pH Gradients in the Stomatal Complex of Tradescantia virginiana

pH of the vacuolar sap, cytoplasm, and apoplast of the cellsof the stomatal complex of Tradescantia virginiana was measuredusing micro-electrodes. Marked differences in vacuolar and apoplasticpH were observed between leaves with open and closed stomata.Cytoplasmic pH appeared to be uniform in all the cells and didnot change with stomatal aperture. The information obtainedenabled proton motive force across the plasmalemma and tonoplastof the guard cells to be calculated. The results are discussedin relation to the accumulation of potassium by the guard cellon stomatal opening. Key words: Stomata, pH gradients, Proton pumps     

An Electrophysiological Study of the Stomatal Complex of Tradescantia virginiana

Measurements of electrical potential difference (PD) and potassiumactivity were carried out on the intact leaf of Tradescantiavirginiana. PD gradients across the stomatal complex were observedwith both open and closed stomata. The guard cell PD appearedto be linearly related to stomatal aperture. With the stomataopen a gradient of potassium activity across the stomatal complexwas observed which became reversed on stomatal closure. Calculationof the driving forces on potassium suggested that it was distributedpassively between the vacuoles of the cells of the stomatalcomplex. The electrophysiological data obtained from this investigationenabled potassium activity in the apoplast to be calculated.The results showed that on stomatal closure there was a massiveincrease in the potassium activity in the guard cell wall. Key words: Stomata, Ionic gradients, Electrical potentials     

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.     

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.     

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in <Emphasis Type="Italic">Pisum sativum</Emphasis>

Main conclusion

Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N’,N’-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

     

New fava bean guard cell signaling mutant impaired in ABA-induced stomatal closure

We isolated a mutant from Vicia faba L. cv. House Ryousai. Itwilts easily under strong light and high temperature conditions,suggesting that its stomatal movement may be disturbed. We determinedresponses of mutant guard cells to some environmental stimuli.Mutant guard cells demonstrated an impaired ability to respondto ABA in 0.1 mM CaCl₂ and stomata did not close in thepresence of up to 1 mM ABA, whereas wild-type stomata closedwhen exposed to 10 µM ABA. Elevating external Ca²⁺caused a similar degree of stomatal closure in the wild typeand the mutant. A high concentration of CO₂ (700 µlliter^–1) induced stomatal closure in the wild type, butnot in the mutant. On the basis of these results, we proposethe working hypothesis that the mutation occurs in the regiondownstream of CO₂ and ABA sensing and in the region upstreamof Ca²⁺ elevation. The mutant is named fia (fava bean impairedin ABA-induced stomatal closure). ³ Corresponding author: E-mail, smoiwai{at}agri.kagoshima-u.ac.jp;Fax, +81-99-285-8556.     

Functional Stomata in a Variegated Leaf Chimera of Pelargonium zonale L. without Guard Cell Chloroplasts

Fluorescence microscopy indicated that chlorophyll was absentfrom epidermal and guard cells overlying all white areas andgreen areas (of certain leaves) in variegated leaves of Pelargoniumzonale, cv. Chelsea Gem. Stomata with chlorophyll-free guardcells, in general, responded normally to light and CO₂ as gaugedby direct measurements of stomatal aperture and by transpirationalwater loss studies, although stomata from white regions of variegatedleaves were more reluctant to open than stomata from green regionsof the leaves. Thus, functional stomata without guard cell chloroplastshave been discovered in another genus, namely Pelargonium, besidesthat originally discovered in Paphiopedilum. When stomata withchlorophyll-free guard cells opened, K⁺ accumulated in the guardcells. This indicates that chloroplasts are not essential forthe normal functioning of stomata and that the energy sourcefor driving stomatal movements can come from sources other thanphotophosphorylation. Key words: Guard cell chloroplasts, Leaf chimera, Pelargonium, Stomata     

Jasmonates Induce Intracellular Alkalinization and Closure ofPaphiopedilumGuard Cells

Jasmonates (jasmonic acid or methyl jasmonate) promote stomatalclosure inPaphiopedilum Supersuk(RHS, 1973) andP. tonsum(Rchb.f)Stein. Studies on guard cells loaded with pH dependent fluorescentdyes show that jasmonates cause intracellular alkalinizationof up to 0.5 pH units within 5 to 15 min. Jasmonate-inducedalkalinization always preceded stomatal closure and where alkalinizationwas not detected no closure occurred. Propionic acid inhibitedjasmonate-induced stomatal closure, suggesting that jasmonate-inducedintracellular alkalinization is involved in guard cell movements. BCECF; confocal microscopy; cytosolic pH; guard cells; jasmonic acid; methyl jasmonate; Paphiopedilum Supersuk(R. H. S.); Snarf-1; stomatal movements     

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     

Properties of the Signal Transduction Pathway in the Blue Light Response of Stomatal Guard Cells of Vicia faba and Commelina benghalensis

