Cell wall anisotropy plays a key role in Zea mays stomatal complex movement: the possible role of the cell wall matrix.

The opening of the stomatal pore in Zea mays is accomplished by the lateral displacement of the central canals of the dumbbell-shaped guard cells (GCs) towards their adjacent deflating subsidiary cells that retreat locally. During this process, the central canals swell, and their cell wall thickenings become thinner. The mechanical forces driving the outward displacement of the central canal are applied by the asymmetrically swollen bulbous ends of the GCs via the rigid terminal cell wall thickenings of the central canal and the polar ventral cell wall (VW) ends. During stomatal pore closure, the shrinking bulbous GC ends no longer exert the mechanical forces on the central canals, allowing them to be pushed back inwards, towards their initial position, by the now swelling subsidiary cells. During this process, the cell walls of the central canal thicken. Examination of immunolabeled specimens revealed that important cell wall matrix materials are differentially distributed across the walls of Z. mays stomatal complexes. The cell walls of the bulbous ends and of the central canal of the GCs, as well as the cell walls of the subsidiary cells were shown to be rich in methylesterified homogalacturonans (HGs) and hemicelluloses. Demethylesterified HGs were, in turn, mainly located at the terminal cell wall thickenings of the central canal, at the polar ends of the VW, at the lateral walls of the GCs and at the periclinal cell walls of the central canal. During stomatal function, a spatiotemporal change on the distribution of some of the cell wall matrix materials is observed. The participation of the above cell wall matrix polysaccharides in the well-orchestrated response of the cell wall during the reversible movements of the stomatal complexes is discussed.

Callose and homogalacturonan epitope distribution in stomatal complexes of Zea mays and Vigna sinensis.

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function.

The intracellular and intercellular cross-talk during subsidiary cell formation in Zea mays: existing and novel components orchestrating cell polarization and asymmetric division.

Background: Formation of stomatal complexes in Poaceae is the outcome of three asymmetric and one symmetric cell division occurring in particular leaf protodermal cells. In this definite sequence of cell division events, the generation of subsidiary cells is of particular importance and constitutes an attractive model for studying local intercellular stimulation. In brief, an induction stimulus emitted by the guard cell mother cells (GMCs) triggers a series of polarization events in their laterally adjacent protodermal cells. This signal determines the fate of the latter cells, forcing them to divide asymmetrically and become committed to subsidiary cell mother cells (SMCs). Scope: This article summarizes old and recent structural and molecular data mostly derived from Zea mays, focusing on the interplay between GMCs and SMCs, and on the unique polarization sequence occurring in both cell types. Recent evidence suggests that auxin operates as an inducer of SMC polarization/asymmetric division. The intercellular auxin transport is facilitated by the distribution of a specific transmembrane auxin carrier and requires reactive oxygen species (ROS). Interestingly, the local differentiation of the common cell wall between SMCs and GMCs is one of the earliest features of SMC polarization. Leucine-rich repeat receptor-like kinases, Rho-like plant GTPases as well as the SCAR/WAVE regulatory complex also participate in the perception of the morphogenetic stimulus and have been implicated in certain polarization events in SMCs. Moreover, the transduction of the auxin signal and its function are assisted by phosphatidylinositol-3-kinase and the products of the catalytic activity of phospholipases C and D. Conclusion: In the present review, the possible role(s) of each of the components in SMC polarization and asymmetric division are discussed, and an overall perspective on the mechanisms beyond these phenomena is provided.

Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape.

The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells.

Spatio-temporal diversification of the cell wall matrix materials in the developing stomatal complexes of Zea mays.

