Microtubule organization and morphogenesis of stomata in caffeine-affected seedlings ofZea mays

Summary Treatment ofZea mays seedlings with a 5 mM caffeine solution inhibits cytokinesis in guard cell mother cells (GMCs), producing unicellular, binucleate aberrant stomata (a-stomata). Ventral wall (VW) strips of limited length, which usually meet the wall portions of GMCs adjoining the cortical zone of the preprophase microtubule band (PMB), are laid down in many a-stomata.In a-stomata with or without VW-strips, the periclinal walls are lined by numerous microtubules (Mts) converging on their mid-region, where local wall thickenings are deposited. When the VW-strips reach the mid-region of the periclinal walls, thickenings lined by numerous Mts rise at their free margins. In certain a-stomata an anticlinal wall column, surrounded by a dense Mt bundle, grows centripetally from either or both of the periclinal wall thickenings. In wall thickenings, the cellulose microfibrils are co-aligned with the adjacent Mts. Pore formation is initiated in all a-stomata. Deposition of an electron dense intra-wall material followed by lysis precedes pore opening. This process is closely related to the a-stornata morphogenesis. These observations show that the primary morphogenetic phenomenon in a-stomata is the establishment of an intense and stable polarity in the cytoplasm abutting on the mid-region of the periclinal walls and/or the adjacent plasmalemma area. Prime morphogenetic factor(s), including microtubule organizing centres (MTOCs), seem to function in these sites. Morphogenesis in a-stomata is a Mt-dependent process that is carried out as in normal stomata but in the absence of a VW.Abbreviations a-stomata unicellular binucleate aberrant stomata - CIPC chlorisopropyl-N-phenyl carbamate - GC guard cell - GMC guard cell mother cell - Mt microtubule - MTOC microtubule organizing centre - PMB preprophase microtubule band - VW ventral wall     

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.     

Experimental studies on the function of the cortical cytoplasmic zone of the preprophase microtubule band

Summary Centrifugation of young seedlings ofTriticum durum andTriticum aestivum for 8–10 hours at 1,500–2,000 x g causes a serious disorder of the spatial organelle relationships in the interphase as well as the preprophase and mitotic subsidiary cell mother cells (SMCs). The nucleus, most organelles and cytoplasm are displaced to the centrifugal end of the cell, while the vacuoles lie at the other end. However, after centrifugation, the preprophase microtubule bands (PMBs) are nucleated and remain at the expected position close to the guard cell mother cells (GMCs). In some elongated SMCs the PMBs become completely separated from the nucleus. The mitotic spindle exhibits variable orientation and is usually formed at some distance from the PMB cortical zone.Cytokinesis in SMCs is spatially highly disturbed and the cell plate shows a variety of unpredictable dispositions, which seem to be determined by: 1. the position of the preprophase-prophase nucleus and the orientation of the mitotic spindle as well as their spatial relationships to the PMB cortical zone, and 2. the space available for cell plate growth. Many of the daughter cells exhibit a highly variable shape and size in different planes. Usually one edge of the cell plate partly or totally joins the anticlinal parent wall adjacent to the PMB cortical zone.In some SMCs ofZea mays andTriticum aestivum, the junction regions of the periclinal walls with the anticlinal ones, lined by the PMB cortical zone in normal SMCs, are detectably thickened after the arrest of mitosis and the prevention of interphase microtubule formation by a prolonged colchicine treatment. In a small number of protodermal cells of the same plants, participating in the development of stomatal complexes, irregular wall bodies or incomplete wall sheets were formed at wall regions lined by the PMB cortical zone.The presented observations are in line with the following hypotheses: 1. the PMB cortical zone interacts with the growing edges of the cell plate attracting it to fuse with the underlying parent wall when the latter approaches the former at a critical distance, and 2. in SMCs particular regions of the PMB cortical zone and/or the adjacent plasmalemma promote the local wall deposition in the absence of microtubules.     

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     

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.     

