Effects of natural ventilation on leaf ultrastructure of Dianthus caryophyllus L. cultured in vitro

Summary Vessel closure configurations exert direct and indirect control over factors pertaining to the physical boundaries of the mieroenvironment, and induce a typical phenotype in in vitro plant production. Upon modification of the in vitro environment, carnation explants showed a gradation of their ultrastructural characteristics from hyperhydric to normal. A higher degree of development was observed in plants from vessels with higher ventilation compared to ones from vessels with low ventilation rates. The cell walls of epidermal cells from both normal and hyperhydric plants grown in nonventilated vessels were less developed than those from plants grown in ventilated vessels. Cytoplasm of normal plants was dense and generally located in parietal areas. The cytoplasm was even more dense in plants grown in ventilated vessels and acclimated. The degree of thylakoid stacking and distribution were affected by ventilation conditions, being poorly developed with low ventilation. Ultrastructurally, stomata from in vitro plants are ready to carry out their task, although there are significant differences in guard cell size and vacuolar area between acclimated and in vitro plants.     

Some new observations and interpretations on the vital staining of leaf epidermal tissue by neutral red

Summary The lower leaf epidermis from 5 plant species was stained with neutral red at 2 pH's (7.1 and 5.6) in the light and dark when the stomata were open or closed. At pH 5.6 no globule (= droplet) formation was observed in the guard cells whether the stomata were open or closed and cell walls possessed a high affinity for the stain. At pH 7.1 globules appeared in guard cells of open stomata, but not closed stomata, within 15 minutes. Anaerobic conditions prevented this globule formation. InZea mays, globules also appeared in subsidiary cells when the stomata were closed and in certain epidermal cells. Where globule formation did not occur increased diffuse staining of certain epidermal cells was considered to be the indication of cell integrity. In old leaf material very large numbers of dark blue globules appeared in epidermal cells ofCommelina diffusa, C. communis andSenecio odoris and this was associated with cell senescence.The staining characteristics were discussed in terms of cellular K⁺, Cl^–, tannin and flavonoid content and vacuolar pH.     

Membrane trafficking in guard cells during stomatal movement: Application of an image processing technique

Pairs of guard cells form small pores called stoma in the epidermis, and the reversible swelling and shrinking of these guard cells regulate the stomatal apertures. The well-documented changes in guard cell volume have been associated with their vacuolar structures. To investigate the contribution of the guard cell vacuoles to stomatal movement, the dynamics of these vacuolar structures were recently monitored during stomatal movement in vacuolar-membrane visualized Arabidopsis plants. Calculation of the vacuolar volume and surface area after reconstruction of three-dimensional images revealed a decrease in the vacuolar volume but an increase in the vacuolar surface area upon stomatal closure. These results implied the possible acceleration of membrane trafficking to the vacuole upon stomatal closure and membrane recycling from the vacuole to the plasma membrane upon stomatal opening. To clarify and quantify membrane trafficking during stomatal movement, we describe in this addendum our development of an improved image processing system.Key words: stomata, guard cells, vacuole, membrane traffic, image processing     

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Background and Aims

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods

Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key Results

Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions

This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.     

Artichoke Leaf Morphology and Surface Features in Different Micropropagation Stages

Artichoke (Cynara scolymus L.) leaf size and shape, glandular and covering trichomes, stomatal density, stomata shape, pore area and epicuticular waxes during micropropagation stages were studied by scanning electron microscopy (SEM) and morphometric analysis with the aim to improve the survival rate after transfer to greenhouse conditions. Leaves from in vitro shoots at the proliferation stage showed a spatular shape, ring-shaped stomata, a large number of glandular trichomes and juvenile covering hairs, but failed to show any epicuticular waxes. Leaves from in vitro plants at the root elongation stage showed a lanceolated elliptic shape with a serrated border, elliptical stomata, decreased pore area percentage, stomatal density, and mature covering trichomes. One week after transfer to ex vitro conditions, epicuticular waxes appeared on the leaf surface and stomata and pore area were smaller as compared to in vitro plants. Artichoke acclimatization may be improved by hormonal stimulation of root development, since useful morphological changes on leaves occurred during root elongation.     

Abaxial and adaxial stomata from Pima cotton (Gossypium barbadense L.) differ in their pigment content and sensitivity to light quality