MAIN CONCLUSION: The matrix cell wall materials, in developing Zea mays stomatal complexes are asymmetrically distributed, a phenomenon appearing related to the local cell wall expansion and deformation, the establishment of cell polarity, and determination of the cell division plane. In cells of developing Zea mays stomatal complexes, definite cell wall regions expand determinately and become locally deformed. This differential cell wall behavior is obvious in the guard cell mother cells (GMCs) and the subsidiary cell mother cells (SMCs) that locally protrude towards the adjacent GMCs. The latter, emitting a morphogenetic stimulus, induce polarization/asymmetrical division in SMCs. Examination of immunolabeled specimens revealed that homogalacturonans (HGAs) with a high degree of de-esterification (2F4- and JIM5-HGA epitopes) and arabinogalactan proteins are selectively distributed in the extending and deformed cell wall regions, while their margins are enriched with rhamnogalacturonans (RGAs) containing highly branched arabinans (LM6-RGA epitope). In SMCs, the local cell wall matrix differentiation constitutes the first structural event, indicating the establishment of cell polarity. Moreover, in the premitotic GMCs and SMCs, non-esterified HGAs (2F4-HGA epitope) are preferentially localized in the cell wall areas outlining the cytoplasm where the preprophase band is formed. In these areas, the forthcoming cell plate fuses with the parent cell walls. These data suggest that the described heterogeneity in matrix cell wall materials is probably involved in: (a) local cell wall expansion and deformation, (b) the transduction of the inductive GMC stimulus, and

Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

BACKGROUND AND AIMS: This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. METHODS: Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. RESULTS: In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. CONCLUSIONS: The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril alignment, spatially controlled cell wall expansion, allowing MCs to acquire their particular shape.

Polarized endoplasmic reticulum aggregations in the establishing division plane of protodermal cells of the fern Asplenium nidus.

The determination of the division plane in protodermal cells of the fern Asplenium nidus occurs during interphase with the formation of the phragmosome, the organization of which is controlled by the actomyosin system. Usually, the phragmosomes between adjacent cells were oriented on the same plane. In the phragmosomal cortical cytoplasm, an interphase microtubule (MT) ring was formed and large quantities of endoplasmic reticulum (ER) membranes were gathered, forming an interphase U-like ER bundle. During preprophase/prophase, the interphase MT ring and the U-like ER bundle were transformed into a MT and an ER preprophase band (PPB), respectively. Parts of the ER-PPB were maintained during mitosis. Furthermore, the plasmalemma as well as the nuclear envelope displayed local polarization on the phragmosome plane, while the cytoplasm between them was occupied by distinct ER aggregations. These consistent findings suggest that Α. nidus protodermal cells constitute a unique system in which three elements of the endomembrane system (ER, plasmalemma, and nuclear envelope) show specific characteristics in the establishing division plane. Our experimental data support that the organization of the U-like ER bundle is controlled on a cellular level by the actomyosin system and intercellularly by factors emitted from the leaf apex. The possible role of the above endomembrane system elements on the mechanism that coordinates the determination of the division plane between adjacent cells in protodermal tissue of A. nidus is discussed.

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

BACKGROUND AND AIMS: The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-ß-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. METHODS: Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. RESULTS: Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. CONCLUSIONS: The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.

Callose implication in stomatal opening and closure in the fern Asplenium nidus.

The involvement of callose in the mechanism of stomatal pore opening and closing in the fern Asplenium nidus was investigated by examination of the pattern of callose deposition in open and closed stomata, and by examination of the effects of callose degradation and inhibition or induction of callose synthesis in stomatal movement. Callose was identified with aniline blue staining and a callose antibody and degraded via beta-1,3-D-glucanase. Callose synthesis was inhibited with 2-deoxy-D-glucose and induced by coumarin or dichlobenil. Stomatal pore opening and closing were assessed by estimation of the stomatal pore width. The open stomata entirely lacked callose, while the closed ones displayed distinct radial fibrillar callose arrays in the external periclinal walls. The latter displayed local bending at the region of callose deposition, a deformation that was absent in the open stomata. Both callose degradation and inhibition of callose synthesis reduced the stomatal ability to open in white light and close in darkness. By contrast, callose synthesis induction considerably improved stomatal pore opening and reduced stomatal closure in same conditions. The present data revealed that: during stomatal closure the external periclinal guard cell walls experience a strong mechanical stress, probably triggering callose synthesis; and that callose participates in stomatal movement.

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)-beta-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 Ca(2+) 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.

Aluminium causes variable responses in actin filament cytoskeleton of the root tip cells of Triticum turgidum.