Microtubule and actin filament organization during stomatal morphogenesis in the fernAsplenium nidus

Summary The newly-formed guard cell mother cells (GMCs) ofAsplenium nidus are small, lens-shaped and are formed by one or two asymmetrical divisions. Their growth axis is parallel to the plane of their future division, a process during which the internal periclinal wall (IPW) is detached from the partner wall of the underlying cell(s). This oriented GMC expansion occurs transversely to a microfibril bundle, which is deposited externally to a U-like microtubule (Mt) bundle and a co-localized actin filament (Af) bundle. They line the IPW and the major part of the anticlinal walls. The deposition of the microfibril bundle is followed by the slight constriction of the internal part of the GMCs and the broadening of the substomatal cavity. The IPW forms a distinct bulging distal to the neighbouring leaf margin, as well as a less defined proximal one. During the IPW bulging, the Mts and Afs under the external periclinal wall (EPW) attain a radial organization. This is followed by thinning of the central EPW region, which becomes impregnated with a callose-like glucan. The rest of the EPW becomes unequally thickened. The disintegration of the U-like Mt bundle is succeeded by the organization of radial Mt and Af arrays under the IPW. The radial Mt systems, controlling the alignment of the newly-deposited microfibrils, allow the GMC to assume a round paradermal profile. The GMCs form a preprophase Mt band (PPB) perpendicular to the interphase U-like Mt bundle. The anticlinal PPB portions appear first and those lining the periclinal walls later. The cytoplasm adjacent to the latter walls retain the radial Mt systems during early preprophase, simultaneously with the anticlinal PPB portions. The observations suggest that the GMCs of the fernA. nidus obtain a unique form, as a result of a particular polarity established in the cortical cytoplasm of the periclinal walls, in which Mts and Afs appear involved. This polarity persists in cell division and is inherited to guard cells (GCs). It provides primary morphogenetic information not only to GMCs but also to GCs.Abbreviations Af actin filament - EPW external periclinal wall - GC guard cell - GMC guard cell mother cell - IPW internal periclinal wall - Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band     

Cellulose microfibril orientation and cell shaping in developing guard cells of Allium: The role of microtubules and ion accumulation

Summary The role of microtubules and ions in cell shaping was investigated in differentiating guard cells of Allium using light and electron microscopy and cytochemistry. Microtubules appear soon after cytokinesis in a discrete zone close to the plasmalemma adjacent to the common wall between guard cells. The microtubules fan out from this zone, which corresponds to the future pore site, towards the other sides of the cell. Soon new cellulose microfibrils are deposited on the wall adjacent to the microtubules and oriented parallel to them. As the wall thickens, the shape of the cell shifts from cylindrical to kidney-like. Studies with polarized light show that guard cells gradually assume a birefringence pattern during development characteristic of wall microfibrils radiating away from the pore site. Retardation increases from 10 Å when cells just begin to take shape, to 80–100 Å at maturity. Both microfibril and microtubule orientation remain constant during development. Observations on aberrant cells including those produced under the influence of drugs such as colchicine, which leads to loss of microtubules, abnormal wall thickenings and disruption of wall birefringence, further support the role of microtubules in cell shaping through their function in the localization of wall deposition and the orientation of cellulose microfibrils in the new wall layer. Potassium first appears in guard mother cells before division and rapidly accumulates afterwards during cell shaping, as judged by the cobaltinitrite reaction. Some chloride and perhaps organic acid anions also accumulate. Thus, these ions, which are known to play a role in the function of mature guard cells, also seem to be important in the early growth and shaping of these cells.Abbreviations IPC isopropyl-N-phenylcarbamate - CB cytochalasin B - GMC guard mother cell - MTOC microtubule organizing center     

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     

Protoxylem: the deposition of a network containing glycine-rich cell wall proteins starts in the cell corners in close association with the pectins of the middle lamella

Antibodies were used to localise polysaccharide and protein networks in the protoxylem of etiolated soybean (Glycine max L.) hypocotyls. The deposition of glycine-rich proteins (GRPs) starts in the cell corners between protoxylem elements and xylem parenchyma cells. Finally, the GRPs form a network between two mature protoxylem elements. The network also interconnects the ring- and spiral-shaped secondary wall thickenings, as well as the thickenings with the middle lamellae of living xylem parenchyma cells. In addition to the GRP network, a polysaccharide network composed mainly of pectins is involved in the attachment of the secondary wall thickenings to the middle lamellae of xylem parenchyma cells.     

