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.     

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.     

Effect of different sugars on stomatal behaviour inMerremia aegyptia (L.) Urban andM. dissecta Hallier F.

The effect of mannitol, glucose and sucrose on the stomatal behaviour of two desert species,Merremia aegyptia andM. dissecta has been studied. Stomatal opening did not uniformly depend on the decrease in turgor of the epidermal and subsidiary cells caused by the different osmotic potential of the sugars. Sucrose caused plasmolysis of the subsidiary cells only but this was not accompanied by the opening of the stomatal pore. InM. aegyptia, no plasmolysis was seen either in epidermal or subsidiary cells, even the stomata opened; inM. dissecta, on the other hand, plasmolysis occurred in these cells without any stomatal opening, after incubation in glucose or mannitol. Mannitol is least absorbed, glucose slightly more and sucrose is absorbed to a very large extent in the guard cells when the materials were inoubated in the respective sugar solutions. However, the absorption of these three sugars was almost always larger in isolated epidermal strips than in discs; in detached intact leaves it was still more reduced.     

Water Supply, Evaporation, and Vapour Diffusion in Leaves

On the basis of experimental results published during the last25 years, but more particularly during the last 5 years andincluding some results presented here, the hypothesis is proposedthat an important portion of the water supply from major veinsin leaves travels within the epidermal tissue to sites of evaporationclose to the stomatal pores. These evaporation sites are innerepidermal walls especially subsidiary and guard cell walls becausethese are closest to air spaces with the highest water vapourdeficits. Less water than is traditionally supposed evaporatesfrom mesophyll cell walls. Low osmotic potentials of guard cells(large negative) are not required in building up high turgorpressures. However, they are required in competing for wateragainst the process of evaporation which causes low matric potentialsto develop in subsidiary and guard cell walls so that guardcolls can maintain the comparatively low turgor pressures whichhave been shown to operate the stomatal apparatus. Traditionalviews about leaf water relations and methods of estimating mesophyllresistances for carbon dioxide diffusion into leaves must bemodified.     

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     

Stomatal Responses of Tradescantia albiflora to Changing Air Humidity in Light and in Darkness

Tradescantia albiflora has green variegated and white leaves.Its stomatal apparatus consists of the guard cells and two pairsof subsidiary cells. Investigations were carried out by observingthe stomata microscopically by means of a video system in situin a CO₂ exchange chamber and by simultaneously measuring thegas exchange of the leaves. In response to air humidity changes,stomatal movements in T. albiflora begin, owing to turgor changes,in the polar and lateral subsidiary cells. The stomatal responseof green leaves to changes of air humidity showed typical transientand oscillatory phases prior to steady-state reactions. In darkness,stomata closed when air humidity decreased; however, they didnot reopen when air humidity was raised again. Stomata of illuminatedwhite leaves responded like those of green leaves in darkness.With increasing soil water stress stomata responded to changingair humidity with reductions of the transient phases and a decreasingtendency to reopen when air humidity became high again. CO₂deficiency of the air caused the stomata to open in the dark,and interacted with the air humidity effect in such a way thatstomata of green leaves responded to air humidity changes indarkness in a similar way as they did in light. Key words: Stomata, humidity response, green and white leaf areas, CO₂ deficient air     

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     

In Situ Measurement of Epidermal Cell Turgor, Leaf Water Potential, and Gas Exchange in Tradescantia virginiana L

A combined system has been developed in which epidermal cell turgor, leaf water potential, and gas exchange were determined for transpiring leaves of Tradescantia virginiana L. Uniform and stable values of turgor were observed in epidermal cells (stomatal complex cells were not studied) under stable environmental conditions for both upper and lower epidermises. The changes in epidermal cell turgor that were associated with changes in leaf transpiration were larger than the changes in leaf water potential, indicating the presence of transpirationally induced within-leaf water potential gradients. Estimates of 3 to 5 millimoles per square meter per second per megapascal were obtained for the value of within-leaf hydraulic conductivity. Step changes in atmospheric humidity caused rapid changes in epidermal cell turgor with little or no initial change in stomatal conductance, indicating little direct relation between stomatal humidity response and epidermal water status. The significance of within-leaf water potential gradients to measurements of plant water potential and to current hypotheses regarding stomatal response to humidity is discussed.     

