Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

Diurnal and seasonal responses of stomatal conductance for cowpea plants subjected to different levels of environmental drought

Summary Previously we reported that leaf conductance of cowpea (Vigna unguiculata) decreased with small changes in soil water status without associated changes in leaf water status. In these studies a larger range of soil water deficits was imposed in a rain-free environment by prolonged soil drying, and by weekly irrigation with different amounts of water. With progressive soil water deficits, leaf conductance and xylem pressure potential both declined, but in a manner which indicated that they were not related. Diurnal courses of leaf conductance usually indicated that stomatal opening occurred in the morning, and partial or complete stomatal closure occurred during midday and afternoon. This stomatal closure was associated with increases in air vapor pressure deficit. Day-to-day increases in leaf conductance, at times when radiation was not limiting stomatal opening, were associated with decreases in air vapor pressure deficits. However, maximum leaf conductances and their responses to vapor pressure deficit were generally smaller for plants subjected to greater depletion of soil water.     

The responses of stomata and leaf gas exchange to vapour pressure deficits and soil water content

The responses of leaf conductance, leaf water potential and rates of transpiration and net photosynthesis at different vapour pressure deficits ranging from 10 to 30 Pa kPa^-1 were followed in the sclerophyllous woody shrub Nerium oleander L. as the extractable soil water content decreased. When the vapour pressure deficit around a plant was kept constant at 25 Pa kPa^-1 as the soil water content decreased, the leaf conductance and transpiration rate showed a marked closing response to leaf water potential at-1.1 to-1.2 MPa, whereas when the vapour pressure deficit around the plant was kept constant at 10 Pa kPa^-1, leaf conductance decreased almost linearly from-0.4 to-1.1 MPa. Increasing the vapour pressure deficit from 10 to 30 Pa kPa^-1 in 5 Pa kPa^-1 steps, decreased leaf conductance at all exchangeable soil water contents. Changing the leaf water potential in a single leaf by exposing the remainder of the plant to a high rate of transpiration decreased the water potential of that leaf, but did not influence leaf conductance when the soil water content was high. As the soil water content was decreased, leaf conductances and photosynthetic rates were higher at equal levels of water potential when the decrease in potential was caused by short-term increases in transpiration than when the potential was decreased by soil drying.As the soil dried and the stomata closed, the rate of photosynthesis decreased with a decrease in the internal carbon dioxide partial pressure, but neither the net photosynthetic rate nor the internal CO₂ partial pressure were affected by low water potentials resulting from short-term increases in the rate of transpiration. Leaf conductance, transpiration rate and net photosynthetic rate showed no unique relationship to leaf water potential, but in all experiments the leaf gas exchange decreased when about one half of the extractable soil water had been utilized. We conclude that soil water status rather than leaf water status controls leaf gas exchange in N. oleander.     

Hydraulic control of stomatal conductance in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and alder [Alnus rubra (Bong)] seedlings

Experiments were conducted on 1-year-old Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and 2- to 3-month-old alder [Alnus rubra (Bong)] seedlings growing in drying soils to determine the relative influence of root and leaf water status on stomatal conductance (g_c). The water status of shoots was manipulated independently of that of the roots using a pressure chamber that enclosed the root system. Pressurizing the chamber increases the turgor of cells in the shoot but not in the roots. Seedling shoots were enclosed in a whole-plant cuvette and transpiration and net photosynthesis rates measured continuously. In both species, stomatal closure in response to soil drying was progressively reversed with increasing pressurization. Responses occurred within minutes of pressurization and measurements almost immediately returned to pre-pressurization levels when the pressure was released. Even in wet soils there was a significant increase in g_c with pressurization. In Douglas fir, the stomatal response to pressurization was the same for seedlings grown in dry soils for up to 120 d as for those subjected to drought stress over 40 to 60 d. The stomatal conductance of both Douglas fir and alder seedlings was less sensitive to root chamber pressure at higher vapour pressure deficits (D), and stomatal closure in response to increasing D from 1.04 to 2.06 kPa was only partially reversed by pressurization. Our results are in contrast to those of other studies on herbaceous species, even though we followed the same experimental approach. They suggest that it is not always appropriate to invoke a ‘feedforward’ model of short-term stomatal response to soil drying, whereby chemical messengers from the roots bring about stomatal closure.     

