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     

Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L.

This study investigated the interaction of NaCl-salinity andelevated atmospheric CO₂ concentration on gas exchange, leafpigment composition, and leaf ultrastructure of the potentialcash crop halophyte Aster tripolium. The plants were irrigatedwith five different salinity levels (0, 25, 50, 75, 100% seawatersalinity) under ambient and elevated (520 ppm) CO₂. Under salineconditions (ambient CO₂) stomatal and mesophyll resistance increased,leading to a significant decrease in photosynthesis and wateruse efficiency (WUE) and to an increase in oxidative stress.The latter was indicated by dilations of the thylakoid membranesand an increase in superoxide dismutase (SOD) activity. Oxidativestress could be counteracted by thicker epidermal cell wallsof the leaves, a thicker cuticle, a reduced chlorophyll content,an increase in the chlorophyll a/b ratio and a transient declineof the photosynthetic efficiency. Elevated CO₂ led to a significantincrease in photosynthesis and WUE. The improved water and energysupply was used to increase the investment in mechanisms reducingwater loss and oxidative stress (thicker cell walls and cuticles,a higher chlorophyll and carotenoid content, higher SOD activity),resulting in more intact thylakoids. As these mechanisms canimprove survival under salinity, A. tripolium seems to be apromising cash crop halophyte which can help in desalinizingand reclaiming degraded land. Key words: Aster tripolium, cash crop halophyte, elevated CO₂, gas exchange, oxidative stress, photosynthesis, salt tolerance, ultrastructure, water use efficiency Received 29 July 2008; Revised 8 October 2008 Accepted 9 October 2008     

The Circadian Rhythm of Stomatal Opening: Evidence for the Involvement of Potassium and Chloride Fluxes

Epidermal strips were taken from plants of Commelina communisat opposite phases of an entrained circadian rhythm, at timeswhich corresponded to the middle of the day phase and the middleof the night phase. Earlier observations of much reduced openingin response to external stimuli in the night phase were confirmed. Evidence is now presented that uptake or retention of specificions by the guard cells is affected by the phase of the rhythm.The overt expression of the rhythm was reduced if the epidermiswas presented with potassium iminodiacetate instead of potassiumchloride. Iminodiacetate is a non-absorbable anion, and thisresult suggested that chloride uptake is important for the fullexpression of the rhythm. The use of an anion channel inhibitor(DIDS) gave strong support for this conclusion. Reduced uptakeof chloride would explain an earlier observation that formationof malate is greater during the night phase. When the guard cells were presented with sodium chloride insteadof potassium chloride there was still an overt expression ofthe rhythm, but this was of reduced magnitude. This suggeststhat potassium movements might contribute to the rhythm, andstudies of ionic fluxes using ⁸⁶Rb as a tracer showed greatereffluxes during the night phase. It is concluded that the circadian rhythm in stomatal openingmay be the result of a varying ability of the guard cells toaccumulate or retain both chloride and potassium ions. Key words: Circadian rhythm, stomatal opening, Commelina communis, potassium and chloride fluxes     

Stomatal responses to sodium ions in Aster tripolium: a new hypothesis to explain salinity regulation in above-ground tissues

A study has been made of the ionic relations of stomata of Aster tripolium L., a maritime halophyte which colonizes coastal saltmarshes. The results obtained allow us to add this species to the growing list for which an involvement of K⁺ transport in stomatal movements has been demonstrated. However, an additional and ecologically important characteristic was found: there was a suppression of stomatal opening by increasing NaCl concentrations. A new hypothesis is offered of the mechanism for controlling salt and water relations in A. tripolium, a species which does not possess glands or other means of excreting salt. It is suggested that when the capacity of the tissues to accumulate salt in cell vacuoles is exceeded, the concentration of Na⁺ ions in the apoplast around the guard cells begins to rise. This causes partial stomatal closure, reduces transpiration and increases water-use-efficiency. Therefore, the flow of salt into the leaves is reduced but growth (and the manufacture of the new photosynthates required to support it) can continue. Aster tripolium can be added to the small list of known species which readily yield isolated epidermis suitable for detailed stomatal studies. Throughout this study, we have compared its stomatal physiology with C. communis, which has been thoroughly investigated in the past.     

