Guard Mother Cells Research Articles

Actin in the developing stomatal complex of winter rye: A comparison of actin antibodies and Rh‐phalloidin labeling of control and CB‐treated tissues

AbstractActin localization during stomatal complex formation in rye leaf epidermis was compared by three different labeling procedures. When leaf segments are fixed with formaldehyde prior to staining microfilament (MF) patterns visualized with actin antibodies and those with rhodamine‐phalloidin (Rh‐ph) are basically identical in controls. Likewise, on tissues treated with cytochalasin B (CB), actin antibodies and Rh‐ph produce very similar labeling patterns. Compared to MF alignments in fixed samples, additional sets of MFs are observed at the very cortical regions of epidermal cells that are stained with Rh‐ph without aldehyde fixation. Cortical MFs are also present in a variety of mitotic cells; MFs of meristematic cells and guard mother cells are more concentrated near the walls facing spindle poles, whereas a fine meshwork of MFs is observed along the entire periclinal surface of subsidiary mother cells. Although exactly how MFs are involved in control of the division site in higher plant cells is still to be determined, the presence of MFs during mitosis and the abnormal division observed in some stomatal cells after treatment with CB suggest that MFs are necessary for normal orientation of division in these cells, and thus normal morphogenesis.

Development of the Stomatal Complexes During Ontogeny in Eucalyptus and Angophora (Myrtaceae)

The mode of stomatal development is studied in cotyledons, seedling and adult leaves of species of eucalypts and three species of Angophora. In the cotyledons of all species examined the early stomatal initials are unilabrate or dolabrate. The stomatal initials in seedling leaves of species of the Corymbosae and Clavigerae are anisocytic. In the 4th seedling leaf in species of a group we have previously called Monocalyptus the stomatal initials are also anisocytic. All other eucalypts retain the early cotyledonary mode of origin of stomata throughout life. These two modes of origin, whether anisocytic or by unilabrate and dolabrate initials, are set in all eucalypts from the 4th seedling leaf onward. Secondary characteristics of the adult stomata, e.g. number of subsidiary cells, are more complex than those of the seedling leaves; rarely, the relatively simple pattern of the seedling leaves may persist in the adult leaves of a given species. In species in which the initials in adult leaves are unilabrate or dolabrate, groups of stomata may share one or more subsidiary cells or be juxtaposed without an intervening subsidiary cell. The sister cell(s) of the guard mother cell may precociously develop a thicker cuticle than ordinary epidermal cells, and this may be apparent at maturity. The abaxial stomata of the cotyledons (but not of seedling or adult leaves) are regularly aligned parallel to the main venation. The existence of three main types of origin of stomata characteristic of three large non-interbreeding groups of eucalypts is of interest in the taxonomy of the genus.

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

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.

Microtubules in plant cell division

Immunofluorescence microscopy has proven to be a valuable accompaniment to electron microscopy for study of the cytoskeleton of plant cells. Whereas electron microscopy provides greater resolution and details of the spatial relationships of the cytoskeleton to other cellular components, fluorescence visualization makes it possible to see the three-dimensional organization of cytoskeletal elements without laborious reconstruction of views from serial sections. An area in which immunofluorescence microscopy has been useful is the investigation of how plant cells organize and position the various microtubule arrays that are utilized during mitosis, cytokinesis and cell expansion phases. One of the earliest indications of an impending division in a meristematic plant cell is the formation of a preprophase band of microtubules in the cell cortex, at the site where the new wall will be placed at the subsequent cytokinesis. At its later stages, the band is narrower than when first identifiable. In most cells, preprophase band microtubules have the same general orientation as the preceding interphase microtubules, and so preprophase band formation here could, in theory, be achieved by lateral bundling of microtubules.Cells in which the division site and the preprophase band that marks it are not oriented parallel to interphase microtubules are found in stomatal complexes of grass leaves . Fig. 1 illustrates the arrangement of two such cell types: the guard mother cell, which divides lengthwise to form two guard cells, side-by-side, and the subsidiary mother cell, which undergoes a very asymmetric division to produce one of the pair of lens-shaped subsidiary cells that flank the guard cells. Interphase and preprophase arrangements of microtubules for each cell type are diagrammed in Figs. 2-4. In order to examine how these cell types achieve the reorientation of microtubules that is necessary to progress from interphase to preprophase, sheets of epidermis containing actively dividing stomatal complex cells were examined with immunofluorescence microscopy using antibodies to tubulin. Thin epidermal slices of leaves were fixed and glued down to a slide, whereupon cell walls were enzymatically weakened so that unwanted cell layers could be removed . Because waves of division pass along grass leaves, cells of the same type in a given file tend to be at similar stages, which facilitates deduction of the developmental pattern.

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.

Microtubule organization during development of stomatal complexes inLolium rigidum

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.

Developmental changes in the arrangement of cortical microtubules in stomatal cells of oat (Avena sativaL.)