Elevated CO2and Temperature have Different Effects on Leaf Anatomy of Perennial Ryegrass in Spring and Summer

Mature second leaves of Lolium perenne L. cv. Vigor, were sampledin a spring and summer regrowth period. Effects of CO₂enrichmentand increased air temperature on stomatal density, stomatalindex, guard cell length, epidermal cell density, epidermalcell length and mesophyll cell area were examined for differentpositions on the leaf and seasons of growth. Leaf stomatal density was smaller in spring but greater in summerin elevated CO₂and higher in both seasons in elevated temperatureand in elevated CO₂xtemperature relative to the respective controls.In spring, leaf stomatal index was reduced in elevated CO₂butin summer it varied with position on the leaf. In elevated temperature,stomatal index in both seasons was lower at the tip/middle ofthe leaf but slightly higher at the base. In elevated CO₂xtemperature,stomatal index varied with position on the leaf and betweenseasons. Leaf epidermal cell density was higher in all treatmentsrelative to controls except in elevated CO₂(spring) and elevatedCO₂xtemperature (summer), it was reduced at the leaf base. Inall treatments, stomatal density and epidermal cell densitydeclined from leaf tip to base, whilst guard cell length showedan inverse relationship, increasing towards the base. Leaf epidermalcell length and mesophyll cell area increased in elevated CO₂inspring and decreased in summer. In elevated CO₂xtemperatureleaf epidermal cell length remained unaltered in spring comparedto the control but decreased in summer. Stomatal conductancewas lower in all treatments except in summer in elevated CO₂itwas higher than in the ambient CO₂. These contrasting responses in anatomy to elevated CO₂and temperatureprovide information that might account for differences in seasonalleaf area development observed in L. perenne under the sameconditions. Lolium perenne ; perennial ryegrass; elevated CO₂and temperature; stomatal density; stomatal index; cell size     

STRUCTURE AND ONTOGENY OF THE STOMATAL COMPLEX IN PINUS STROBUS L. AND PINUS BANKSIANA LAMB.

The morphology of the stomatal complex in Pinus strobus L. and P. banksiana Lamb, is described and it is proposed that the stomatal complex should be considered an eight-celled complex consisting of two guard cells, and two polar, two lateral, and two hypodermal subsidiary cells. An ontogenetic study found these cells closely related developmentally. It was also found that the stomatal complex in these two pines could not readily be classified as haplocheilic because a polar subsidiary cell arises from the same protodermal cell as does the guard cell mother cell. A modification of the classical concept of stomatal development was necessary to describe the stomata as eumesoperigenous.     

Stomatal Responses to Sulphur Dioxide and Vapour Pressure Deficit

Stomatal conductances (g_s) of plants of Vicia faba, Raphanussativus, Phaseolus vulgaris, Heilanthus annuus, and Nicotianatabacum were measured in chambers containing either clean airor air containing between 18 and 1000 parts 10^–9 SO₂ atwater vapour pressure deficits (vpd) ranging from 1·0to 1·8 kPa. When vpd was low (<1·3 kPa at 22 °C) and stomatawere open, exposure to SO₂ induced rapid and irreversible increasesin g_s in V. faba. This response persisted throughout the exposure(3 d). The increase in g_s, 20–30% compared with cleanair, was independent of SO₂ concentration up to 350 parts 10^–9Stomatal conductances of polluted plants at night were greaterthan controls. When stomata were closed before exposure to SO₂,there was no effect on g_s. When vpd was varied, g_s of unpolluted plants of P. vulgarisshowed no response, but that of R. sativus increased slightlywith increasing vpd. In both species exposure to SO₂ causedan increase in g_s at all vpd values. g_s of unpolluted plantsof V. faba, H. annuus, and N. tabacum decreased with increasingvpd. At low vpd values exposure to SO₂ in these species causedan increase in g_s, but, above a certain value of vpd, dependingon species, g_s decreased with exposure to SO₂. It is postulated that SO₂, once in the substomatal cavity, entersthe stomatal complex via adjacent epidermal cells and at lowconcentrations leads to a reduction in turgor in these cellsand consequently to stomatal opening. In vpd-sensitive species,increased transpiration from guard cells or epidermal cellsadjacent to the stomata induced by SO₂ may lead to stomatalclosure at large vpd levels. Stomatal sensitivity to vpd insuch cases may be enhanced because adjacent epidermal cell turgoris lowered by SO₂. At high SO₂ concentrations direct disruptionof guard cell structure may lead to a loss of turgor and stomatalclosure.     

