Evidence for an Extracellular Reception Site for Abscisic Acid in Commelina Guard Cells

The phytohormone abscisic acid (ABA) triggers stomatal closing as a physiological response to drought stress. Several basic questions limit an understanding of the mechanism of ABA reception in guard cells. Whether primary ABA receptors are located on the extracellular side of the plasma membrane, within the intracellular space of guard cells, or both remains unknown. Furthermore, it is not clear whether ABA must be transported into guard cells to exert control over stomatal movements. In the present study, a combination of microinjection into guard cells and physiological assays of stomatal movements have been performed to determine primary sites of ABA reception in guard cells. Microinjection of ABA into guard cells of Commelina communis L. resulted in injected cytosolic concentrations of 50 to 200 [mu]M ABA and in additional experiments in lower concentrations of approximately 1 [mu]M ABA. Stomata with ABA-loaded guard cells (n > 180) showed opening similar to stomata with uninjected guard cells. The viability of guard cells following ABA injection was demonstrated by neutral red staining as well as monitoring of stomatal opening. Extracellular application of 10 [mu]M ABA inhibited stomatal opening by 98% at pH 6.15 and by 57% at pH 8.0. The pH dependence of extracellular ABA action may suggest a contribution of an intracellular ABA receptor to stomatal regulation. The findings presented here show that intracellular ABA alone does not suffice to inhibit stomatal opening under the imposed conditions. Furthermore, these data provide evidence that a reception site for ABA-mediated inhibition of stomatal opening is on the extracellular side of the plasma membrane of guard cells.     

ABA regulation of K(+)-permeable channels in maize subsidiary cells

An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K(+) ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K(+) uptake and K(+) release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K(+) channels. ABA caused a pronounced rise in time-dependent outward-rectifying K(+) currents (K(out)) at alkaline pH and decreased inward-rectifying K(+) currents (K(in)) in a Ca(2+)-dependent manner. Our results show that the ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K(+) transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K(+) channels.     

狭基巢蕨叶表皮的结构和气孔器发育的观察

狭基巢蕨Ｎｅｏｔｏｐｔｅｒｉｓａｎｔｒｏｐｈｙｏｉｄｅｓ（Ｃｈｒｉｓｔ）Ｃｈｉｎｇ叶片的上表皮无气孔器,仅具表皮细胞,下表皮由表皮细胞和气孔器组成,气孔指数为２．５。上下表皮细胞和气孔器的细胞中均含有叶绿体。每个气孔器由２个肾形的保卫细胞和２～６个副卫细胞组成,其中以３个和４个副卫细胞的占绝大多数（３细胞的占４５．１％,４细胞的占４３．５％）。从发育上看,气孔器原始细胞进行２次分裂,产生２个保卫细胞和１个同源的副卫细胞。气孔器的发育过程大体可分为４个时期：（１）气孔器原始细胞的分化和分裂期;（２）保卫细胞母细胞成熟期;（３）保卫细胞母细胞分裂和气孔器幼期;（４）气孔器成熟期。狭基巢蕨的气孔器属于中周型     

Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum

The role of nitric oxide (NO) during bicarbonate-induced stomatal closure was studied in the abaxial epidermis of Pisum sativum . A few experiments were done with 10 μ M ABA, for comparison. The presence of 2 m M sodium bicarbonate or 10 μ M ABA induced an increase of NO in guard cells. Elevation of NO by sodium nitroprusside induced stomatal closure and enhanced further the closure by bicarbonate. The bicarbonate induced increase in NO of guard cells, or stomatal closure was prevented partially by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide, an NO scavenger and N -nitro- l -Arg-methyl ester, an inhibitor of NO synthase (NOS). These results suggested that guard cells generated NO on exposure to bicarbonate and that NOS was involved at least partially in such NO production. Time course experiments revealed that on exposure to bicarbonate or ABA, the rise in guard cell NO production peaked within 10 min. Experiments using pharmacological compounds like wortmannin/LY294002 (phosphatidylinositol 3 kinase inhibitors), 1 H -(1,2,4)-oxadiazole-[4,3 a ]quinoxalin-1-one (guanylyl cyclase inhibitor), nicotinamide (cyclic adenosine diphosphate ribose antagonist), guanosine 5'-O-(2-thiodiphosphate) (G-protein antagonist) suggested a role of phosphatidylinositol 3-phosphate or G-proteins during bicarbonate-induced stomatal closure.     

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.     

Effects of external potassium supply on stomatal closure induced by abscisic acid

Abstract Epidermal strips of Commelina communis with ‘isolated’ stomata were incubated on Trizma-maleate buffer containing 0-500 mM KCL, with or without 10^?4 M ABA, for 2.5 h. The resulting stomatal apertures indicate that there is no absolute requirement for live epidermal and subsidiary cells for ABA-mediated closure. This implies that ABA has a direct effect on influx or efflux of K⁺ into or out of the guard cells rather than on uptake of K⁺ by the subsidiary cells. The possible in vivo role of subsidiary cells in stomatal closure is discussed.     

