more fibrillar layers. The internal wall layer has sublayers with fibrils in axial and radial orientations with respect to the pore. During substomatal cavity formation, the middle lamella is stretched into an electron dense network and into strands and sheets. After stomatal pore formation, the subsidiary cell walls close to the guard cell become strikingly thickened. The functional implications of these results are discussed.     

Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell     

Structure,ontogeny and taxonomic significance of trichomes and stomata in some Capparidaceae

The present work embodies epidermal structure, structure and ontogeny of stomata in five genera embracing sixteen species of the Capparidaceae namely Cleome (8 species) Capparis (5 species), Cadaba (1 species), Crataeva (1 species) and Maerua (1 species). The epidermal cells are polygonal, isodiametric or elongated arranged irregularly, with evenly or unevenly thickened, sinuous, straight or arched anticlinal walls. Two main types of trichomes: glandular (four types) and eglandular (five types) are noticed. The stomatal types include cyclocytic, triacytic, staurocytic, tetracytic, anomocytic, anisocytic, paracytic and with a single subsidiary cell. The ontogeny of stomata with a single subsidiary cell is perigenous or mesoperigenous, of paracytic mesoperigenous or mesogenous, of anisocytic is mesoperigenous or mesogenous, while that of the other types is perigenous. Abnormalities observed are: single guard cell; aborted guard cells; complete or incomplete division of guard cells; contiguous stomata; giant stomata and cytoplasmic connections. The present observations do not support the separation of Cleomaceae from the Capparidaceae.     

Cortical actin filament organization in developing and functioning stomatal complexes of Zea mays and Triticum turgidum

Cortical actin filament (AF) organization was studied in detail in developing stomatal complexes of the grasses Zea mays and Triticum turgidum. AF arrays during the whole stomatal complex development are dynamic, partly following the pattern of cortical microtubule (MT) organization. They also exhibit particular patterns of organization, spatially and temporarily restricted. Among AF arrays, the radial ones that underlie young guard cell (GC) periclinal walls, those that line the bulbous GC ends and the AF ring at the junction between subsidiary cells (SCs) and GCs are described here for the first time. Although many similarities in cortical AF organization exist among the stomatal cells of both plants studied, considerable differences have also been observed between them. Our data reveal that the expanding areas of stomatal cell walls are lined by distinct cortical AF aggregations that probably protect the plasmalemma against mechanical stresses. Experimental AF disruption does not seem to affect detectably stomatal cell morphogenesis. Moreover, the structural and experimental data of this study revealed that, in contrast to the elliptical stomata, in the dumbbell-shaped ones the AFs and MTs seem not to be involved in the mechanism of opening and closing of the stomatal pore.     

A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species

Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.     

An analytical model of the hydraulic aspects of stomatal dynamics

An analytical model of the hydraulic aspects of stomatal dynamics is formulated in this paper. The model consists of a coupled system of non-linear, ordinary differential equations, written in terms of water potentials, hydrostatic pressures, osmotic potentials, water vapor resistances and water fluxes. The model is validated by comparisons with the experimental literature. Numerical solutions of the model show qualitative agreement with most known stomatal responses.Stomatal opening in the model is dependent on the interaction of the guard and subsidiary cells in the following manner. Pore opening is initiated by a rise in the guard cell hydrostatic pressure. As the stomate opens, transpiration increases, causing the cell wall water potential to drop. The drop in cell wall water potential then causes the subsidiary cell pressure to drop, opening is accelerated, and the stomate literally “pops” open. Simulated opening proceeds in two distinct phases: a stress phase and a motor phase. During the stress phase, guard cell pressure rises but the pore remains closed. The motor phase commences when the guard cell pressure has risen sufficiently to initiate pore opening, beyond which point opening progresses rapidly.Hydropassive stomatal movements are found to be insufficient to regulate water loss at low leaf water potentials. Stable, hydraulically-based oscillations in stomatal aperture are shown in the model by the existence of a stable limit cycle. The period of these oscillations is strongly influenced by the cell membrane hydraulic conductivity. An increased conductivity results in a shorter period oscillation. Environmental conditions promoting oscillatory behavior are in qualitative agreement with the experimental literature.     