Guard cell cation channels are involved in Na+–induced stomatal closure in a halophyte

The halophyte Aster tripolium, unlike well-studied non-halophytic species, partially closes its stomata in response to high Na⁺ concentrations. Since A. tripolium possesses no specific morphological adaptation to salinity, this stomatal response, preventing excessive accumulation of Na⁺ within the shoot via control of the transpiration rate, is probably a principal feature of its salt tolerance within the shoot. The ionic basis of the stomatal response to Na⁺ was studied in guard cell protoplasts from A. tripolium and from a non-halophytic relative, Aster amellus, which exhibits classical stomatal opening on Na⁺. Patch-clamp studies revealed that plasma membrane K⁺ channels (inward and outward rectifiers) of the halophytic and the non-halophytic species are highly selective for K⁺ against Na⁺, and are very similar with respect to unitary conductance and direct sensitivity to Na⁺. On the other hand, both species possess a significant permeability to Na⁺ through non-rectifying cation channels activated by low (physiological) external Ca²⁺ concentrations. Finally, it appeared that the differential stomatal response between the two species is achieved, at least in part, by a Na⁺-sensing system in the halophyte which downregulates K⁺ uptake. Thus, increases in guard cell cytosolic Na⁺ concentration in A. tripolium but not in A. amellus, lead to a delayed (20–30 min) and dramatic deactivation of the K⁺ inward rectifier. This deactivation is probably mediated by an increase in cytosolic Ca²⁺ since buffering it abolishes the response. The possible role of K⁺ inward rectifiers in the response of A. tripolium’s stomata to Na⁺, suggested by patch-clamp studies, was confirmed by experiments demonstrating that specific blockade of inward rectifying channels mimics Na⁺ effects on stomatal aperture, and renders aperture refractory to Na⁺.     

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     

Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis

There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch–sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance.     

Microanalytical Studies of Potassium and Chloride Fluxes and Stomatal Movements of Two Species: Vicia faba and Pelargonium x hortorum

Potassium and chloride exchange kinetics in guard cells werefound to be related to stomatal opening and closing kineticsin Pelargonium x hortorum and Vicia faba by simultaneously measuringion contents and degrees of opening with an electron probe anda hydrogen diffusion porometer. Large shifts between both kinetics occur during opening andclosing. These results show that maximal potassium content isdelayed, which seems to question the primary role of potassiumin stomatal opening     

Elevated carbon dioxide affects the patterning of subsidiary cells in Tradescantia stomatal complexes

The influence of elevated CO₂ concentration (670 ppm) on thestructure, distribution, and patterning of stomata in Tradescantialeaves was studied by making comparisons with plants grown atambient CO₂. Extra subsidiary cells, beyond the normal complementof four per stoma, were associated with nearly half the stomatalcomplexes on leaves grown in elevated CO₂. The extra cells sharedcharacteristics, such as pigmentation and expansion, with thetypical subsidiary cells. The position and shape of the extrasubsidiary cells in face view differed in the green and purplevarieties of Tradescantia. Substomatal cavities of complexeswith extra subsidiary cells appeared larger than those foundin control leaves. Stomatal frequency expressed on the basisof leaf area did not differ from the control. Stomatal frequencybased on cell counts (stomatal index) was greater in leavesgrown in CO₂-enriched air when all subsidiary cells were countedas part of the stomatal complex. This difference was eliminatedwhen subsidiary cells were included in the count of epidermalcells, thereby evaluating the frequency of guard cell pairs.The extra subsidiary cells were, therefore, recruited from theepidermal cell population during development. Stomatal frequencyin plants grown at elevated temperature (29 C) was not significantlydifferent from that of the control (24 C). The linear aggregationsof stomata were similar in plants grown in ambient and elevatedCO₂. Since enriched CO₂ had no effect on the structure or patterningof guard cells, but resulted in the formation of additionalsubsidiary cells, it is likely that separate and independentevents pattern the two cell types. Plants grown at enrichedCO₂ levels had significantly greater internode lengths, butleaf area and the time interval between the appearance of successiveleaves were similar to that of control plants. Porometric measurementsrevealed that stomatal conductance of plants grown under elevatedCO₂ was lower than that of control leaves and those grown atelevated temperature. Tradescantia was capable of regulatingstomatal conductance in response to elevated CO₂ without changingthe relative number of stomata present on the leaf. Key words: Elevated CO₂, stomata, subsidiary cells, patterning     

