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     

Probable involvement of cytoskeleton in stomatal-pore formation inAsplenium nidus L.

Summary Stomatal-pore formation in the fernAsplenium nidus L. commences in postcytokinetic guard cells at the mid-region of the ventral wall, before the deposition of any cellulosic wall material on it, by the local movement of the adjacent plasmalemmata apart from each other. In this way a rudimentary internal stomatal pore is formed. At this stage the ventral wall exhibits an undulated appearance and gives a positive reaction to aniline blue. Detailed study of postcytokinetic guard cells by electron microscopy, as well as after tubulin immunolabeling and actin staining, shows that stomatal pore initiation coincides with the initiation of the organization of the anticlinal microtubule bundles along the middle of the ventral wall and the colocalization of actin filaments at the same sites. Afterwards, the stomatal pore broadens towards the periclinal walls, a phenomenon keeping pace with the further bundling of the cytoskeletal elements beneath the plasmalemmata lining the middle of the ventral wall. At this stage the anticlinal microtubule bundles lining the stomatal pore are very prominent. The above findings, as well as the fact that treatments with antimicrotubule drugs inhibit the internal stomatal-pore formation, denote that the cortical cytoskeleton lining the ventral wall and particularly the microtubules are involved in this process. Afterwards, distinct local wall thickenings are deposited at the sites of junction of the mid-region of the ventral wall with the periclinal walls as well as at the junctions of the polar ventral-wall ends with the external periclinal wall. Along the middle-lamella region of the former wall thickenings the fore- and rear-chambers of the stomatal pore are formed. The final stomatal-pore opening is achieved by disruption of the expanded thin median periclinal wall region inherited from the guard cell mother cell and of the overlying cuticle, which covers the stomatal pore externally and internally. At the same time the fore-chamber of the stomatal pore broadens by a schizogenous opening towards the polar ventral-wall ends. The observations show that the stomatal-pore formation inA. nidus is a unique process, which is probably restricted to ferns.Abbreviations Af actin filament - GC guard cell - Mt microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline - VW ventral wall     

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     

Origin and development of stomatal microanatomy in two species ofEucalyptus

Summary Development of the stomata ofEucalyptus orbifolia (in which they are relatively superficial) andE. incrassata (in which they are deeply sunken) is described from light microscopy of thin sections of resin-embedded material. The envelope of the guard mother cell is retained intact while in the daughter cells (guard cells) the inner and outer thickenings are formed. The mother cell envelope may even remain discrete and intact during early stages of formation of the separation spaces, precursors of the future stomatal pore, between the thickenings. Remnants of the guard mother cell wall may be retained as parts of at least the inner stomatal ledges. Likewise, remnants of the wall which divides the mother cell persist on the maturing guard cells.Sudan III-positive materials, probably cutin, are removed from the cuticle over the mother cell soon after it is formed. The cuticle above the guard cell is finally perforated by enzymic attack forming, inE. incrassata, a large cavity outside the developing stoma into which the outer stomatal ledges grow as extensions of the upper guard cell walls.The termostiole is suggested for the aperture in the cuticle. The flanges of cuticle seen in section to bound it are termedostiolar ledges. The ostiolar ledges are to be distinguished from the outer stomatal ledges, which develop from the upper thickenings of the guard cell initials. The distinction is clear inE. incrassata (and other species with deeply sunken stomata) but not in mesophytic plants or species with superficial stomata such asE. orbifolia in which the outer stomatal ledges are fused with the cuticle.Growth of the outer stomatal ledges inE. incrassata involves transport of wall materials through an annular space, the equivalent of an ectocythode.The relevance of the observations to stomatal development in other genera is discussed.     

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.     

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.     

Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow

Lucifer yellow has been microinjected into stomatal cells of Allium cepa L. epidermal slices and Commelina communis L. epidermal peels and the symplastic spread of dye to neighboring cells monitored by fluorescence microscopy. Dye does not move out of injected mature guard cells, nor does it spread into the guard cells when adjacent epidermal or subsidiary cells are injected. Dye does spread from injected subsidiary cells to other subsidiary cells. These results are consistent with the reported absence of plasmodesmata in the walls of mature guard cells. Microinjection was also used to ascertain when dye coupling ceases during stomatal differentiation in Allium. Dye rapidly moves into and out of guard mother cells and young guard cells. Hovewer, dye movement ceases midway through development as the guard cells begin to swell but well before a pore first opens. Since plasmodesmata are still present at this stage, the loss of symplastic transport may result from changes in these structures well in advance of their actual disappearance from the guard cell wall.Abbreviations DIC differential interference contrast - GMC guard mother cell - LY Lucifer yellow - Pd plasmodesmata You can observe a lot by watching Lawrence Berra, as quoted in Sports Illustrated, vol. 60 (No. 14), p. 94, 2 April 1984     

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.     