Direct turgor pressure measurements in individual leaf cells of Tradescantia virginiana

The water relations of leaves of Tradescantia virginiana were studied using the miniaturized pressure probe (Hüsken, E. Steudle, Zimmermann, 1978 Plant Physiol. 61, 158–163). Under well-watered conditions cell turgor pressures, P _o, ranged from 2 to 8 bar in epidermal cells. In subsidiary cells P _o was about 1.5 to 4.5 bar and in mesophyll cells about 2 to 3.5 bar. From the turgor pressure, relaxation induced in individual cells by changing the turgor pressure directly by means of the pressure probe, the half-time of water exchange was measured to be between 3 and 100 s for the epidermal, subsidiary, and mesophyll cells. The volumetric elastic modulus, , of individual cells was determined by changing the cell volume by a defined amount and simultaneously measuring the corresponding change in cell turgor pressure. The values for the elastic modulus for epidermal, subsidiary, and mesophyll cells are in the range of 40 to 240 bar, 30 to 200 bar, and 6 to 14 bar, respectively. Using these values, the hydraulic conductivity, L _p, for the epidermal, subsidiary, and mesophyll cells is calculated from the turgor pressure relaxation process (on the basis of the thermodynamics of irreversible processes) to be between 1 and 55·10^-7 cm s^-1 bar^-1. The data for the volumetric elastic modulus of epidermal and subsidiary cells indicate that the corresponding elastic modulus for the guard cells should be considerably lower due to the large volume changes of these cells during opening or closing. Recalculation of experimental data obtained by K. Raschke (1979, Encycl. Plant Physiol. N.S., vol. 7, pp 383–441) on epidermal strips of Vicia faba indicates that the elastic modulus of guard cells of V. faba is in the order of 40–80 bar for closed stomata. However, with increasing stomatal opening, i.e., increasing guard cell volume, decreases. Therefore, in our opinion Raschke's results would indicate a relationship between guard cell volume and which would be inverse to that for plant cells known in the literature. assumes values between 20–40 bar when the guard cell colume is soubled.     

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.     

Cell water potential,osmotic potential,and turgor in the epidermis and mesophyll of transpiring leaves

Water potential, osmotic potential and turgor measurements obtained by using a cell pressure probe together with a nanoliter osmometer were compared with measurements obtained with an isopiestic psychrometer. Both types of measurements were conducted in the mature region of Tradescantia virginiana L. leaves under non-transpiring conditions in the dark, and gave similar values of all potentials. This finding indicates that the pressure probe and the osmometer provide accurate measurements of turgor, osmotic potentials and water potentials. Because the pressure probe does not require long equilibration times and can measure turgor of single cells in intact plants, the pressure probe together with the osmometer was used to determine in-situ cell water potentials, osmotic potentials and turgor of epidermal and mesophyll cells of transpiring leaves as functions of stomatal aperture and xylem water potential. When the xylem water potential was-0.1 MPa, the stomatal aperture was at its maximum, but turgor of both epidermal and mesophyll cells was relatively low. As the xylem water potential decreased, the stomatal aperture became gradually smaller, whereas turgor of both epidermal and mesophyll cells first increased and afterward decreased. Water potentials of the mesophyll cells were always lower than those of the epidermal cells. These findings indicate that evaporation of water is mainly occurring from mesophyll cells and that peristomatal transpiration could be less important than it has been proposed previously, although peristomatal transpiration may be directly related to regulation of turgor in the guard cells.     