Stomatal development in Arabidopsis and grasses: differences and commonalities

Stomata, found on the epidermis of all terrestrial plants, consist of two specialized cells called guard cells, which surround a tiny pore. Major advances have been made in our understanding of the genetic control of stomatal development in Arabidopsis and grasses. In Arabidopsis, three basic-helix-loop-helix (bHLH) genes control the successive steps that lead to stomatal formation. SPEECHLESS (SPCH) drives the cell division that initiates the stomatal cell lineage, MUTE induces the formation of the immediate stomatal precursor cell, and FAMA causes the stomatal precursor cell to divide into the two guard cells. Recent results demonstrate that these genes share functions with their grass homologs, and that MUTE is expressed later in development than its grass counterparts. Other differences in stomatal development between these two plant groups are exemplified by the PANGLOSS1 (PAN1) gene of maize. PAN1, which encodes a leucine-rich repeat receptor-like kinase with an inactive kinase domain, promotes polarization of the subsidiary mother cell and orients its cell division plane. Because such events do not exist in Arabidopsis, it is likely that the PAN1-like genes of Arabidopsis and PAN1 are paralogs. Together, these results indicate that distinctions in the regulation of gene expression and protein function are both responsible for the divergence of stomatal development between Arabidopsis and grasses.     

Direct Measurements of Turgor Pressure Potentials of Guard Cells: II. THE MECHANICAL ADVANTAGE OF SUBSIDIARY CELLS, THE SPANNUNQSPHASE, AND THE OPTIMUM LEAF WATER DEFICIT

Evidence of the mechanical advantage of subsidiary cells wasobtained by simultaneous measurements of turgor pressure potentialsin adjacent subsidiary and guard cells using injection circuitswith two separate needles. In Tradescantia virginiana the mechanicaladvantage approaches two. Using the same technique evidencewas obtained that the Spannungsphase is, in the first place,a turgor relations phenomenon due to the mechanical advantageof epidermal or subsidiary cells. In addition, the evidenceindicated that the elastic properties of guard cell walls mayundergo changes during the Spannungsphase when potassium iontransport commences. During these measurements it was confirmedthat the optimum leaf water deficit for maximum stomatal openingoccurs when the epidermal turgor is near zero. Under these conditionsthe width of the stomatal pore is a function of the turgor pressureof the guard cells, since at zero turgor of the subsidiary cellstheir mechanical advantage has disappeared.     

The mechanical diversity of stomata and its significance in gas-exchange control

Given that stomatal movement is ultimately a mechanical process and that stomata are morphologically and mechanically diverse, we explored the influence of stomatal mechanical diversity on leaf gas exchange and considered some of the constraints. Mechanical measurements were conducted on the guard cells of four different species exhibiting different stomatal morphologies, including three variants on the classical "kidney" form and one "dumb-bell" type; this information, together with gas-exchange measurements, was used to model and compare their respective operational characteristics. Based on evidence from scanning electron microscope images of cryo-sectioned leaves that were sampled under full sun and high humidity and from pressure probe measurements of the stomatal aperture versus guard cell turgor relationship at maximum and zero epidermal turgor, it was concluded that maximum stomatal apertures (and maximum leaf diffusive conductance) could not be obtained in at least one of the species (the grass Triticum aestivum) without a substantial reduction in subsidiary cell osmotic (and hence turgor) pressure during stomatal opening to overcome the large mechanical advantage of subsidiary cells. A mechanism for this is proposed, with a corollary being greatly accelerated stomatal opening and closure. Gas-exchange measurements on T. aestivum revealed the capability of very rapid stomatal movements, which may be explained by the unique morphology and mechanics of its dumb-bell-shaped stomata coupled with "see-sawing" of osmotic and turgor pressure between guard and subsidiary cells during stomatal opening or closure. Such properties might underlie the success of grasses.     

STRUCTURE AND ONTOGENY OF THE STOMATAL COMPLEX IN PINUS STROBUS L. AND PINUS BANKSIANA LAMB.

The morphology of the stomatal complex in Pinus strobus L. and P. banksiana Lamb, is described and it is proposed that the stomatal complex should be considered an eight-celled complex consisting of two guard cells, and two polar, two lateral, and two hypodermal subsidiary cells. An ontogenetic study found these cells closely related developmentally. It was also found that the stomatal complex in these two pines could not readily be classified as haplocheilic because a polar subsidiary cell arises from the same protodermal cell as does the guard cell mother cell. A modification of the classical concept of stomatal development was necessary to describe the stomata as eumesoperigenous.     

Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma)

Summary The dynamics of actin distribution during stomatal complex formation in leaves of winter rye was examined by means of immunofluorescence microscopy of epidermal sheets. This method results in actin localization patterns that are the same as those seen with rhodamine-phalloidin staining, but are more stable. During stomatal development MFs are extensively rearranged, and most of the time the orientation or placement of MFs is distinctly different from that of MTs, the exception being co-localization of MTs and MFs in phragmoplasts. Although MFs show an orientation similar to that of MTs in interphase guard mother cells, no banding of MFs into anything resembling the interphase MT band is observed. From prophase to telophase, a distinct, dense concentration of MFs is found in subsidiary cell mother cells (SMCs) between the nucleus and the region of the cell cortex facing the guard mother cell. Cytochalasin B treatment causes incorrect positioning of the SMC nucleus/daughter nuclei and abarrent placement and orientation of the new cell wall that forms the boundary of the subsidiary cell at cytokinesis. These results suggest that MFs are involved in maintaining the SMC nucleus in its correct position and the SMC spindle in the correct orientation relative to the division site previously delineated by the preprophase band. Because these MFs thus appear to assure that the SMC phragmoplast begins to form in the correct orientation near the division site to which it needs to grow, we suggest that MFs are involved in control of correct placement and orientation of the new cell wall of the subsidiary cell.Abbreviations CB cytochalasin B - DIC differential interference contrast - DMSO dimethylsulfoxide - MBS m-maleimidobenzoyl-N-hydroxylsuccinimide ester - MF microfilament - MT microtubule - PBS phosphate buffered saline - SMC subsidiary cell mother cell Dedicated to the memory of Professor Oswald Kiermayer     

Morphological evolution in the graminid clade: comparative floral anatomy of the grass relatives Flagellariaceae and Joinvilleaceae

New comparative data are presented on the reproductive morphology and anatomy of two genera closely related to grasses, Flagellaria and Joinvillea, in which the flowers are superficially similar, especially in stamen morphology. This investigation demonstrates some anatomical differences between the two genera. For example, both genera depart from the ‘typical’ condition of tepal vasculature (three‐traced outer tepals and one‐traced inner tepals): in Flagellaria, each tepal receives a single vascular bundle and, in Joinvillea, each tepal is supplied by three vascular bundles. Joinvillea possesses supernumerary carpel bundles, as also found in the related family Ecdeiocoleaceae, but not in Flagellaria or grasses. In the anther, the tapetum degenerates early in Flagellaria, and is relatively persistent in Joinvillea, in which the pollen grains remain closely associated with the tapetum inside the anther locule, indicating a correlation between peripheral pollen (a feature that is common in grasses) and a persistent tapetum. This study highlights the presence of a pollen‐tube transmitting tissue (PTTT) or solid style in the gynoecium of Flagellaria, as also in many Poaceae, but not in Joinvillea or Ecdeiocoleaceae. We speculate that the presence of a PTTT could represent one of the factors that facilitated the subsequent evolution of the intimately connected gynoecia that characterize grasses. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 393–404.     

Form,development and function of grass stomata

Stomata are cellular breathing pores on leaves that open and close to absorb photosynthetic carbon dioxide and to restrict water loss through transpiration, respectively. Grasses (Poaceae) form morphologically innovative stomata, which consist of two dumbbell‐shaped guard cells flanked by two lateral subsidiary cells (SCs). This ‘graminoid’ morphology is associated with faster stomatal movements leading to more water‐efficient gas exchange in changing environments. Here, we offer a genetic and mechanistic perspective on the unique graminoid form of grass stomata and the developmental innovations during stomatal cell lineage initiation, recruitment of SCs and stomatal morphogenesis. Furthermore, the functional consequences of the four‐celled, graminoid stomatal morphology are summarized. We compile the identified players relevant for stomatal opening and closing in grasses, and discuss possible mechanisms leading to cell‐type‐specific regulation of osmotic potential and turgor. In conclusion, we propose that the investigation of functionally superior grass stomata might reveal routes to improve water‐stress resilience of agriculturally relevant plants in a changing climate.     