The effects of aluminium on the actin filament (AF) cytoskeleton of Triticum turgidum meristematic root tip cells were examined. In short treatments (up to 2 h) with 50-1000 microM AlCl3.6H2O, interphase cells displayed numerous AFs arrayed in thick bundles that lined the plasmalemma and traversed the endoplasm in different directions. Measurements using digital image analysis and assessment of the overall AF fluorescence revealed that, in short treatments, the affected cells possessed 25-30% more AFs than the untreated ones. The thick AF bundles were not formed in the Al-treated cells in the presence of the myosin inhibitors 2,3-butanedione monoxime (BDM) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), a fact suggesting that myosins are involved in AF bundling. In longer Al treatments, the AF bundles were disorganised, forming granular actin accumulations, a process that was completed after 4 h of treatment. In the Al-treated cells, increased amounts of callose were uniformly deposited along the whole surface of the cell walls. In contrast, callose formed local deposits in the Al-treated cells in the presence of cytochalasin B, BDM, or ML-7. These results favour the hypothesis that the actomyosin system in the Al-treated cells, among other roles, participates in the mechanism controlling callose deposition.

Hyperosmotically induced accumulation of a phosphorylated p38-like MAPK involved in protoplast volume regulation of plasmolyzed wheat root cells.

A 46 kDa protein resembling immunochemically to the mammalian dually phosphorylated p38-MAPK was detected in wheat root cells under hyperosmotic conditions, using Western blot analysis. This protein accumulated in a time- and dose-dependent fashion and exhibited pharmacological sensitivity similar to the activated p38-MAPK. The application of a highly specific p38-MAPK inhibitor revealed that the p38-like MAPK is probably implicated in hyperosmotically induced tubulin cytoskeleton reorganization as well as in protoplast volume regulation and osmotic tolerance of wheat root cells. As far as we know, the p38-MAPK has not been previously reported in higher plants.

Actomyosin is involved in the plasmolytic cycle: gliding movement of the deplasmolyzing protoplast.

The leaf cells of Chlorophytum comosum seem to have the ability to regulate their protoplast volume and shape during the plasmolytic cycle. This phenomenon was morphologically expressed by the stabilization of the plasmolyzed protoplast volume and shape within 1-5 min after the immersion of the leaf segments in the plasmolytic fluid and temporarily at the onset of deplasmolysis. During the latter stage the plasmolyzed protoplast rounded up and assumed a perfectly convex shape and glided into the cell lumen along the cell axis. This gliding movement was active, nonsaltatory, and conducted with a constant velocity and lasted for a short time. During this movement the protoplast volume did not change appreciably. As far as we know, this movement has not been described so far. Deplasmolysis proceeded and was rapidly completed when the protoplast stopped moving. Leaf cells which have been affected by an antiactin filament drug or myosin inhibitors lost their ability to regulate the volume and shape of the plasmolyzing protoplast. In addition, the gliding protoplast movement was also inhibited in the treated cells. These data show for the first time that the actomyosin system is involved in the mechanism of volume regulation during the plasmolytic cycle and that it underlies the gliding movement of the deplasmolyzing protoplast.

Organization of the endoplasmic reticulum in dividing cells of the gymnosperms Pinus brutia and Pinus nigra, and of the pterophyte Asplenium nidus.

Endoplasmic reticulum (ER) organization in the dividing cells of the pterophyte Asplenium nidus and of the gymnosperms Pinus brutia and Pinus nigra has been studied by immunolocalization techniques using the monoclonal antibody 2E7, which recognizes luminar ER resident proteins containing C-terminal HDEL sequences. In the pterophyte, the ER reorganization during cell cycle is similar to that in angiosperms. Among others, prominent ER gatherings were found at the mitotic spindle poles and in the phragmoplast during cytokinesis. However, in the gymnosperms examined, the ER displays a unique pattern of reorganization not described so far. In both the Pinus species, well-defined ER patterns are successively formed during cell cycle. They are the preprophase ER-band, the prophase- metaphase- and anaphase ER-spindle, the interzonal ER-system, the ER-phragmoplast and an ER-system lining the daughter cell wall. The ER patterns are closely similar to that of the correspondent microtubule (MT) arrangements with which they are co-organized. Observations made on P. nigra root-cells affected by oryzalin, colchicine and cytochalasin D favour the conclusion that the pattern of ER organization is controlled during mitosis and cytokinesis by the MT cytoskeleton.

Hyperosmotic stress-induced actin filament reorganization in leaf cells of Chlorophyton comosum.