Short-term and long-term effects of plant water deficits on stomatal response to humidity in Corylus avellana L.

Short-term (hours) changes in plant water status were induced in hazel (Corylus avellana L.) by changing the evaporative demand on a major portion of the shoot while maintaining a branch in a constant environment. Stomatal conductance of leaves on the branch was influenced little by these short-term changes in water status even with changes in leaf water potential as great as 8 bars. Long-term (days) changes in plant water status were imposed by soil drying cycles. Stomatal conductance progessively decreased with increases in long-term water stress. Stomata still responded to humidity with long-term water stress but the range of the conductance response decreased. Threshold responses of stomata to leaf water potential were not observed with either short-term or long-term changes in plant water status even when leaves wilted. It is suggested that concurrent measurements of plant water status may not be sufficient for explaining stomatal and other plant responses to drought.     

Water relations and leaf expansion: importance of time scale

The role of leaf water relations in controlling cell expansion in leaves of water-stressed maize and barley depends on time scale. Sudden changes in leaf water status, induced by sudden changes in humidity, light and soil salinity, greatly affect leaf elongation rate, but often only transiently. With sufficiently large changes in salinity, leaf elongation rates are persistently reduced. When plants are kept fully turgid throughout such sudden environmental changes, by placing their roots in a pressure chamber and raising the pressure so that the leaf xylem sap is maintained at atmospheric pressure, both the transient and persistent changes in leaf elongation rate disappear. All these responses show that water relations are responsible for the sudden changes in leaf elongation rate resulting from sudden changes in water stress and putative root signals play no part. However, at a time scale of days, pressurization fails to maintain high rates of leaf elongation of plants in either saline or drying soil, indicating that root signals are overriding water relations effects. In both saline and drying soil, pressurization does raise the growth rate during the light period, but a subsequent decrease during the dark results in no net effect on leaf growth over a 24 h period. When transpirational demand is very high, however, growth-promoting effects of pressurization during the light period outweigh any reductions in the dark, resulting in a net increase in growth of pressurized plants over 24 h. Thus leaf water status can limit leaf expansion rates during periods of high transpiration despite the control exercised by hormonal effects on a 24 h basis.     

The apparent feed-forward response to vapour pressure deficit of stomata in droughted, field-grown Eucalyptus globulus Labill

This study tested a multiplicative model of stomatal response to environment for drought‐affected trees of Eucalyptus globulus Labill. growing in southern Australia. The model incorporates a feed‐forward response to vapour pressure deficit of ambient air (δe_a) and performed well if evaluated using reduced major axis regression and log‐transformed data. There was strong evidence from gas‐exchange data, leaf water potentials and sapflow measurements of the feed‐forward response by stomata to leaf‐to‐air vapour pressure deficit (δe_l). The response of stomata to δe_l was irreversible. Stomatal conductance and the rate of net photosynthesis were highly correlated and declined, together with the rate of transpiration, throughout the afternoon as δe_a increased despite increasing leaf water potentials. The concentration of CO₂ inside leaves (c_i) increased as stomatal conductance declined indicating increasing non‐stomatal limitations to photosynthesis. The stomatal response to δe_l of E. globulus in the field is best described as an ‘apparent feed‐forward response’ that probably results from both slowly reversible depression of net photosynthesis and abscisic acid accumulation in guard cells. We suggest that the stomatal response to c_i may strengthen the link between photosynthetic capacity and stomatal conductance during leaf drying as a result of either drought or large δ e_l.     