AbstractChanges in microtubule organization were monitored in the stomatal complexes ofAvena sativausing tubulin immunocytochemistry. Radial arrays of cortical microtubules, previously thought to be characteristic of guard cells, also appear in adjacent subsidiary cells early in development. The subsidiary cell arrays are evident even before guard cells form via division of precursor guard mother cells. Thus, before the stomatal pore opens between sister guard cells, each complex contains four similar microtubule arrays. As the pore opens, however, the subsidiary cell system is reorganized into a network of microtubules distributed along the length of the cell. A similar change is effected in the guard cells after the pore opens. Subsidiary cells and guard cells elongate during later stages of differentiation, and a thickened wall is deposited int he narrow midzone of the latter. At the same time, microtubules in both cells assume a more axial orientation. The results are discussed in terms of developmental symmetry and the control of microtubule organization and cell wall deposition.

Formation of the oblique spindle in dividing guard mother cells ofAllium

Microtubule organization in dividing guard mother cells ofAllium cepa was investigated using tubulin immunofluorescence, in order to determine when the spindle assumes its characteristic oblique orientation. The prophase spindle is transverse, perpendicular to the preprophase band and the future cell plate. Its orientation then shifts, until the majority of spindles are oblique by late metaphase. These observations show that the early spindle conforms to positional information determining the division plane, but becomes oblique during later stages of division, perhaps in response to space constraints.

Division plane determination in guard mother cells of Allium: Video time-lapse analysis of nuclear movements and phragmoplast rotation in the cortex

In guard mother cells of Allium, the formation of a longitudinal cell wall oriented 90° to all other division planes in the epidermis is programmed prior to mitosis. However, the spindle axis is oblique and daughter chromosomes separate toward opposite corners of the cell. The correct division plane is achieved in late anaphase/telophase when a reorientation process moves the chromosome masses and the cell plate into their proper positions. Video time-lapse methods have now been used to analyze these movements. At least two processes are involved. In phase 1, movement of the daughter chromosomes along the side walls accounts for some (or all in the case of short GMCs) of reorientation before the cell plate forms. In most cells, this initial phase ceases before the oblique orientation of the spindle is corrected. As a result, the new cell plate maintains a skewed position until the phragmoplast reaches the preprophase band site near the paradermal walls. The plate edge than rotates into the midlongitudinal plane, marking the start of phase 2. Realignment continues around the rest of the plate edge as the phragmoplast expands cross the cell, and eventually the center of the plate also turns due to the action at the edges. The daughter nuclei also resume movement as the plate turns. Phase 2 ceases when the plate is longitudinal and the nuclei face each other midway along the side walls. These events are seen in green as well as white light. Blue light does not affect phase 1 but it does interfere with edge rotation in phase 2. Edge rotation is also sensitive to sodium azide. Thus, data obtained from live cells show that the phragmoplast interacts with the cortex/plasmalemma at the preprophase band site to achieve the correct plane of division.

Metabolic inhibitors block anaphase A in vivo.

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.

Development of Stomata in Selaginella: Division Polarity and Plastid Movements

The young guard cell of Selaginella inherits a single plastid from the division of the stomatal guard mother cell (GMC). During early stomatal development the single plastid undergoes a complex series of migrations and divisions. The regular pattern of plastid behavior appears to be an expression of the genetic program controlling division plane and cytomorphogenesis. The plastid in the GMC becomes precisely aligned with its midconstriction intersected by the plane of a preprophase band of microtubules (PPB) oriented parallel to the long axis of the leaf. This alignment with respect to the future division plane of the cytoplasm ensures equal plastid distribution to the daughter cells. Cytokinesis occurs in the plane previously marked by the PPB and the plastid in each daughter cell lies between the lateral wall and the newly formed nucleus. Following cytokinesis the plastid in each young guard cell develops a median constriction and migrates to the common ventral wall where the isthmus is associated with a system of microtubules in the vicinity of the developing pore region. Plastid division is completed while the plastid is adjacent to the common ventral wall. Following division, the two daughter plastids move back toward the lateral wall. Each plastid may divide again during guard cell maturation but no further migrations occur.

DEVELOPMENT OF STOMATA IN SELAGINELLA: DIVISION POLARITY AND PLASTID MOVEMENTS

The young guard cell of Selaginella inherits a single plastid from the division of the stomatal guard mother cell (GMC). During early stomatal development the single plastid undergoes a complex series of migrations and divisions. The regular pattern of plastid behavior appears to be an expression of the genetic program controlling division plane and cytomorphogenesis. The plastid in the GMC becomes precisely aligned with its midconstriction intersected by the plane of a preprophase band of microtubules (PPB) oriented parallel to the long axis of the leaf. This alignment with respect to the future division plane of the cytoplasm ensures equal plastid distribution to the daughter cells. Cytokinesis occurs in the plane previously marked by the PPB and the plastid in each daughter cell lies between the lateral wall and the newly formed nucleus. Following cytokinesis the plastid in each young guard cell develops a median constriction and migrates to the common ventral wall where the isthmus is associated with a system of microtubules in the vicinity of the developing pore region. Plastid division is completed while the plastid is adjacent to the common ventral wall. Following division, the two daughter plastids move back toward the lateral wall. Each plastid may divide again during guard cell maturation but no further migrations occur.