STOMATAL MOVEMENTS ASSOCIATED WITH POTASSIUM FLUXES

Association of potassium ions with stomatal movements is reported here for 22 different plants. These include Ophioglossum engelmanni, Ginkgo biloba, and Pinus sylvestris. In all 22 plants potassium migrates into the guard cells when stomata open in response to light. In addition, potassium migration into guard cells also occurs with night opening in Crassula argentea and with rhythmic opening in Mimosa pudica. Potassium inside the guard or subsidiary cells, conventionally detected through light microscopic observations of epidermal peels treated with sodium cobaltinitrite reagent, may also be mapped by x-ray microanalysis of such histochemically treated peels, as was the case in this study. In addition to the potassium migration, we also show the movement of chloride as an accompanying anion in Ophioglossum engelmanni, Ginkgo biloba, Plantago rugelii, Begonia sp., and Avena sativa. Eight plants are shown to accumulate potassium inside the stomatal initials or cells of immature stomatal apparatuses; ordinary, immature epidermal cells do not show such an accumulation of potassium. A list in the discussion indicates all the plants in which potassium fluxes associated with stomatal movements have so far been established, including the new examples reported in this paper.     

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.     

Humidity Responses of Stomata and the Potassium Content of Guard Cells

Humidity responses of stomata and changes in the potassium contentof their guard cells were investigated in intact plants anddetached epidermal strips of Valerianella locusta (L.) Betcke.Potassium content was determined by Macallum‘s stain.It was found that changes in stomatal aperture caused by decreasingor increasing humidity were followed only after a delay by changesin the potassium content of the guard cells. By comparison,if stomatal movements occurred in response to changes in illuminationthe relative potassium content of the guard cells correlatedcontinuously with the changes in stomatal aperture. Since thepotassium content of the guard cells changed only after mostof the stomatal movements in response to changes in humiditywere completed changes in potassium content and humidity responsesof stomata can be described as following a hysteresis curve.     

Salt Tolerance in the Halophyte Suaeda maritima L. Dum.: Intracellular Compartmentation of Ions

The time-course of exchange of sodium and potassium ions fromroot and leaf material of the halophyte Suaeda maritima hasbeen followed and the data analysed according to the phenomenologyof efflux, or compartmental, analysis. Sodium ions were exchangedmuch more slowly (c. 4 times) from the vacuoles of leaf cellsof plants grown in sodium chloride than were potassium ionsfrom the vacuoles of leaf cells of plants grown either in similarconcentrations of potassium chloride or in low concentrationsof potassium. In plants grown in sodium chloride, sodium ionswere exchanged 9 times more slowly from the vacuoles of leafcells than from the vacuoles of root cells. The concentration of sodium ions in the cytoplasm of leaf cellsof plants growing in 340 mol m^–3 sodium chloride was estimatedto be 165 mol m^–3 when the average concentration in theleaf tissue was about 600 mol m^–3. As measured by movement from mature to developing leaves inintact plants; there was less in vivo retranslocation of ²²Naand ³⁶CI in plants growing in sodium chloride than there wasof ⁸⁶Rb in plants growing either in potassium chloride or innon-saline conditions. The results are discussed in terms of the concept and energeticsof compartmentation of ions in the cells of halophytes.     

Direct Measurements of Turgor Pressure Potentials of Guard Cells: II. THE MECHANICAL ADVANTAGE OF SUBSIDIARY CELLS, THE SPANNUNQSPHASE, AND THE OPTIMUM LEAF WATER DEFICIT

Evidence of the mechanical advantage of subsidiary cells wasobtained by simultaneous measurements of turgor pressure potentialsin adjacent subsidiary and guard cells using injection circuitswith two separate needles. In Tradescantia virginiana the mechanicaladvantage approaches two. Using the same technique evidencewas obtained that the Spannungsphase is, in the first place,a turgor relations phenomenon due to the mechanical advantageof epidermal or subsidiary cells. In addition, the evidenceindicated that the elastic properties of guard cell walls mayundergo changes during the Spannungsphase when potassium iontransport commences. During these measurements it was confirmedthat the optimum leaf water deficit for maximum stomatal openingoccurs when the epidermal turgor is near zero. Under these conditionsthe width of the stomatal pore is a function of the turgor pressureof the guard cells, since at zero turgor of the subsidiary cellstheir mechanical advantage has disappeared.     