Blue light-dependent proton extrusion in guard cell protoplastsfrom Vicia faba and light-dependent stomatal opening in theepidermis of Commelina benghalensis are inhibited by the calmodulin(CaM) antagonist, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfononamide(W-7) and the myosin light chain kinase (MLCK) inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-lH-hexahydro-1,4-diazepine (ML-7) [Shimazaki, K., Kinoshita, T.and Nishimura, M. (1992) Plant Physiol. 99: 1416]. We now suggestthat the inhibition occurs in the blue light signaling pathwaywithout affecting the proton pump. Addition of fusicoccin (FC),an activator of H⁺-ATPase, to the protoplasts and the epidermiswhose blue light-dependent proton extrusion and light-dependentstomatal opening had been inhibited by W-7 and ML-7, inducedboth proton extrusion and stomatal opening, respectively. Bluelight-dependent proton extrusion was inhibited by K-252a, awide-range inhibitor of protein kinases, and KT5926, a selectiveinhibitor of MLCK. FC induced proton extrusion in the presenceof K-252a and KT5926. In contrast, phenylmercuric acetate (PMA),carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) inhibited both the proton extrusion and stomatal opening,but FC did not induce the responses. These results suggest thatW-7, ML-7, K-252a and KT5926 inhibit the signal transductionprocess by which the perception of blue light is transducedinto activation of the proton pump in guard cells, and thatMLCK or MLCK-like protein is involved in the blue light responseof stomata. The possibility that calcium-dependent, calmodulinindependent protein kinase [Harper, J.F. et al. (1991) Science252: 951] functions rather than MLCK in the blue light responseof stomata should be noted, however. (Received July 23, 1993; Accepted September 30, 1993)     

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     

Responses of the stomata of Aster tripolium to calcium and sodium ions in relation to salinity tolerance

In previous work, the stomata of the maritime halophyte Astertripolium L. were shown to close when NaCl concentrations risein the vicinity of the guard cells. Further studies have nowrevealed important effects of calcium on the ionic responsesof the stomata. When the guard cells were presented with KCl,Ca²⁺ suppressed opening in a manner similar to that which hasbecome familiar in other species such as Commelina communisL. However, in the presence of NaCl, Ca²⁺ had the opposite effect,reducing the closing response to NaCl. This pattern of behaviouris discussed in relation to known salt effects on membranes,but the underlying physiological basis remains obscure. A previous study led to the hypothesis that the closing responseof the stomata to Na⁺ ions may make an important contributionto the salinity tolerance of this species. Here we report thatincreasing supplies of Ca²⁺ ions reduce the effect of salinityon stomatal conductance in the whole plant as well as in theisolated epidermis. This finding is consistent with the wellestablished role of calcium in increasing resistance to salinity:in the presence of high calcium the plant can tolerate a greatersalt intake, and hence there is a reduced need for transpirationto be restricted by partial stomatal closure. Key words: Sodium, calcium, Aster tripolium, stomata, salinity tolerance     

Acid-Induced Stomatal Opening in Commelina communis and Vicia faba

The effects of H^$ and fusicoccin (FC) on stomatal opening inthe dark were investigated using epidermal strips of Commelinacommunis and Vicia faba cv. Ryosai Issun. Citrate-phosphatebuffer induced maximal opening of stomata at pH 3.0 when testedover the range of 2.7 to 5.0. HCl at 1 mM also induced stomatalopening without appreciable accumulation of K^$ in the guardcells. After 4 hr treatment with 10 µM FC, stomata openedwith concomitant accumulation of K^$ in the guard cells, although1–2 hr treatment caused opening without concomitant K^$increase. These results suggest that stomatal opening can be caused bysalt accumulation and/or changes of the physicochemical conditionsin the cell wall of the guard cells due to high acidity. ¹ Present address: Biological Laboratory, Faculty of Education,Nagasaki University, Nagasski 852, Japan. (Received April 30, 1982; Accepted July 17, 1982)     

Rapid Structural Changes and Acidification of Guard Cell Vacuoles during Stomatal Closure Require Phosphatidylinositol 3,5-Bisphosphate

Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)–induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H⁺-ATPase vha-a2 vha-a3 and vacuolar H⁺-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P₂, which is essential for vacuolar acidification and convolution.     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

Potassium Involvement in Stomatal Movements of Paphiopedilum

There are conflicting reports about whether guard cells of Paphiopedilumspp. accumulate K during stomatal opening. In this study X-raymicroprobe analysis and histochemical staining for K indicatedthat K accumulated in guard cells in leaves of Paphiopedilumspp. when stomata opened. Additionally, stomatal opening inepidermal strips of P. harrisseanum could only be induced inan MES buffered KC1 medium. Element analysis ofP. harrisseanumepidermis also indicated substantial levels of K, Na and Cawithin the tissue. We conclude that K is involved in the stomatal movements ofPaphiopedilum. Key words: K⁺ transport, Paphiopedilum, Stomatal movements     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

An Unusual Feature of Stomatal Microanatomy in Certain Taxonomically- related Eucalyptus spp

The line of closure in the stoma in fully grown adult leavesof certain eucalypts is formed by special ridges of cuticlecalled ‘stomatal bars’ developed at the line ofclosure itself or from upgrowth of the cuticle of the lowersurface of the guard cells. Stomatal bars, previously discoveredin three members of the informal group ‘Bisectae’are shown to be restricted to certain species in that group.Possession of stomatal bars may affect stomatal performancebut does not appear to be a general adaptive response to thehabitats of the species which possess them. More probably, thepossession of stomatal bars is an inherited character with taxonomicvalue. The presence of stomatal bars in other genera is discussed. Eucalyptus spp, stomata, microanatomy, guard cells, stomatal bars