Breaking the silence: three bHLH proteins direct cell-fate decisions during stomatal development

Stomata are microscopic pores on the surface of land plants used for gas and water vapor exchange. A pair of highly specialized guard cells surround the pore and adjust pore size. Studies in Arabidopsis have revealed that cell-cell communication is essential to coordinate the asymmetric cell divisions required for proper stomatal patterning. Initial research in this area identified signaling molecules that negatively regulate stomatal differentiation. However, genes promoting cell-fate transition leading to mature guard cells remained elusive. Now, three closely related basic helix-loop-helix (bHLH) proteins, SPEECHLESS, MUTE and FAMA have been identified as positive regulators that direct three consecutive cell-fate decisions during stomatal development. The identification of these genes opens a new direction to investigate the evolution of stomatal development and the conserved functions of bHLH proteins in cell type differentiation adopted by plants and animals.     

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.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

Structure of the stomatal complex of the monocot Flagellaria indica

The structure of the mature stomatal complex of Flagellaria indica L. was studied since the Flagellariaceae is reported to be one of a handful of nongrass families with a grass-type stoma, and since relatively little is known about stomatal ultrastructure in monocots other than grasses. Both the grass guard cell and its nucleus are dumbbell-shaped, and the walls that separate adjacent grass guard cells are perforated. Electron and fluorescence microscopy reveal that the Flagellaria guard cell lacks these features. Instead, the Flagellaria guard cell is neither dumbbellnor kidney-shaped, its nucleus is roughly kidney-shaped, and the end walls are thickened and imperforate. Additional structural features of the stomatal apparatus of Flagellaria include: 1) the subsidiary cells have a protuberance that underlies the middle of the guard cell and that forms an additional and innermost aperture of the pore; 2) guard and subsidiary cell walls are thickened differentially and are layered; and 3) organelles in both cell types appear to be confined to specific domains. Although Flagellaria is closely related to grasses, it does not have a grass or dumbbell-shaped type of stomate. This suggests that the grass type of stomate may be less widespread than reported.     

TOO MANY MOUTHS promotes cell fate progression in stomatal development of <Emphasis Type="Italic">Arabidopsis</Emphasis> stems

Mutations in TOO MANY MOUTHS (TMM), which encodes a receptor-like protein, cause stomatal patterning defects in Arabidopsis leaves but eliminate stomatal formation in stems. Stomatal development in wild-type and tmm stems was analyzed to define TMM function. Epidermal cells in young tmm stems underwent many asymmetric divisions characteristic of entry into the stomatal pathway. The resulting precursor cells, meristemoids, appropriately expressed cell fate markers such as pTMM:GFP. However, instead of progressing developmentally by forming a guard mother cell, the meristemoids arrested, dedifferentiated, and enlarged. Thus asymmetric divisions are necessary but not sufficient for stomatal formation in stems, and TMM promotes the fate and developmental progression of early precursor cells. Comparable developmental and mature stomatal phenotypes were also found in tmm hypocotyls and in the proximal flower stalk. TMM is also a positive regulator of meristemoid division in leaves suggesting that TMM generally promotes meristemoid activity. Our results are consistent with a model in which TMM interacts with other proteins to modulate precursor cell fate and progression in an organ and domain-specific manner. Finally, the consistent presence of a small number of dedifferentiated meristemoids in mature wild-type stems suggests that precursor cell arrest is a normal feature of Arabidopsis stem development.     

The too many mouths and four lips mutations affect stomatal production in Arabidopsis

Stomata regulate gas exchange through the aerial plant epidermis by controlling the width of a pore bordered by two guard cells. Little is known about the genes that regulate stomatal development. We screened cotyledons from ethyl methanesulfonate-mutagenized seeds of Arabidopsis by light microscopy to identify mutants with altered stomatal morphology. Two mutants, designated too many mouths (tmm) and four lips (flp), were isolated with extra adjacent stomata. The tmm mutation results in stomatal clustering and increased precursor cell formation in cotyledons and a virtual absence of stomata in the inflorescence stem. The flp mutation results in many paired stomata and a small percentage of unpaired guard cells in cotyledons. The double mutant (tmm flp) exhibits aspects of both parental phenotypes. Both mutations appear to affect stomatal production more than patterning or differentiation. tmm regulates stomatal production by controlling the formation, and probably the activity, of the stomatal precursor cell.     

