蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖,花粉萌发,特定基因表达等生理过程,气孔能灵敏地对外界刺激作出反应,快速开闭,本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素;外源纯化钙调素能促进气孔关闭,抑制气孔开放,最适浓度为10^-8mol/L;不能透过质膜的大分子钙调素拮抗剂W—-agarose和钙调素抗血清都能抑制气孔关闭,促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭,抑制开放。而且只能在细胞外起作用,推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用。     

蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖、花粉萌发、特定基因表达等生理过程.气孔能灵敏地对外界刺激作出反应,快速开闭.本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素.外源纯化钙调素能促进气孔关闭、抑制气孔开放,最适浓度为10-8mol/L;不能透过质膜的大分子钙调素拮抗剂W7-agarose和钙调素抗血清都能抑制气孔关闭、促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭、抑制开放,而且只能在细胞外起作用.推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用.     

Stomatal deregulation in Plasmopara viticola-infected grapevine leaves

In grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA) treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation.     

The mechanical diversity of stomata and its significance in gas-exchange control

Given that stomatal movement is ultimately a mechanical process and that stomata are morphologically and mechanically diverse, we explored the influence of stomatal mechanical diversity on leaf gas exchange and considered some of the constraints. Mechanical measurements were conducted on the guard cells of four different species exhibiting different stomatal morphologies, including three variants on the classical "kidney" form and one "dumb-bell" type; this information, together with gas-exchange measurements, was used to model and compare their respective operational characteristics. Based on evidence from scanning electron microscope images of cryo-sectioned leaves that were sampled under full sun and high humidity and from pressure probe measurements of the stomatal aperture versus guard cell turgor relationship at maximum and zero epidermal turgor, it was concluded that maximum stomatal apertures (and maximum leaf diffusive conductance) could not be obtained in at least one of the species (the grass Triticum aestivum) without a substantial reduction in subsidiary cell osmotic (and hence turgor) pressure during stomatal opening to overcome the large mechanical advantage of subsidiary cells. A mechanism for this is proposed, with a corollary being greatly accelerated stomatal opening and closure. Gas-exchange measurements on T. aestivum revealed the capability of very rapid stomatal movements, which may be explained by the unique morphology and mechanics of its dumb-bell-shaped stomata coupled with "see-sawing" of osmotic and turgor pressure between guard and subsidiary cells during stomatal opening or closure. Such properties might underlie the success of grasses.     

Responses of Stomata to IAA and Fusicoccin at the Opposite Phases of an Entrained Rhythm

The stomata of Commelma communis showed reduced opening responsesto light and low CO₂ concentrations during the night phase oftheir entrained circadian rhythm. Increased supplies of potassiumions, and treatments with indol-3-ylacetic acid and fusicoccin,failed to promote opening during the night phase to a levelequivalent to that in the day phase. The inability of fusiccocinto overcome the suppression of opening during the night phasecontrasts with its ability to counteract the closure inducedby agents such as CO₂, darkness and abscisic acid. It is concludedthat there are at least two basic mechanisms by which the turgorof guard cells can be regulated, one which is susceptible tooverriding control by fusicoccin and another which is unaffectedby fusicoccin. Several previous studies had shown a positive correlation betweenmalate in the epidermis (mainly located in guard cells) andstomatal opening. In the present experiments the aperture/malatecorrelation was broken in epidermis treated with fusicoccinduring the night phase of the rhythm. The amount of malate presentexceeded that associated with the same stomatal aperture inthe day phase. Possible explanations are (1) that fusicoccinstimulates similar proton fluxes out of the guard cells duringboth phases of the rhythm, but an unknown factor imposes a restrictionon stomatal opening during the night phase; (2) that there arelower proton fluxes in the night phase (limited, for example,by a reduced supply of ATP) but chloride availability or transportis reduced to an even greater extent so that a larger productionof malate in the guard cells is required. Key words: Stomata, IAA, Fusicoccin, Rhythms     

The stomata frontline of plant interaction with the environment-perspectives from hormone regulation

Plants have evolved elaborate mechanisms to perceive and integrate signals from various environmental conditions.On leaf surface,stomata formed by pairs of guard cells mediate gas exchange,water transp...     