Microtubule arrays and Arabidopsis stomatal development

Microtubule arrays in living cells were analysed during Arabidopsis stomatal development in order to more closely define stages in the pathway and contexts where intercellular signalling might operate. Arabidopsis stomata are patterned iteratively via the orientation of an asymmetric division in a cell located next to an existing stoma. It was found that preprophase bands of microtubules (PPBs) were correctly placed away from stomata and from two types of precursor cells. This suggests that all three cell types participate in an intercellular signalling pathway that orients the division site. These and other asymmetric divisions in the pathway were preceded by a polarized cytoplasm, with the PPB around the nucleus at one end, and the vacuole at the other. PPBs before symmetric divisions of guard mother cells (GMCs) were broader than those in asymmetric divisions, and the GMC division site was marked by unusual end-wall thickenings. This work identifies an accessible system for studying cytoskeletal function and provides a foundation for analysing the role of genes involved in stomatal development.     

The development of stomata and other epidermal cells on the rice leaves

In the leaves of rice (Oryza sativa), stomatal initials arose from two asymmetric cell divisions and a symmetric division. Guard mother cells (GMCs) and long cells in stomatal files (LCSs) were formed through the first asymmetric division of the precursor cell of GMCs. Subsidiary cells (SCs) were produced by the second asymmetric division of subsidiary mother cells or LCSs. Following SC formation, GMCs divided once symmetrically to generate guard cells and then differentiated terminally to form mature stomata. The developmental patterns of long cells, prickle hairs and short cells (phellem cells and silica cells) were also examined. Interestingly, we found that the different developmental stages of stomata and epidermal cells occurred in the similar location of immature leaves of the same phyllotaxis. In addition, two spacing patterns (“one stoma, one long cell” and “one short cell row”) probably exist in rice leaves.     

Rearrangements of F-actin during Stomatogenesis Visualised by Confocal Microscopy in Fixed and Permeabilised Tradescantia Leaf Epidermis

Abstract: New details of F-actin organisation in leaf epidermal and stomatal cells were revealed by rhodamine — and fluorescein — phalloidin staining of fixed epidermal peels of Tradescantia virginiana and visualisation by confocal microscopy. Non-specialised epidermal cells contain highly organised arrays of fine cortical actin filaments aligned in transverse or oblique orientations. In interphase guard mother cells (GMCs), the arrangement of cortical F-actin changes on the periclinal and anticlinal cell walls at different times during differentiation. Initially, cortical F-actin on the periclinal surfaces is oriented transversely and F-actin is evenly distributed around the anticlinal walls. Following polarisation of the adjacent subsidiary mother cells (SMCs), actin in GMCs concentrates on the lateral anticlinal walls, but not on the transverse walls. Subsequently, F-actin on the periclinal walls reorients to radial and then longitudinal. Organisation of F-actin in SMCs appears to be influenced by the adjacent GMCs and co-ordination in F-actin arrangements in cells of the stomatal complex continues through to the formation of the guard cell pair. Our studies indicate that actin bands marking the division site in prophase cells, and detected in microinjected living material, are a particularly labile subset of F-actin. Actin bands were difficult to preserve, even when aldehyde fixation was avoided, in contrast to all interphase and mitotic F-actin.     