Sensitivity to light quality and pigment composition were analysed and compared in abaxial and adaxial stomata of Gossypium barbadense L. (Pima cotton). In most plants, abaxial (lower) stomatal conductances are higher than adaxial (upper) ones, and stomatal opening is more sensitive to blue light than to red. In greenhouse-grown Pima cotton, abaxial stomatal conductances were two to three times higher than adaxial ones. In contrast, adaxial stomatal conductances were 1·5 to two times higher than abaxial ones in leaves from growth chamber-grown plants. To establish whether light quality was a factor in the regulation of the relationship between abaxial and adaxial stomatal conductances, growth-chamber-grown plants were exposed to solar radiation outdoors and to increased red light in the growth chamber. In both cases, the ratios of adaxial to abaxial stomatal conductance reverted to those typical of greenhouse plants. We investigated the hypothesis that adaxial stomata are more sensitive to blue light and abaxial stomata are more sensitive to red light. Measurements of stomatal apertures in mechanically isolated epidermal peels from growth chamber and greenhouse plants showed that adaxial stomata opened more under blue light than under red light, while abaxial stomata had the opposite response. Using HPLC, we quantified the chlorophylls and carotenoids extracted from isolated adaxial and abaxial guard cells. All pigments analysed were more abundant in the adaxial than in the abaxial guard cells. Antheraxanthin and β-carotene contents were 2·3 times higher in adaxial than in abaxial guard cells, comparing with ad/ab ratios of 1·5–1·9 for the other pigments. We conclude that adaxial and abaxial stomata from Pima cotton have a differential sensitivity to light quality and their distinct responses are correlated with different pigment content.     

ABNORMAL GUARD CELL DEVELOPMENT IN AN OLIVE NECROTIC MUTANT OF MAIZE

A study of a mutant variety of Zea mays (ON8147) revealed that the mutant plants, in contrast with normal maize plants, do not exhibit a light-induced increase in the rate of transpiration, and that the ontogeny of the stomatal complex is abnormal. In later stages of differentiation, the guard cells of mutant plants deteriorate, leaving the mature stomata with only the two subsidiary cells. The subsidiary cells in stomata of mutant leaves are similar to those of normal leaves with respect to their capacity to accumulate K⁺ in the dark, but they do not lose K⁺ in the light, as do subsidiary cells of stomata of nonmutant plants. It is suggested that impairment of guard cell function causes death of the mutant plant seedlings primarily by restricting CO₂ entry into the leaf.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.     

Stomatal characteristics during micropropagation of Wrightia tomentosa

A deviation from usually found characteristics of stomata in Wrightia tomentosa was noted during in vitro propagation. Increase in stomatal frequency in leaves of plants grown in vitro was observed with 29.4 % malformed stomata. The stomata were spherical, wide open, did not close in detached leaves even after 3 h. The leaves exhibited 93.4 % total water loss during 3-h period. Stomatal frequency, percentage of malformed stomata and rate of water loss declined in subsequent rooting phase. Nevertheless, for high survival rate plantlets were hardened under gradually decreasing air humidity either in partially opened glass bottles containing Soilrite™ moistened with 1/4 Murashige and Skoog nutrients or in pots covered with polyethylene bags. The stomatal characteristics of hardened plants were comparable to seedlings. Survival rate was more than 95 %.     

Use of Potassium and Sucrose by Onion Guard Cells during a Daily Cycle of Osmoregulation

Solute content of stomata from intact onion cotyledons grownunder either greenhouse or growth chamber conditions was followedover the course of a daily light cycle to determine patternsof osmoregulation. Initial opening of stomata was well correlatedwith guard cell potassium accumulation under both growth conditions.Subsequently, however, there was a consistent decrease in guardcell potassium content despite constant or increasing aperture.Although a secondary increase in potassium was sometimes observedduring the second half of the light cycle, guard cell potassiumcontent was poorly correlated with aperture. Sucrose levelsin guard cells increased 60% during the period of decliningpotassium content, suggesting its use as an alternate osmoticum.Guard cells are postulated to use multiple pathways for theproduction of osmotica over the course of a complete daily cycleof stomatal movements. (Received December 5, 1995; Accepted April 9, 1996)     

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.     

Effects of infection by Phytophthora injestans (Mont.) de Bary on the stomata of potato leaves

Phytophthora injestans infection of potato leaves causes abnormal opening of the stomata in the tissue colonized by the fungus before sporangiophores emerge through them. The affected area, which may be up to 7 mm wide, extends from the necrotic tissue to within 1–2 mm of the advancing edge of the colonizing mycelium. These stomata open wider than those in uninfected parts of the leaf, and do not close in the dark, but closure can be induced experimentally by a high water deficit. Affected guard cells have an increased osmotic value, reduced starch content, and show degradation of the chloro-plasts. No direct infection of the guard cells has been observed. Autoradiography of infected leaflets which had been exposed to ¹⁴CO₂ in the light, showed that a zone of increased photosynthesis occurs in the region of wide-open stomata.     

The relationship between guard cell water potential and the aperture of stomata in Populus

Abstract Previous work with clones of Populus trichocarpa demonstrated that the water vapour conductance of leaves from well-watered cuttings of this species does not decline with loss of turgor from the bulk leaf. In the present study, stomatal responses to water potential in Populus were examined with detached epidermal strips. Stomata in epidermal strips from well-watered plants of P. trichocarpa did not close at low water potentials which led to plasmolysis of the guard cells. In contrast, stomata of P. deltoides and a P. trichocarpa×deltoides hybrid closed when the guard cells lost turgor. A period of water stress preconditioning resulted in modified stomatal responses in P. trichocarpa such that stomata of stressed and re-watered plants nearly closed when guard cell turgor was lost.     