Interactive effects of NaCl salinity and elevated atmospheric CO2 concentration on growth,photosynthesis, water relations and chemical composition of the potential cash crop halophyte Aster tripolium L.

This study was aimed at obtaining detailed information about the interaction of NaCl-salinity and elevated atmospheric CO₂ concentration in the potential cash crop halophyte Aster tripolium. Plants were irrigated with 5 different salinity levels (0, 25, 50, 75 and 100% sws) under ambient and elevated (520 ppm) CO₂. Under saline conditions leaf water potential decreased to a value below the one of the nutrient solution. Osmotic adjustment was mainly due to the accumulation of sodium and chloride (includer type). However, the salt was unequally distributed within the plants. K/Na selectivity was high in the lateral roots and low in the petioles, so that these organs served as “salt filters” which prevented an excessive salt accumulation and ion toxicity in the leaf blades and in the main root, the storage organ for organic substances. Despite some signs of ion toxicity and nutrial imbalance, these factors do not seem to be predominantly responsible for the limited salinity tolerance of A. tripolium. In order to maintain a positive water balance the salt treated plants increased stomatal resistance. But at the same time stomata closure led to a significant decrease in photosynthesis and thus in WUE. The impaired assimilation rate contributed to the significant growth depression (50% reduction of the maximum yield between 50% and 75% sws), together with the higher energy consumption needed for various salinity tolerance mechanisms, e.g. for an enhanced synthesis of compatible solutes (proline, carbohydrates) and stress-induced proteins. Elevated atmospheric CO₂ concentration led to a significant increase in photosynthesis and in WUE. The latter indicates, together with a higher water potential, that the water relations of the plants had improved. By reducing stomatal resistance energy gain was maximized. The additional supply with energy-rich organic substances was not employed for producing more biomass but for increasing the investment in salinity tolerance mechanisms, e.g. for an enhanced synthesis of proline, carbohydrates and proteins. These mechanisms led to a higher survival rate under saline conditions, i.e. to an improved salt tolerance. The results of the study indicate that A. tripolium is a promising cash crop halophyte which will probably benefit from rising atmospheric CO₂ concentrations in future.     

Analysis of Guard Cell Viability and Action in Senescing Leaves of Nicotiana glauca (Graham), Tree Tobacco

In an attempt to determine whether low epidermal conductances to water vapor diffusion of senescing leaves were caused by internal changes in guard cells or by factors external to guard cells, stomatal behavior was examined in intact senescing and nonsenescing leaves of Nicotiana glauca (Graham), tree tobacco, grown in the field or in an environmental chamber. Conductances of senescing leaves were 5 to 10% of the maximum conductances of nonsenescing leaves of the same plant, yet guard cell duplexes isolated from epidermal peels of senescing leaves developed full turgor in the light in solutions containing KCl, and sodium cobaltinitrite staining showed that K⁺ accumulated as turgor developed. Ninety-five per cent of the guard cells isolated from senescing leaves concentrated neutral red and excluded trypan blue. Intercellular leaf CO₂ concentrations of senescing and nonsenescing leaves of chamber-grown plants were not significantly different (about 240 microliters per liter), but the potassium contents of adaxial and abaxial epidermes of senescing leaves taken from plants grown in the field were less than half those of nonsenescing leaves. We conclude that guard cells do not undergo the orderly senescence process that characteristically takes place in mesophyll tissue during whole-leaf senescence and that the reduced conductances of senescing leaves are produced by factors external to guard cells.     