Effects of abscisic acid on potassium uptake and starch content of stomatal guard cells

Summary Abscisic acid (ABA) at a concentration of 100 m reduced the mean stomatal aperture on isolated epidermis of Commelina communis from 9.5 to 3.1 m. This closure resulted from a fall in osmotic pressure of the guard cells from 14.1 to 9.8 bars; the osmotic pressure of the subsidiary cells did not change significantly. Histochemical tests showed that the potassium concentration in guard cells was reduced by ABA-treatment, while the starch content of the chloroplasts increased. ABA was found to exert a significant effect on Rb⁸⁶ uptake into leaf discs, but this was relatively small in magnitude. It is concluded that ABA has a greater effect on ion uptake into guard cells than into the leaf tissues as a whole.Recent hypotheses of the stomatal mechanism are discussed in relation to these new observations, and the rejection by some writers of any major role for starch hydrolysis is challenged. Evidence from several sources suggests that starch disappearance occurs simultaneously with K⁺ entry into guard cells. Breakdown of starch may lead to formation of organic anions, with which K⁺ uptake may be associated. In this case starch breakdown would contribute as much to the increased osmotic pressure as does K⁺ uptake.     

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.     

The Effects of Temperature and ABA on Stomata of Zea mays L.

Epidermal fragments were removed from maize leaves by tearingparallel to the veins. These were incubated at a number of differenttemperatures in several concentrations of ABA. The sensitivityof stomata to temperature was dependent upon the technique usedto incubate epidermis. Generally, the widest apertures wererecorded at around 25°C. In all experiments, stomata incubatedat low (10°C) temperatures on 5.6 x 10^–4 M ABA showedwider apertures than those incubated on distilled water. ThisABA-stimulated stomatal opening was accompanied by an increasein the intensity of potassium staining in the guard cells. At25 °C, epidermis incubated on several concentrations ofABA showed some stomatal closure, decreased potassium stainingin the guard cells and increased potassium staining in the subsidiarycells.     

脱落酸诱导气孔关闭的信号转导研究

气孔保卫细胞是单个细胞水平研究ABA信号转导机制的一个模式系统。脱落酸(ABA)通过对保卫细胞生理生化状态、胞质Ca^2 浓度及其离子通道调节诱导气孔关闭过程。这个过程涉及的因素有：ROS、IP3、cADPR、蛋白质的可逆磷酸化等。     

The Effect of Osmotic Stress on the Solutes in Guard Cells of Vicia faba L.

We investigated the changes in the levels of solutes in guardcells under osmotic stress. Epidermal strips peeled from Viciafaba L. leaflets were sonicated and incubated in 0.4 M mannitolsolution (osmotic stress) in either light or dark. Stomata wereclosed by osmotic stress. Under osmotic stress, malate accumulatedlight-dependently and sucrose accumulated light-independentlyin the guard cells. The level of K⁺ in guard cells increasedslightly under osmotic stress in the light, although withoutstatistical significance. The levels of all these solutes werereduced by 10 µM ABA treatment. These results suggestthat osmotic stress affects carbon metabolism in guard cells;this metabolic change is different from that caused by ABA alone.Respiratory activity of guard cells decreased under osmoticstress. Therefore, the accumulation of malate and sucrose maybe caused by reduced respiration under osmotic stress. Accumulationof solutes in guard cells by osmotic stress may result in increasedosmotic pressure of guard cells and may play a role in protectionof guard cells from osmotic stress. (Received December 17, 1998; Accepted May 28, 1999)     

Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure

Petioles of water‐sufficient intact Vicia faba L. plants were infused with 1 µm abscisic acid (ABA) to simulate the import of root‐source ABA. This protocol permitted quantitative ABA delivery, up to 300 pmol ABA over 60 min, to the leaf without ambiguities associated with perturbations in plant–water status. The ABA concentrations in whole‐leaf samples and in apoplastic sap increased with the amount infused; ABA degradation was not detected. The ABA concentration in apoplastic sap was consistent with uptake of imported ABA into the leaf symplast, but this interpretation is qualified. Our focus was quantitative cellular compartmentation of imported ABA in guard cells. Unlike when leaves are stressed, the guard‐cell symplast ABA content did not increase because of ABA infusion (P = 0·48; 3·0 ± 0·5 versus 4·0 ± 1·2 fg guard‐cell‐pair^?1). However, the guard‐cell apoplast ABA content increased linearly (R² = 0·98) from ?0·2 ± 0·5 to 3·1 ± 1·3 fg guard‐cell‐pair^?1 (≈ 3·1 µm ) and was inversely related to leaf conductance (R² = 0·82). Apparently, xylem ABA accumulates in the guard‐cell wall as a result of evaporation of the apoplast solution. This mechanism provides for integrating transpiration rate and ABA concentration in the xylem solution.     