Tissue distribution of phenylpropanoid metabolism in cotyledons of Raphanus sativus L.

The tissue distributions of sinapic acid esters (1-sinapoylglucose, sinapolyl-L-malate, 6,3'-disinapoylsucrose), kaempferol glycosides, free malic acid and of the enzyme involved in the synthesis of sinapoyl-L-malate, 1-sinapoylglucose: L-malate sinapoyltransferase (SMT), have been investigated in cotyledons of Raphanus sativus L. seedlings. The kaempferol glycosides were mainly localized in the upper epidermis. The sinapoyl esters were found in all tissues, but differed markedly in their concentrations. While disinapoylsucrose was localized predominantly in the mesophyll, most sinapoylmalate was found in the epidermal layers, as was most SMT activity. Ultraviolet microscopy and microfluorospectrophotometry of isolated epidermal peels indicated that the epidermal sinapoyl esters were restricted to guard cells, guard mother cells and adjacent epidermal cells. Upon excitation by UV light (365 nm) these exhibited strong blue fluorescence with an emission maximum at about 480 nm. Our results indicate a highly tissue-and cell-specific secondary metabolism in Raphanus cotyledons and indicate that the biosynthesis of sinapoylmalate is intimately related to the malic-acid metabolism of the guard cells.

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.

Some Observations on the Relationship between Cell Division and Cell Determination in the Epidermis of the Developing Leaf of Corn (Zea mays)

The relationship of cell division and cell elongation in the epidermis of leaves of Zea mays at increasing plastochron index (PI) has been followed. There is a correlation of elongation and cell division in epidermal cells of leaves with PI < 6. Above this PI, the processes of division and elongation are increasingly separated in time. Stomatal complexes develop between PI 3.5 and PI 5.6 in all the varieties observed. The pattern of differentiation is similar to that described in other monocotyledons. Sensitive staining for potassium shows that one of the earliest events in the differentiation of presumptive stomatal guard mother cells is the development of the potassium pump associated with stomatal movement. This is found at PIs as low as 3.2

Microtubules and morphogenesis in stomata of the water fernAzolla: An unusual mode of guard cell and pore development

A mature stomate of the water fernAzolla consists of a single apparently unspecialized annular guard cell (GC) with two nuclei surrounding an elongated pore aligned longitudinally in the leaf. During development, the guard mother cell develops a preprophase band (PPB) of microtubules (MTs) oriented transverse to the leaf axis. This is followed by a cell plate which fuses with the parental walls at the PPB site. Subsequently only the central part of the cell plate is consolidated, while the parts to either side become perforated and tenuous and may disperse completely, forming a single composite GC.

Structure and development of normal and abnormal stomata in the seedlings of some Cruciferae

The structure and ontogeny of normal and abnormal stomata in the seedlings of 16 species of the Cruciferae have been presented. The mature stomata are anomocytic, paracytic, anisocytic, helicocytic and with a single subsidiary cell. The ontogeny of the anisocytic, paracytic, helicocytic and stoma with a single subsidiary cell conforms to the syndetocheilic or mesogenous, while that of anomocytic to haplocheilic or perigenous type. Several types of aberrant stomatal formations such as: single guard cell; contiguoas single-guard cells; contiguous stomatal; degeneration of guard cell/s; persistert stomatal cell; amitotic division of guard mother cell nucleus followed by pore formation; stoma with double pores; binucleate guard cell; division of guard cell; incomplete stoma with unequal guard cells; cytoplasmic connection; ring-shaped division of guard mother cell and uncommon wall thickening have been noticed. Aberrant stomatal developments are naturally occurring and not induced.

Epifluorescence and Video Analysis of Vacuole Motility and Development in Stomatal Cells of Allium

The vacuole in stomatal cells of Allium undergoes major changes in shape during differentiation, switching from a globular form in new guard mother cells to a network of interconnected tubules and chambers, and then back to a globular form as guard cells mature. In addition, vacuolar network elements exhibit characteristic movements and rearrangements.

Comparative effects of phalloidin and cytochalasin B on motility and morphogenesis in Allium

Cytochalasin B (CB), thought to disaggregate F-actin in animal cells, and phalloidin (Phal), known to stabilize F-actin in vivo and in vitro, have nearly identical effects on cotyledon epidermal cells of Allium cepa. Both drugs rapidly induce cessation of streaming and both, by preventing normal telophase reorientation movement, lead to abnormal division planes in dividing guard mother cells. Neither, however, prevents normal microtubule deposition, wall thickening, and cellulose orientation during guard cell differentiation. Furthermore, both drugs have no effect on spindle formation and anaphase chromosome motion. Examination of Nitella and Chara cells, in which streaming had been stopped by either agent, shows that microfilament cables are still present. With both drugs, the minimum effective concentrations were routinely used (CB, 2 μM; Phal, 100–200 μM). Our results are discussed in terms of the mode of action of these drugs and their possible role in host-fungus interactions. Implications for the mechanisms underlying cell plate alignment, cellulose orientation, and cytoplasmic streaming are discussed.