Direct Measurements of Turgor Pressure Potentials of Guard Cells, I.

Measurements were made of pressures applied in subsidiary andguard cells which caused closure and opening of stomatal pores.These pressures were lower than would be expected from plasmolyticallydetermined osmotic potentials of guard cell saps. The pressuresneeded in guard cells to open almost closed stomata were practicallythe same as those required to close open stomata when appliedin adjacent subsidiary cells. Completely closed stomata couldnot be opened by this technique. The implications for the understandingof the mechanism of guard cell deformation, the Spannungsphase,and the pressure potentials of guard cells are discussed.     

Roles of ion channels and transporters in guard cell signal transduction

Stomatal complexes consist of pairs of guard cells and the pore they enclose. Reversible changes in guard cell volume alter the aperture of the pore and provide the major regulatory mechanism for control of gas exchange between the plant and the environment. Stomatal movement is facilitated by the activity of ion channels and ion transporters found in the plasma membrane and vacuolar membrane of guard cells. Progress in recent years has elucidated the molecular identities of many guard cell transport proteins, and described their modulation by various cellular signal transduction components during stomatal opening and closure prompted by environmental and endogenous stimuli.     

Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in Arabidopsis

CaM ubiquitously presents inside eukaryotic cells. CaM抯 gene expression and its subcellular localization are regulated by light, osmotic stress, pathogens, plant hormones, etc.[1]. Intracellular CaM of plant displays important functions in pathogenesis and wounding reaction[2] and hypersensitive response[3]. CaM has been found extracellular spaces in many plant species, such as soluble extracts of oat coleoptile cell walls[4], the wheat coleoptile cell walls[5], maize root tips cell walls[6…     

Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels

Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K(+) extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K(+) salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.     

Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Extracellular calmodulin(CaM)plays significant roles in many physiological processes,but little is known about its mechanism of regulating stomatal movements.In this paper,whether CaM exists in the guard cell walls of Arabidopsis and whether depolymerization of actin cytoskeleton is involved in extracellular CaM-induced stomatal closing are investigated.It is found that CaM exists in guard cell walls of Arabidopsis,and its molecular weight is about 17 kD.Bioassay using CaM antagonists W7-agarose and anti-CaM serum shows that the endogenous extracellular CaM promotes stomatal closure and delays stomatal opening.The long radial actin filaments in guard cells undergo disruption in a time-dependent manner during exogenous CaM-induced stomatal closing.Pharmacological experiments show that depolymerization of actin cytoskeleton enhances the effect of exogenous CaM-induced stomatal closing and polymerization reduces the effect.We also find that exogenous CaM triggers an increase in [Ca2+]cyt of guard cells.If [Ca2+]cyt increase is blocked with EGTA,exogenous CaM-induced stomatal closure is inhibited.These results indicate that extracellular CaM causes elevation of [Ca2+]cyt in guard cells,subsequently resulting in disruption of actin filaments and finally leading to guard cells closure.     

An analysis of the mechanics of guard cell motion

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

Potassium Loss from Stomatal Guard Cells at Low Water Potentials

The potassium content of guard cells and the resistance to viscousflow of air through the leaf were determined in sunflower (Helianthusannuus) subjected to low leaf water potentials under illuminatedconditions. In intact plants desiccated slowly by withholdingwater from the soil, large losses in guard cell K occurred asleaf water potentials decreased. Leaf viscous resistance increased,indicating stomatal closure. Similar results were obtained whendetached leaf segments were desiccated rapidly. Upon rehydrationof leaves, no stomatal opening was observed initially, despiteleaf water potentials at predesiccated levels. After severalhours, however, re-entry of K occurred and stomata became fullyopen. Turgid leaf segments floated on an ABA solution showedlosses of guard cell K and closure of stomata as rapidly andcompletely as those brought about by desiccation. It is concludedthat stomatal closure at low water potentials under illuminatedconditions is not controlled solely by water loss from the tissuebut involves the loss of osmoticum from the guard cells as well.This in turn decreases the turgor difference between the guardcells and the surrounding cells, and closing occurs.     