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     

Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

Guard cells emit an alkali-induced, blue fluorescence upon excitationby ultraviolet radiation (emission maximum energy at 365 nm).Fluorescence emission of guard cells was brighter than thatof the neighbouring epidermal cells in a number of wild andcultivated plants including conifers, but the relative fluorescenceintensity and quality was species-dependent. Three representativeplants possessing stomatal complexes which differed morphologicallywere studied: Olea europaea, Vicia faba and Triticum aestivum.Immersing leaves of these plants in chloroform for 30 s (therebyremoving epicuticular waxes) significantly reduced the intensityof the fluorescence emitted by guard cells. This indicates thatguard cell fluorescence could be due to either an increasedconcentration of fluorescing compounds (probably wax-bound phenolics),or a thicker cuticular layer covering the guard cells. Giventhat the alkali-induced blue fluorescence of the guard cellsis a common characteristic of all plants examined, it couldbe used as a rapid and convenient method for in situ measurementsof the number, distribution and size of stomatal complexes.The proposed experimental procedure includes a single coatingof the leaf surface by, or immersion of the whole leaf in, a10% solution of KOH for 2 min, washing with distilled water,and direct observation of the leaf surface under the fluorescencemicroscope. Fluorescence images were suitable for digital imageanalysis and methodology was developed for stomatal countingusing Olea europaea as a model species. Copyright 2001 Annalsof Botany Company Cuticle, epicuticular waxes, fluorescence microscopy, image analysis, phenolics, stomata     

Induction of CAM in Mesembryanthemum crystallinum Abolishes the Stomatal Response to Blue Light and Light-Dependent Zeaxanthin Formation in Guard Cell Chloroplasts

Facultative CAM plants such as Mesembryanthemum crystallinum(ice plant) possess C3 metabolism when unstressed but developCAM under water or salt stress. When ice plants shift from C3metabolism to CAM, their stomata remain closed during the dayand open at night. Recent studies have shown that the stomatalresponse of ice plants in the C3 mode depends solely on theguard cell response to blue light. Recent evidence for a possiblerole of the xanthophyll, zeaxanthin in blue light photoreceptionof guard cells led to the question of whether changes in theregulation of the xanthophyll cycle in guard cells parallelthe shift from diurnal to nocturnal stomatal opening associatedwith CAM induction. In the present study, light-dependent stomatalopening and the operation of the xanthophyll cycle were characterizedin guard cells isolated from ice plants shifting from C3 metabolismto CAM. Stomata in epidermis detached from leaves with C3 metabolismopened in response to white light and blue light, but they didnot open in response to red light. Guard cells from these leavesshowed light-dependent conversion of violaxan-thin to zeaxanthin.Induction of CAM by NaCI abolished both white light- and bluelight-stimulated stomatal opening and light-dependent zeaxanthinformation. When guard cells isolated from leaves with CAM weretreated with 100 mM ascorbate, pH 5.0 for 1 h in darkness, guardcell zeaxanthin content increased at rates equal to or higherthan those stimulated by light in guard cells from leaves inthe C3 mode. The ascorbate effect indicates that chloroplastsin guard cells from leaves with CAM retain their competenceto operate the xanthophyll cycle, but that zeaxanthin formationdoes not take place in the light. The data suggest that inhibitionof light-dependent zeaxanthin formation in guard cells mightbe one of the regulatory steps mediating the shift from diurnalto nocturnal stomatal opening typical of plants with CAM. (Received July 5, 1996; Accepted December 12, 1996)     

Stomatal Responses to Two Herbicidal Auxins

The effects of 1-naphthylacetic acid (NAA) and 2-naphthoxyaceticacid (NOXA) on stomatal opening on illumination of excised,turgid leaves of Stachytarpheta indica were investigated bymicroscopic examination of abaxial epidermises fixed in absoluteethanol. Both chemicals were effective in restricting, but notcompletely preventing, stomatal opening and suppressing starchhydrolysis and potassium accumulation in the guard cells. Therepressive effects were only partly reversed by CO₂-free air.It is concluded that NAA and NOXA do not greatly affect passiveopening mediated by changes in the leaf water balance, but partlysupress photoactive opening by arresting starch hydrolysis andpotassium accumulation in the guard cells and partly by disturbingthe intercellular CO₂ concentration. A possible link betweenstarch hydrolysis and potassium accumulation in the guard cellsis briefly mentioned.