The function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.     

Effects of light, CO2 and humidity on carnation growth, hyperhydration and cuticular wax development in a mist reactor

Summary Plant survival ex vitro requires functioning stomata, adequate cuticular wax composition and deposition, and normal morphological development. Light intensity, CO₂ and relative humidity were altered inside an acoustic window mist reactor to study their effects on carnation (Dianthus caryophyllus) growth, stomata development, hyperhydration and epicuticular wax content. Increasing the light intensity from 65 to 145 μmol m⁻² s⁻¹ and enrichment of the gas phase with CO₂ (1350 ppm) reduced the number of hyperhydrated plants from 75 to 25% and increased the percentage dry weight of normal (healthy) plants from 17 to 25%. Lowering the relative humidity (≈70% RH) surrounding the plants during the mist-off phase for the last 2 wk of culture reduced the number of hyperhydrated plants from 70 to 9% and also increased the percentage of dry weight of normal plants from 16 to 25%. The stomata on plants grown in conditions of high light or low humidity had smaller apertures and appeared sunken when compared to stomata from plants grown in low light and high relative humidity. The epicuticular wax profiles of plants from the greenhouse or Magenta boxes showed a distinct shift in wax compounds with developmental age, plant type (hyperhydrated or normal), and type of box that was used (vented or not). In addition, very different wax profiles were observed from plants grown in reactors with altered CO₂ and light intensities.     

The dynamic changes of tonoplasts in guard cells are important for stomatal movement in Vicia faba

Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxy-succinyl-l-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement.     

Guard cells on adaxial and abaxial epidermes of <Emphasis Type="Italic">Erythrina corallodendron</Emphasis> sepals

This study investigated guard cells on the adaxial and the abaxial epidermes during Erythrina corallodendron sepal development. On the adaxial epidermis, the morphology of guard cells was highly variable and changes in aperture induced by abscisic acid (ABA) were observed in 9.1 % stomata, while on the abaxial epidermis 86.7 % stomata responded to ABA. On the adaxial epidermis, stomata did not close even when guard cell pressure potential was reduced to zero by plasmolysis, even if fluorescein diacetate revealed that guard cells were alive. It was concluded that guard cells on the adaxial and the abaxial epidermes of sepals sensed environmental conditions differently, maybe due to different guard cell wall elasticity.     

Studies on stomatal function, epicuticular wax and stem-root transition region of polyethylene glycol-treated and nontreatedin vitro grape plantlets

Summary Scanning electron microscopy, light microscopy, and gravimetric analysis was used to evaluate stomatal function, epicuticular wax, and the stem-root transition region of grape (Vitis sp. ‘Valiant’) plantlets grownin vitro, polyethylene glycoltreatedin vitro, and greenhouse-grown plants. Scanning electron microscopic studies of leaf surfaces ofin vitro-grown plants showed widely open stomata as compared to leaf stomata of polyethylene glycol-treatedin vitro-cultured and greenhouse-grown plants. Ultrastructurally, leaf epicuticular wax ofin vitro plants was less dense than in their polyethylene-treated and greenhouse counterparts. Quantitatively,in vitro-grown plants had reduced epicuticular was as compared to polyethylene glycol-treated and greenhouse-grown plants. Light microscopic studies showed no obvious differences in the vascular connections in the stem-root transition region ofin vitro-cultured, polyethylene glycol-treatedin vitro-cultured, and greenhouse-grown plants. It is therefore likely that the rapid wilting and desiccation observed after transplantingin vitro grape plantlets is due to their defective stomatal function and reduced epicuticular wax and may not be due to poor water transport associated with vascular connection.     

A STRUCTURAL AND FUNCTIONAL STUDY OF THE COORDINATED REACTIONS OF INDIVIDUAL COMMELINA COMMUNIS L. STOMATA (COMMELINACEAE)

Opening and closure movements of individual stomata were analyzed by light microscopy, scanning electron microscopy, and time-lapse photomicrography. Turgor-pressure-induced changes in pore shape of Commelina communis L. stomata were observed in vivo and in fixed material. The ventral wall of the guard cells undergoes three-dimensional alterations during opening and closing. Stomatal aperture increases with increase in light intensity and with decrease in CO₂ concentration as previously described, while reactions to relative humidity and anaerobiosis are somewhat divergent from common experience. Low humidity induces opening rather than closure of stomata in well watered greenhouse plants, and lack of oxygen induces consecutive opening and closure movements. Individual stomata characteristically open by three distinct steps: (1) rapid opening; (2) immobility or slow closure; (3) relatively rapid opening. Since the timing of the second step varies for individual stomata, it is obscured by methods that integrate the responses of several stomata. The movements of individual stomata are well synchronized, but active communication between stomata in different areas can not be confirmed.     

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

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