Anion-Channel Blockers Inhibit S-Type Anion Channels and Abscisic Acid Responses in Guard Cells

The effects of anion-channel blockers on light-mediated stomatal opening, on the potassium dependence of stomatal opening, on stomatal responses to abscisic acid (ABA), and on current through slow anion channels in the plasma membrane of guard cells were investigated. The anion-channel blockers anthracene-9-carboxylic acid (9-AC) and niflumic acid blocked current through slow anion channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid reversed ABA inhibition of stomatal opening in V. faba L. and Commelina communis L. The anion-channel blocker probenecid also abolished ABA inhibition of stomatal opening in both species. Additional tests of 9-AC effects on stomatal aperture in Commelina revealed that application of this anion-channel blocker allowed wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100 mM) KCI conditions. These results indicate that anion channels can function as a negative regulator of stomatal opening, presumably by allowing anion efflux and depolarization, which prohibits ion up-take in guard cells. Furthermore, 9-AC prevented ABA induction of stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced stomatal closure and inhibition of stomatal opening is discussed.     

Studies of the mechanism of action of fusicoccin,the fungal toxin that induces wilting,and its interaction with abscisic acid

Summary Effects of fusicoccin alone and together with abscisic acid were observed on the stomatal complex of Commelina communis. The experimental material consisted of isolated epidermal strips incubated in a medium containing the ions required for stomatal opening. Fusicoccin stimulated opening and this was accompanied by potassium entry into the guard cells, and hydrolysis of the starch in their chloroplasts. Abscisic acid alone inhibited potassium entry and starch hydrolysis, but these effects could be almost entirely overcome by fusicoccin.Attempts were made to measure the solute potential of the guard cells under the various treatments. Abscisic acid clearly increased their solute potential, but no absolute measurements could be made in the presence of fusicoccin owing to a failure of plasmolysis even with mannitol solutions of solute potential as low as —35 bars. Experiments using isotopically labelled mannitol indicated a massive uptake into the epidermis in the presence of fusicoccin.The mechanism of stimulation of stomatal opening by fusicoccin probably depends in part on a stimulation of the normal processes associated with opening in the guard cells, but may also involve release of pressure due to destruction of the surrounding cells. The effectiveness of this toxin under natural conditions may depend on its ability to counteract effects of abscisic acid, the stress hormone that induces stomatal closure.     

Green light reversal of blue-light-stimulated stomatal opening is found in a diversity of plant species

Reversal by green light of blue-light-stimulated stomatal opening was found across a number of plant species, including leguminous and nonleguminous dicots and grass and nongrass monocots. Simultaneous exposure to equal fluence rates of blue and green light resulted in ～50% reversal of normal blue light opening. Complete reversal occurred when the fluence rate of green light was approximately twice that of blue light. These results suggest that blue-green reversibility of stomatal opening is a basic photobiological property of guard cells. The blue-green reversibility of stomatal opening has been hypothesized to ensue from the cycling of two interconvertible, isomeric forms of the blue-light photoreceptor, zeaxanthin. Testing of blue-green reversibility could provide a valuable diagnostic tool for zeaxanthin-mediated blue-light photoperception.     