Reinstatement of Microtubule Arrays from Cortical Nucleating Sites in Stomatal Complexes of Lolium rigidum Following Depolymerization of Microtubules by Oryzalin and High Pressure

Immunofluorescence visualization of microtubule (MT) arraysin stomatal complexes of Lolium rigidum shows that disassemblyof the arrays can be successfully achieved using oryzalin orhigh pressure treatments. Under conditions allowing for MT recovery,MTs reappear within an hour after oryzalin or within 5 min afterhigh pressure treatment. During recovery guard mother cells(GMCs) nucleate MTs at sites distributed randomly in the cellcortex. Even after 22 h of recovery the MTs are not arrangedinto any configuration found in untreated tissue. This inabilityto reorganize their MTs after treatment makes GMCs more sensitiveto the loss of MTs than are other cells of the leaf. In guardcells (GCs) MTs reappear around the pore at the junction ofthe periclinal and ventral walls. They subsequently appear throughoutmost of the cell cortex and the majority of stomatal complexesrecover fully organized MT arrays indistinguishable from thosein untreated cells. The results support and extend ultrastructuraland immunofluorescence observations that suggest that MTs inGCs of developing stomata are nucleated in the cell cortex. ²Present address: Department of Biology, The University of SouthwesternLouisiana, Lafayette, Louisiana 70504-2451, U.S.A. (Received April 24, 1990; Accepted July 7, 1990)     

The role of callose in guard-cell wall differentiation and stomatal pore formation in the fern Asplenium nidus

Background and Aims

The pattern of callose deposition was followed in developing stomata of the fern Asplenium nidus to investigate the role of this polysaccharide in guard cell (GC) wall differentiation and stomatal pore formation.

Methods

Callose was localized by aniline blue staining and immunolabelling using an antibody against (1 → 3)-β-d-glucan. The study was carried out in stomata of untreated material as well as of material treated with: (1) 2-deoxy-d-glucose (2-DDG) or tunicamycin, which inhibit callose synthesis; (2) coumarin or 2,6-dichlorobenzonitrile (dichlobenil), which block cellulose synthesis; (3) cyclopiazonic acid (CPA), which disturbs cytoplasmic Ca²⁺ homeostasis; and (d) cytochalasin B or oryzalin, which disintegrate actin filaments and microtubules, respectively.

Results

In post-cytokinetic stomata significant amounts of callose persisted in the nascent ventral wall. Callose then began degrading from the mid-region of the ventral wall towards its periphery, a process which kept pace with the formation of an ‘internal stomatal pore’ by local separation of the partner plasmalemmata. In differentiating GCs, callose was consistently localized in the developing cell-wall thickenings. In 2-DDG-, tunicamycin- and CPA-affected stomata, callose deposition and internal stomatal pore formation were inhibited. The affected ventral walls and GC wall thickenings contained membranous elements. Stomata recovering from the above treatments formed a stomatal pore by a mechanism different from that in untreated stomata. After coumarin or dichlobenil treatment, callose was retained in the nascent ventral wall for longer than in control stomata, while internal stomatal pore formation was blocked. Actin filament disintegration inhibited internal stomatal pore formation, without any effect on callose deposition.

Conclusions

In A. nidus stomata the time and pattern of callose deposition and degradation play an essential role in internal stomatal pore formation, and callose participates in deposition of the local GC wall thickenings.     

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.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

The SCARECROW gene's role in asymmetric cell divisions in rice plants

Asymmetric cell division is one of the most important mechanisms in the diversification of cell function and fate. In Arabidopsis, SCARECROW (SCR) is essential for the asymmetric division of the cortex/endodermis progenitor cell in the root. To learn more about how SCR is involved in asymmetric division, we analyzed the rice SCR (OsSCR) expression. In the root tip, OsSCR expression was observed in the endodermal cell layer and downregulated in the daughter cortex cell after asymmetric division, just as with Arabidopsis SCR. In leaf primordia, expression of OsSCR was observed in stomatal and ligule formation. In stomatal development, OsSCR was specifically expressed in the stomatal cell files before formation of guard mother cells (GMCs), and then, its expression was localized in GMCs, when the first asymmetric division occurred to generate the GMCs. Before the second asymmetric division of subsidiary mother cells (SMCs), localized OsSCR expression was observed in SMCs in the area close to the GMCs. Before these asymmetric divisions, the localization of OsSCR mRNA in GMC-forming cells and SMCs was observed in the area of the daughter GMC and subsidiary cells. OsSCR expression was also observed in the initiation area of ligule formation, and its downregulation occurred in the inner L2 cells generated by asymmetric division. Based on these observations, we proposed that OsSCR is involved not only in the asymmetric division of the cortex/endodermis progenitor cell but also during stomata and ligule formation by establishing the polarization of cytoplasm.     