The Effects of Low Concentrations of Sulphur Dioxide on Stomatal Conductance and Epidermal Cell Survival in Field Bean (Vicia faba L.)

Exposure of 3 week old field bean plants to concentrations ofSO₂ from 50–500 µg m^-3 induced comparable 20–25%increases in mean leaf diffusive conductance regardless of whetherthe diffusive conductances were obtained by porometric measurementor calculation from gas exchange data. The stomatal conductancesof the adaxial and abaxial leaf surfaces were both increasedby exposure to SO₂. Microscopic examination of epidermal strips from control andpolluted plants revealed that the stomatal opening observedin treated plants was associated with a sharp reduction in theproportion of living epidermal cells adjacent to the stomata.The proportion of surviving adjacent epidermal cells was invariablysmaller on the lower epidermis and appeared to decrease as theSO₂ concentration was raised from 50 to 500 µg m^–3.Although the guard cells appeared to be undamaged at concentrationsbelow 200 µg m^–3, structural disorganization ordeath of one or both guard cells was observed frequently ator above 500 µg m^–3. The results are discussed in relation to the controversy concerningthe effects of SO₂ on stomatal aperture.     

Responses of Stomata in Epidermal Strips of Vicia fabato Carbon Dioxide and Growth Hormones when Incubated on Potassium Chloride and Potassium Iminodiacetate

The effect of various K⁺ levels in combination with Cl^&minus; or iminodiacetate (IDA^{& minus;}) on stomatal responsesin isolated epidermal strips of Vicia faba L. were examinedin order to determine the role of malate during guard cell movements.Responses of guard cells to ABA, kinetin, and varying CO₂ levelswere similar when epidermal strips were floated on KCL or KIDAat 10 mM; such responses were typical in that ABA caused closure,kinetin stimulated opening in ambient air, and apertures weregreater in CO₂-free than ambient air. Maximal stomatal openingwas observed in both ambient and CO₂-free air with KCL at 100mM. The transfer of epidermal strips from 100 mM KCL to solutionsof 100 mM KCL supplemented with ABA or kinetin did not bringabout changes in stomatal aperture. KCL at 100 mM supporteda greater degree of stomatal opening than did 100 mM KIDA irrespectiveof the CO₂ content of the air. In CO₂-free air transfer of epidermalstrips from 100 mM KIDA to solutions containing 100 mM KIDAsupplemented with ABA or kinetin caused little change in stomatalaperture, whereas, in ambient air, the same treatments resultedin stomatal opening. The results are discussed in relation tothe role of malate during guard cell movements.     

Dynamics of stomatal water relations during the humidity response: implications of two hypothetical mechanisms

The feasibility of two hypothetical mechanisms for the stomatal response to humidity was evaluated by identifying theoretical constraints on these mechanisms and by analysing timecourses of stomatal aperture following a step change in humidity. The two hypothetical mechanisms, which allow guard cell turgor pressure to overcome the epidermal mechanical advantage, are: (1) active regulation of guard cell osmotic pressure, requiring no hydraulic disequilibrium between guard and epidermal cells, and (2) a substantial hydraulic resistance between guard and epidermal cells, resulting in hydraulic disequilibrium between them. Numerical simulations of the system are made possible by recently published empirical relationships between guard cell pressure and volume and between stomatal aperture, guard cell turgor pressure, and epidermal cell turgor pressure; these data allow the hypothetical control variables to be inferred from stomatal aperture and evaporative demand, given physical assumptions that characterize either hypothesis. We show that hypothesis (1) predicts that steady‐state π_g is monotonically related to transpiration rate, whereas hypothesis (2) suggests that the relationship between transpiration rate and the steady‐state guard to epidermal cell hydraulic resistance may be either positive or negative, and that this resistance must change substantially during the transient phase of the stomatal response to humidity.     