An analysis of the mechanics of guard cell motion

This paper presents a mechanical analysis of the cellular deformations which occur during the opening and closing of stomata. The aperture of the stomatal pore is shown to be a result of opposing pressures of the guard and adjacent epidermal cells. The analysis indicates that the epidermal cells have a mechanical advantage over the guard cells. With no mechanical advantage, an equal reduction in the turgor pressure of both guard and epidermal cells would have a neglible effect upon stomatal aperture. However, due to the mechanical advantage of the surrounding cells, the stomatal aperture increases with equal reductions in turgor, until the adjacent epidermal cells become flaccid. The minimum diffusion resistance of the pore occurs at this point. Further reductions in guard cell turgor lead to closure of the pore. The analysis further demonstrates how the shape, size, wall thickness and material properties of the guard cell walls influence their behavior.     

ABNORMAL STOMATAL DEVELOPMENT IN FOLIAGE LEAVES OF BEGONIA ARIDICAULIS

Dehnel , George S. (U. Missouri, Columbia.) Abnormal stomatal development in foliage leaves of Begonia aridicaulis. Amer. Jour. Bot. 48(2): 129–133. Illus. 1961.—The results of 2 types of aberrant stomatal development in abaxial epidermis of normal laminae of B. aridicaulis are discussed and illustrated. The more common is a so-called persistent stomatal initial that is an essentially circular cell (in face view) with chloroplasts and a wall of uniform thickness. The other type of anomalous development yields a single guard cell, which possesses chloroplasts, differential wall thickening, rarely a pore, and a form characteristic of a normally paired guard cell. Both types of cells may either be isolated or occur variably in close relationship to normal stomata.     

BASL and EPF2 act independently to regulate asymmetric divisions during stomatal development

The initiation of stomatal development in the developing Arabidopsis epidermis is characterized by an asymmetric ‘entry’ division in which a small cell, known as a meristemoid, and a larger daughter cell is formed. The meristemoid may undergo further asymmetric divisions, regenerating a meristemoid each time, before differentiating into a guard mother cell which divides symmetrically to form a pair of guard cells surrounding a stomatal pore. Recently EPF2 and BASL have emerged as regulators of these asymmetric divisions and here we present results indicating that these two factors operate independently to control stomatal developmentKey words: stomata, development, meristemoids, asymmetric cell division, leaf epidermis, cell polarity, peptide signal     

Stomata and plasmodesmata

Summary In developing epidermal tissue ofPhaseolus vulgare L. complete plasmodesmatal connections occurred between guard cells and epidermal cells and between sister guard cells of a stoma but they were not seen in fully differentiated tissue. However, incomplete, aborted plasmodesmata were occasionally seen in the common guard/epidermal cell wall, usually connected to the epidermal cell protoplast, in mature tissue. Plasmodesmatal connections between neighbouring epidermal cells were commonly observed in tissue at all stages of development. In all locations, the plasmodesmata were usually unbranched occurring singly or in small pit fields; very rarely branched, incomplete plasmodesmata were also seen in the wall between mature guard and epidermal cells. The significance of these findings were related to stomatal functioning and to the development of plasmodesmata in general.     

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.     

The role of peristomatal transpiration in the mechanism of stomatal movement

Abstract. Peristomatal transpiration is defined as the relative high local rate of cuticular water loss from external and internal surfaces around the stomatal pore and its decisive role in the control of stomatal movement is re-emphasized. As the resistance towards changes in air humidity is low in the pore surroundings, the state of turgor is particularly unsteady there. Due to the inherent instability the guard cell 'senses' fluctuations in the supply-demand relationship of water and is thus the control unit proper. The environmental variables (supply and demand) are cross-correlated within the subsidiary cell and the information is transmitted to the guard cell through the water potential gradient between the two cells. A conceptual segregation of a 'humidity response' by 'passive' stomatal movements is rejected.
As ions always accumulate at the most distant point of the liquid path and as this point varies with pore width according to the prevailing water potential gradients, it is felt that the water stream is causing the characteristic pattern of ion distribution within the epidermis. Passive import of ions is attributed to local concentration gradients which are steepened by continuous supply and by water uptake into the guard cell in response to starch hydrolysis. A mechanistic model supplements the discussion.