七叶树小孢子发生及雄配子体发育研究

用石蜡切片法观察了七叶树花药的发育过程.结果表明:(1)雄蕊花药四室,花药壁完全分化时,从外到内依次是表皮、药室内壁、中层和绒毡层,花药壁发育为基本型.表皮细胞1层,发育过程中始终存在;药室内壁在花药成熟时形成带状纤维层加厚;幼小花药壁的中层3~4层细胞,在花药发育成熟时退化消失;绒毡层1层细胞,发育类型为分泌型,小孢子母细胞减数分裂时绒毡层开始退化解体,花药成熟完全消失,仅剩1层绒毡层膜.每一花药中有多列雄性孢原细胞,发生于幼小花药表皮下方;(2)小孢子母细胞减数分裂为同时型,四分体多呈正四面体排列;减数分裂过程中,小孢子母细胞外方被胼胝质壁所包被,小孢子形成后胼胝质壁逐渐消失.成熟花粉二细胞型,外形呈圆三角状,具三孔沟.     

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.     

Effects of anoxia on pollen tube growth and tube wall formation of Impatiens glandulifera

Summary When pollen of Impatiens glandulifera was cultured in aerated liquid medium for 1 h, 70% of the pollen grains germinated; these attained an average tube length of 1 mm. Subsequently, these aerobic growth conditions were changed to anaerobic by substituting a nitrogen inlet for the air inlet. As a result, the pollen tubes stopped elongating and burst. The ultrastructural changes which occurred upon inducing anoxia were studied with samples taken at 0 s, 45 s, and 4 min after changing the gas. Anoxia caused rapid and considerable changes in the ultrastructure of the dictyosome vesicles involved in cell wall formation. There was an increase in the osmiophyly of the vesicle content, and the presence of fibrillar material became apparent. Simultaneously, the fusion behavior of the dictyosome vesicles changed. Instead of the normal fusion of the dictyosome vesicles with the plasma membrane, there was a premature fusion of the vesicles with each other inside the cytoplasm that resulted in the formation of aggregates. Furthermore, the cell wall precursors that were excreted were not incorporated in their usual configuration into the growing cell wall. Instead of a smooth inner cell wall surface, irregular thickenings were formed.     

Actin filament-organized local cortical endoplasmic reticulum aggregations in developing stomatal complexes of grasses

Endoplasmic reticulum (ER) immunolabeling in developing stomatal complexes and in the intervening cells of the stomatal rows (ICSRs) of Zea mays revealed that the cortical-ER forms distinct aggregations lining locally expanding wall regions. The polarized subsidiary cell mother cells (SMCs), displayed a cortical-ER-patch lining the wall region shared with the inducing guard cell mother cell (GMC), which disorganized during mitosis. In dividing SMCs, ER persisted in the preprophase band region and was unequally distributed in the mitotic spindle poles. The subsidiary cells (SCs) formed initially an ER-patch lining the common wall with the GMC or the young guard cells and afterwards an ER-ring in the junction of the SC wall with the neighboring ones. Distinct ER aggregations lined the ICSR wall regions shared with the SCs. The cortical-ER aggregations in stomatal cells of Z. mays were co-localized with actin filament (AF) arrays but both were absent from the respective cells of Triticum turgidum, which follow a different morphogenetic pattern. Experimental evidence showed that the interphase ER aggregations are organized by the respective AF arrays, while the mitotic ER aggregations by microtubules. These results revealed that AF and ER demarcated “cortical cytoplasmic domains” are activated below the locally expanding stomatal cell wall regions, probably via a mechanosensing mechanism triggered by the locally stressed plasmalemma/cell wall continuum. The probable role(s) of the local ER aggregations are discussed.