Separation and Purification of Protoplast Types from Commelina communis L. Leaf Epidermis

Guard cell and epidermal/subsidiary cell protoplasts obtainedby enzymic digestion of peeled Commelina communis leaf epidermiswere separated and purified by discontinuous density gradientccntrifugation with media based on Percoll (Pharmacia Fine ChemicalsAB, Uppsala, Sweden). The cell types were recovered over 99.9%pure at yields exceeding 50% efficiency, and mesophyll contaminationcould be virtually eliminated when desired. Osmotic characteristicsof the protoplast types were evaluated and compared to in vivovalues, and the viability of the protoplasts, assessed usinga range of criteria, was found to be high. Purified Commelinaguard cell protoplasts were able to evolve O₂ when illuminated,and this was substantially reduced in the presence of the inhibitorDCMU, indicating that they possess photosystem II activity.Specific advantages of this method of protoplast purification,and the potential uses of separate suspensions of guard cellsand epidermal/subsidiary cells in experiments on stomatal physiologyare discussed. Key words: Commelina communis, Protoplasts, Epidermis     

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

Cell membrane potentials have been measured both in epidermalstrips and intact leaf sections of Tradescantia virginiana andCommelina communis, and in epidermal cells over green and overalbino mesophyll cells of T. albiflora var. albovittata. Membranepotentials (_cell) in strips were considerably lower than thosein intact sections and were insensitive to light and to theabsence or presence of calcium. Their response to external cationlevels was indifferent to ionic species. However, in intactleaf sections incubated with calcium present, membrane potentialsresponded to K⁺ levels but not to Na⁺. were more negative thancells in epidermal strips, and responded to changes in illumination. Long-term recordings of _cell and vacuolar K⁺ levels in T. virginianaduring stomatal closure suggest that the fluctuations of _cellwere unrelated to K⁺ movement (which we could not detect) andthus probably to stomatal movement as well. Turgor pressures measured in epidermal cells of intact leafsections of T. virginiana were found to be of the same magnitudeas those previously reported for epidermal strips. It is concludedthat epidermal cells maintain their solute contents during strippingwithout the involvement of an electrophysiological transportsystem. With the possible exception of lateral subsidiary cells,there was no evidence suggesting that ordinary epidermal cellsare capable of osmotic adjustment even when additional KCI wassupplied in the osmoticum. Absolute turgor levels in intactleaf sections kept at constant external KCI were unrelated tosteady state _cell.     

Mechanisms of stomatal movement in response to air humidity,irradiance and xylem water potential

Turgor, and osmotic and water potentials of subsidiary cells, epidermal cells and mesophyll cells were measured with a pressure probe and a nanoliter osmometer in intact transpiring leaves of Tradescantia virginiana L. Xylem water potential was manipulated by changing air humidity, light, and water supply. In a transpiring leaf the water potential of mesophyll cells was lower, but turgor was higher, than in cells surrounding the stomatal cavity owing to the presence of a cuticle layer which covers the internal surface of subsidiary and guard cells. Cuticular transpiration from the outer leaf surface was negligibly small. When stomata closed in dry air, transpiration decreased despite an increasing vapor-pressure difference between leaf and air, and the water potential of subsidiary cells dropped to the level of the water potential in mesophyll cells. We suggest that the observed decrease of transpiration at increasing vapor-pressure difference can be attributed to a shortage of water supply to the guard cells from subsidiary cells, causing turgor to decrease in the former more than in the latter. The leafs internal cuticle appears to play a special role in channelling the internal water flow during a water shortage.Abbreviations and Symbols VPD Vapor-pressure difference between leaf and air - PFD photon flux density - water potential