The evolution,structure and functioning of stomata

Summary

The anatomy and ultrastructure of guard cells from a range of species varying from the primitive types, such as mosses, to the advanced grasses and orchids are described. An attempt is made to trace the lines along which stomata developed and to define what might be considered advanced stomata. Additionally, the differentiation of guard cells from guard mother cells is discussed. Of particular note is the preprophase band of microtubules which marks the zone where the future cell wall will form and the movement of the spindle and developing cell plate through 45 degrees. The structure and function of guard cells are intimately linked. Stomata are turgor regulated valves; the osmotica for absorbing water during opening are K⁺, Cl^- and malate anions which accumulate in the guard cell vacuoles. Upon stomatal closure, K⁺ and Cl^- exit from the guard cells while at least some of the carbon from malate is channelled into starch and there is a resultant loss of guard cell turgor. The Calvin cycle may be absent or of low activity in guard cell chloroplasts and under those circumstances a source of carbon and energy to sustain the guard cells is needed. Hence it is believed that sucrose is transported into the guard cells from mesophyll cells. A brief consideration of the mechanism by which the ions are transported across the plasma membrane and tonoplast is made: the driving force for the K⁺, Cl^- and malate movement across the membranes is the proton motive force set up by proton-pumping ATPases.     

An analytical model of the hydraulic aspects of stomatal dynamics

An analytical model of the hydraulic aspects of stomatal dynamics is formulated in this paper. The model consists of a coupled system of non-linear, ordinary differential equations, written in terms of water potentials, hydrostatic pressures, osmotic potentials, water vapor resistances and water fluxes. The model is validated by comparisons with the experimental literature. Numerical solutions of the model show qualitative agreement with most known stomatal responses.Stomatal opening in the model is dependent on the interaction of the guard and subsidiary cells in the following manner. Pore opening is initiated by a rise in the guard cell hydrostatic pressure. As the stomate opens, transpiration increases, causing the cell wall water potential to drop. The drop in cell wall water potential then causes the subsidiary cell pressure to drop, opening is accelerated, and the stomate literally “pops” open. Simulated opening proceeds in two distinct phases: a stress phase and a motor phase. During the stress phase, guard cell pressure rises but the pore remains closed. The motor phase commences when the guard cell pressure has risen sufficiently to initiate pore opening, beyond which point opening progresses rapidly.Hydropassive stomatal movements are found to be insufficient to regulate water loss at low leaf water potentials. Stable, hydraulically-based oscillations in stomatal aperture are shown in the model by the existence of a stable limit cycle. The period of these oscillations is strongly influenced by the cell membrane hydraulic conductivity. An increased conductivity results in a shorter period oscillation. Environmental conditions promoting oscillatory behavior are in qualitative agreement with the experimental literature.     

ACTION OF FUSICOCCIN ON THE POTASSIUM BALANCE OF GUARD CELLS OF PHASEOLUS VULGARIS

Fusicoccin induces stomatal opening in both the light and dark. The stomatal aperture and K content of guard cells was measured to determine whether the action of fusicoccin in inducing stomatal opening is directly related to the uptake of K by the guard cells. Both detached and attached epidermis was treated with fusicoccin and the K content was determined by staining with cobalt sodium nitrite or by electron probe microanalysis. The K content of guard cells in detached epidermal strips floated on 10 μm fusicoccin in 10 mm KCl and aqueous CH₃OH (0.02%, v/v) increased in the light and dark as the stomata opened. After exposure to fusicoccin for 6 hr in the light, however, the stomata were closed and no K could be detected in the guard cells. The K content of guard cells of attached epidermis painted with fusicoccin also increased as the stomata opened, but the concentration of K in the subsidiary cells was not significantly altered by fusicoccin-stimulated opening. Moreover, painting with fusicoccin did not significantly change the Ca and P content of the guard or subsidiary cells. Stomata of epidermal strips, opened to their maximum width by fusicoccin, showed only a small and temporary closure when transferred to a solution of 10 μm abscisic acid. The use of metabolic inhibitors suggested that energy for the uptake of the K may be provided by both photophosphorylation and oxidative phosphorylation.