Abscisic Acid Accumulation by in Situ and Isolated Guard Cells of Pisum sativum L. and Vicia faba L. in Relation to Water Stress

Isolated guard cells, prepared by sonication of epidermal peels, were used to investigate the endogenous level of abscisic acid (ABA) in the guard cells of turgid and stressed leaves of Vicia faba L. and the argenteum (arg) mutant of Pisum sativum L. The guard cells of V. faba and arg were found to contain 18 and 8 times more ABA, respectively, when isolated from stressed leaves than from turgid leaves. Isolated guard cells of V. faba were also directly stressed with the osmoticum Aquacide III. These guard cells were capable of producing stress-induced ABA to at least 3 times their ABA level when non-stressed.     

海南莎草属植物叶下表皮微形态的研究

利用光学显微镜对海南莎草属(Cyperus Linnaeus)15个种1个变种的叶下表皮微形态进行了研究。结果显示:(1)该属叶下表皮微形态基本特征如下:长细胞为长筒形、短筒形,少数为近方形,边缘波状、深波状;无短细胞的存在;副卫细胞为高圆屋顶形、圆屋顶形、圆屋顶至三角形或三角形;(2)根据保卫细胞的形态将海南莎草属分为两大类型即保卫细胞两端明显加厚型和保卫细胞两端不明显加厚型;(3)海南莎草属叶下表皮微形态一致,表明莎草属是一个自然类群,但在莎草属种间叶下表皮的长细胞形态、保卫细胞及副卫细胞形态、气孔长宽等存在一定的差异,可作为本属种间分类鉴别的参考依据。     

Metabolic Inhibitors Block ABA-Induced Stomatal Closure

Closure of stomata of Commelina communis L. leaf epidermis causedby abscisic acid (ABA) was inhibited by sodium azide, potassiumcyanide and hypoxic conditions. Azide was more effective thancyanide at low concentrations, but the cyanide effect couldbe enhanced by addition of salicylhydroxamic acid, providingindirect evidence for cyanide-resistant respiration in epidermaltissue. Azide also inhibited ABA-induced closure of ‘isolated’ stomata and shrinkage of guard cell protoplasts.The results indicate that metabolic energy is required for ABAaction involving solute loss from the guard cells. Possiblemechanisms of action are discussed.     

Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging

Guard cells, which form stomata on the leaf epidermis, play important roles in plant gas exchange and defense against pathogens. Abscisic acid (ABA) is a phytohormone that can be induced by drought and leads to stomatal closure. Guard cells have been a premier model system for studying ABA signal transduction. Despite significant progress on the identification of molecular components in the ABA signaling pathway, our knowledge of the protein components is very limited. Here, we employ a recently developed multiplexed isobaric tagging technology to identify ABA-responsive proteins in Brassica napus guard cells. A total of 431 unique proteins were identified with relative quantitative information in control and ABA-treated samples. Proteins involved in stress and defense constituted a major group among the 66 proteins with increased abundance. Thirty-eight proteins were decreased in abundance and fell into several functional groups including metabolism and protein synthesis. Many of the proteins have not been reported as being ABA responsive or involved in stomatal movement. A large percentage of the protein-coding genes contained ABA-responsive elements. This study not only established a comprehensive inventory of ABA-responsive proteins, but also identified new proteins for further investigation of their functions in guard cell ABA signaling.     

Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells

Blue light (BL) receptor phototropins activate the plasma membrane H(+)-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H?-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H?-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h. Here, we show that BL-induced phosphorylation of guard cell H?-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H?-ATPase. BL-induced phosphorylation of the H?-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H?-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H?-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H?-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.     

水分胁迫下ABA由蚕豆根向地上部的运输及其在叶片组织中的分布

蚕豆根装载的３Ｈ－ＡＢＡ可经５．６ｃｍ／ｍｉｎ以上的速率向冠部运输。短时间内（５ｍｉｎ）根运来的ＡＢＡ主要分布在有大量气孔密布的下表皮,但长时间内（３ｈ）则主要分布在对内组织中。抑制蒸腾可降低ＡＢＡ向叶片中的运输积累。光镜放射自显影术显示,根运来的ＡＢＡ可有效地在表皮细胞及保卫细胞的质外体积累。３Ｈ－ＡＢＡ由根向地上部快速运输及其在作用部位的有效积累,说明水分胁迫下蚕豆根部可以通过ＡＢＡ信号的传递控制气孔的行为。