Drought-induced H2O2 accumulation in subsidiary cells is involved in regulatory signaling of stomatal closure in maize leaves

Increasing H₂O₂ levels in guard cells in response to environmental stimuli are recently considered a general messenger involved in the signaling cascade for the induction of stomatal closure. But little is known as to whether subsidiary cells participate in the H₂O₂-mediated stomatal closure of grass plants. In the present study, 2-week-old seedlings of maize (Zea mays) were exposed to different degrees of soil water deficit for 3 weeks. The effects of soil water contents on leaf ABA and H₂O₂ levels and stomatal aperture were investigated using physiological, biochemical, and histochemical approaches. The results showed that even under well-watered conditions, significant amounts of H₂O₂ were observed in guard cells, whereas H₂O₂ concentrations in the subsidiary cells were negligible. Decreasing soil water contents led to a significant increase in leaf ABA levels associated with significantly enhanced O₂ ^? and H₂O₂ contents, consistent with reduced degrees of stomatal conductance and aperture. The significant increase in H₂O₂ appeared in both guard cells and subsidiary cells of the stomatal complex, and H₂O₂ levels increased with decreasing soil water contents. Drought-induced increase in the activity of antioxidative enzymes could not counteract the significant increase in H₂O₂ levels in guard cells and subsidiary cells. These results indicate that subsidiary cells participate in H₂O₂-mediated stomatal closure, and drought-induced H₂O₂ accumulation in subsidiary cells is involved in the signaling cascade regulating stomatal aperture of grass plants such as maize.     

Differentiation of stomatal meristemoids and guard cell mother cells into guard-like cells in Vigna sinensis leaves after colchicine treatment

The temporary development of Vigna sinensis seedlings in the presence of colchicine results in the inhibition of stomata generation and the formation of numerous persistent stomatal meristemoids (P-SM) and guard cell mother cells (P-GMC). Before dividing differentially or becoming GMC, the untreated meristemoiidsundergo a preparatory differentiation, during which a synthesis of new densely ribosomal cytoplasm, an increase of nuclear size, and a detectable proliferation of all the organelles are observed. The same process appears depressed and delayed in treated meristemoids; the cells have usually undergone only part of it when they reach the C mitosis. After the inhibition of their division, the bulged meristemoids II and GMC increase further in size, synthesize new nonribosomal cytoplasm, and start vacuolating slowly. The plastids also increase in size, change in shape, and become able to synthesize large quantities of starch. The cells retain a ribosomal cytoplasm, rough ER membranes, and active dictyosomes for a long time. At the advanced stages of differentiation, the microtubules reappear in the cells even when the plant remains under colchicine treatment. When mature, the P-GMC and P-SM are quite similar to the guard cells and possess considerably thickened periclinal walls, numerous mitochondria, and small vacuoles, while the nucleus, the plastids, and the cytoplasm occupy significant parts of the cell volume. In the epidermis displaying open stomata in light, significant K⁺ quantities are detectable in guard cells and P-GMC or P-SM, while they are almost absent from their surrounding cells. When the stomata close in darkness, K⁺ is accumulated primarily in the subsidiary or typical epidermal cells surrounding these idioblasts and only minimally inside them. Besides, the P-GMC and P-SM, like the guard cells, retain the starch for a long time and build up considerable starch quantities from exogenously supplied sugars.Abbreviations P-GMC persistent guard cell mother cell - PSM persistent stomatal meristemoid - ER endoplasmic reticulum     

Observations on the structure of epidermal cells, particularly the cork and silica cells, from the flowering stem internode of Lolium temulentum L. (Gramineae)

Features of the epidermis such as stomata, hairs, cork and silica cells are described from both light and electron microscope studies. The stomatal complex consists of two guard cells and two subsidiary cells. After division of the guard mother cell a pore is left at each end of the dividing wall. The cork and silica cells arise from a single another cell and develop differentially. The silica cell enlarges more than the cork cell and finally becomes filled with solidified silica. The outer tangential and radial walls of the cork cells become very thick-walled, whereas the inner tangential and radial walls of the silica cells become thickened. The outer tangential wall of the silica cell remains thin and is covered with a thin layer- of cuticle. This wall frequently collapses in old cells leaving a depression in the surface of the stem. The change in the ultrastructure of the cork and silica cells are described and the possible functions of these cells discussed.     

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.