Actin filament (AF) organization was studied during the plasmolytic cycle in leaf cells of Chlorophyton comosum Thunb. In most cells the hyperosmotic treatment induced convex or concave plasmolysis and intense reorganization of the AF cytoskeleton. Thin cortical AFs disappeared and numerous cortical, subcortical and endoplasmic AFs arranged in thick and well-organized bundles were formed. Plasmolysed cells displayed a significant increase in the overall AF content compared with the control cells. Cortical AF bundles were preferentially localized in the shrunken protoplast areas, lining the detached plasmalemma regions. The endoplasmic AF bundles were mainly found in the perinuclear cytoplasm and on the tonoplast surface. AFs also traversed some of the Hechtian strands. AF disorganization after cytochalasin B (CB) treatment induced dramatic changes in the pattern of plasmolysis, which lasted for a longer time and led to a greater decrease of the protoplast volume compared to the untreated cells. In many of the above cells the protoplasts assumed an 'amoeboid' form and were often subdivided into sub-protoplasts. Soon after the removal of the plasmolytic solution both CB-treated and untreated cells were deplasmolysed, while the AF cytoskeleton gradually reassumed the organization observed in the control cells. The findings of this study revealed for the first time in angiosperm cells that plasmolysis triggers an extensive reorganization of the AF cytoskeleton, which is involved in the regulation of protoplast shape and volume. The probable mechanism(s) leading to AF reorganization as well as the function(s) of the atypical AF arrays in plasmolysed cells are discussed.

Endoplasmic reticulum preprophase band in dividing root-tip cells of Pinus brutia.

In dividing root-tip cells of Pinus brutia Ten., immunolocalization of the luminal endoplasmic reticulum (ER) proteins, which have the C-terminal HDEL sequence, reveals that the ER is reorganized during the preprophase/prophase stage. Portions of ER were arrayed into a ring-like structure at the site of the microtubule preprophase band (Mt-PPB). This preprophase ER band (ER-PPB) resembles that of the Mt-PPB. The former undergoes a maturation process closely similar to that of the latter. Our data show that the PPB region has a more complex organization than is currently believed. The probable function(s) of the ER-PPB is discussed.

Systemic lupus erythematosus, a disease associated with low levels of clusterin/apoJ, an antiinflammatory protein.

OBJECTIVE: To measure the serum levels of clusterin, an antiinflammatory protein, which binds and inactivates complement, in patients with systemic lupus erythematosus (SLE) to determine whether the levels correlate with disease. METHODS: The levels of serum clusterin were measured by ELISA in 80 patients with SLE (76 female, 4 male). Clinical and serological information was gathered on 115 visits. Overall disease activity scores were determined using the Systemic Lupus Activity Measure-Revised. RESULTS: Serum clusterin levels were significantly decreased in patients with SLE and correlated inversely with disease activity (p < 0.00001). Low clusterin levels were significantly associated with skin ulcers (p < 0.0001), loss of hair (p = 0.002), proteinuria (p = 0.018), low platelet count (p = 0.03), and arthritis (p < 0.0001). The clusterin levels did not correlate with either systemic complement consumption, as measured by C3 or C4, or with prednisone use. CONCLUSION: A highly significant correlation was observed between low levels of serum clusterin and a number of SLE disease features. This deficiency of clusterin could directly or indirectly affect the disease process. Individuals lacking sufficient amounts of clusterin systemically likely have poor control of antibody mediated inflammation at sites of apoptosis where autoantigens are exposed.

Microtubule and actin filament organization during stomatal morphogenesis in the fern Asplenium nidus. II. Guard cells.

The post-cytokinetic guard cells of Asplenium nidus display a prominent perinuclear microtubule system and a few microtubules under the periclinal walls. Afterwards, microtubules appear on the whole surface of the ventral wall, whereas those below the periclinal walls proliferate and tend to become parallel with the ventral wall. The perinuclear microtubules gradually diminish but persist in later stages of guard cell differentiation. In post-cytokinetic guard cells, actin is found in the perinuclear cytoplasm and in the cortical cytoplasm lining all the walls. In differentiating guard cells, the following cortical microtubules and actin filament 'systems' appear in succession: (a) radial microtubule and actin filament arrays beneath the periclinal walls converging on the stomatal pore region, (b) anticlinal microtubule bundles, which are co-localized with actin filaments, along the ventral wall outlining the region of the stomatal pore, (c) periclinal microtubules and actin filaments on the polar ventral wall ends. These cytoskeletal systems, except for the radial actin filaments, persist in advanced stages of guard cell differentiation. Instead of the radial actin filaments, a prominent actin filament reticulum is organized under the margins of the developing wall thickenings of the stomatal pore. In addition, an extensive endoplasmic actin filament reticulum develops around the plastids. It seems likely that the successive microtubule systems in guard cells are formed by putative microtubule organizing centres operating in a definite spatial and temporal succession. Guard cell morphogenesis is the outcome of a definite process, in which the cortical microtubule cytoskeleton plays the primary role, implicated in the deposition of cellulose microfibrils and probably of the local wall thickenings. Callose or a callose-like glucan is deposited on the whole surface of the nascent ventral wall and in the wall regions where thickenings are deposited. Finally, the guard cells of Asplenium assume a kidney shape and display polar hypostomatic swellings. Particular structural features established in guard cell mother cells affect guard cell morphogenesis.

Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice.

In order to understand better the origins of the elevated levels of the glycoform of IgG that lacks galactose on both arms of the oligosaccharide chain (G0%) located in the Fc, which occurs in man and mouse with age, and in particular in autoimmune disease, we investigated the clearance of two glycosylated forms of IgG2a and IgG1 in normal (BALB/c) and autoimmune-prone (MRL/1pr, MRL/+, and non-obese diabetic (NOD)) mice. To investigate the possibility of different rates of catabolism, enzymatically generated glycoforms of monomeric IgG1 and IgG2a (fully glycosylated or G0%), were iodinated and injected into the tail vein of the mice. We found that the G0% IgG2a remained in circulation significantly longer than the fully glycosylated variants, in all of the mouse strains tested. In contrast, the two forms of IgG1 had similar kinetics in all the autoimmune-prone mice, whereas in BALB/c, there was a longer half-life (t1/2) for G0% IgG1. These data suggest that there may be differences in the ability of the IgG glycoforms to bind to the Fc gamma receptors, in particular Fc gamma RI. The clearance rates were found to vary among the strains studied, with MRL/1pr having the fastest catabolic rates for all glycoforms and IgG subclasses tested. This appeared to be due to the presence of circulating IgG and IgM rheumatoid factors (RF). There were significantly increased frequencies and titres for both IgM and IgG RF in MRL/1pr mice compared with the other strains. In contrast, interferon-gamma, known to induce the Fc gamma RI, was found to be similar in the sera, in all of the strains of mice examined. These results suggest that RF probably play an important biological function in the MRL/1pr mice and aid in the clearance of circulating IgG. Our study shows that the state of glycosylation of IgG affects the t1/2 in vivo, and that by removing the terminal sugars (sialic acid and galactose), the antibody (IgG2a) will remain in circulation significantly longer. These observations may thus provide a partial explanation for the increase in relative percentage of this glycoform that occurs with age.

Sinuous ordinary epidermal cells: behind several patterns of waviness, a common morphogenetic mechanism.

Morphogenesis of sinuous epidermal cells in leaves of the fern Asplenium nidus and the monocotyledonous Cyperus papyrus, petals of the dicotyledonous Begonia lucerna. and in-vitro-grown leaves of the fern Adantum capillus-veneris is controlled by the local differentiation of their walls. In all these cases wall pads, including radial cellulose microfibrils, arc deposited at the junctions of the external periclinal wall with the anticlinal ones. Moreover, in Asplenium nidus, similar wall pads form at the junctions of the internal periclinal wall with the anticlinal ones. The wall pads are connected to anticlinal cellulose microfibril bundles running the whole depth of the anticlinal walls nr part of it. This wall differentiation imposes a highly controlled cell wall expansion, a consequence of which is the waviness of the epidermal cell anticlinal, walls. The pattern of wall reinforcement varies among different species, resulting in differences in the pattern of waviness. Cortical microtubule arrays mirror the orientated deposition of cellulose microfibrils in the epidermal Cells. These findings, derived from plants from different major groups, show a common epidermal cell morphogenetic mechanism depending on radial cellulose microfibrils and cellulose microfibril bundles. The facts that (a) epidermal cell morphogenesis in Adiantum copillus-veneris leaves grown in vitro differs considerably from that of typical leaves and (b) petal epidermal cells in Begonia lucerna are sinuous, while leaf epidermal cells are not, suggest that this mechanism may he affected by epigenetic factors.