Stomatal physiology of a micropropagated CAM plant; Agave tequilana (Weber)

Experiments were designed to assess the capacity of an in vitro cultured CAM plant to control water loss and to examine the response of their stomata to various factors. Detached leaves of micropropagated Agave tequilana plants lost water at similar rates as did field-grown plantlets when dehydrated in air. This was consistent with the fact that stomata from micropropagated plants show similar morphology than field-grown plantlets. In addition, stomata from micropropagated plants responded to various factors in a manner similar to those from field-grown plantlets. It appears that in vitro culture does not affect the capacity of leaves to control water loss nor does it alters the nocturnal stomatal opening of this CAM plant.     

The temperature dependence of shoot hydraulic resistance: implications for stomatal behaviour and hydraulic limitation

Recent soil pressurization experiments have shown that stomatal closure in response to high leaf–air humidity gradients can be explained by direct feedback from leaf water potential. The more complex temperature‐by‐humidity interactive effects on stomatal conductance have not yet been explained fully. Measurements of the change in shoot conductance with temperature were made on Phaseolus vulgaris (common bean) to test whether temperature‐induced changes in the liquid‐phase transport capacity could explain these temperature‐ by‐humidity effects. In addition, shoot hydraulic resistances were partitioned within the stem and leaves to determine whether or not leaves exhibit a greater resistance. Changes in hydraulic conductance were calculated based on an Ohm’s law analogy. Whole‐plant gas exchange was used to determine steady‐ state transpiration rates. A combination of in situ psychrometer measurements, Scholander pressure chamber measurements and psychrometric measurements of leaf punches was used to determine water potential differences within the shoot. Hydraulic conductance for each portion of the pathway was estimated as the total flow divided by the water potential difference. Temperature‐induced changes in stomatal conductance were correlated linearly with temperature‐induced changes in hydraulic conductance. The magnitude of the temperature‐induced changes in whole‐plant hydraulic conductance was sufficient to account for the interactive effects of temperature and humidity on stomatal conductance.     

Unraveling the Effects of Plant Hydraulics on Stomatal Closure during Water Stress in Walnut

The objectives of the study were to identify the relevant hydraulic parameters associated with stomatal regulation during water stress and to test the hypothesis of a stomatal control of xylem embolism in walnut (Juglans regia x nigra) trees. The hydraulic characteristics of the sap pathway were experimentally altered with different methods to alter plant transpiration (Eplant) and stomatal conductance (gs). Potted trees were exposed to a soil water depletion to alter soil water potential (Psisoil), soil resistance (Rsoil), and root hydraulic resistances (Rroot). Soil temperature was changed to alter Rroot alone. Embolism was created in the trunk to increase shoot resistance (Rshoot). Stomata closed in response to these stresses with the effect of maintaining the water pressure in the leaf rachis xylem (P(rachis)) above -1.4 MPa and the leaf water potential (Psileaf) above -1.6 MPa. The same dependence of Eplant and gs on P(rachis) or Psileaf was always observed. This suggested that stomata were not responding to changes in Psisoil, Rsoil, Rroot, or Rshoot per se but rather to their impact on P(rachis) and/or Psileaf. Leaf rachis was the most vulnerable organ, with a threshold P(rachis) for embolism induction of -1.4 MPa. The minimum Psileaf values corresponded to leaf turgor loss point. This suggested that stomata are responding to leaf water status as determined by transpiration rate and plant hydraulics and that P(rachis) might be the physiological parameter regulated by stomatal closure during water stress, which would have the effect of preventing extensive developments of cavitation during water stress.     

The influence of plant water stress on stomatal control of gas exchange at different levels of atmospheric humidity

Summary Leaves of well-watered and mildly water-stressed seedlings of Betula pendula Roth. and Gmelina aroborea L. were subjected to a range of vapour pressure deficits (VPD) between 10 and 24 kPa. The stomatal conductance of birch seedlings decreased as VPD was increased and at least in mildly-stressed seedlings this response seemed to be closely linked to the water status of the air rather than to the bulk water status of the plant. Mild water stressing enhanced the degree of the stomatal humidity-response and resulted in a significant increase in the efficiency of water use at high VPD. Stomata of Gmelina were apparently insensitive to variation in VPD, but were more sensitive to a decrease in bulk leaf water status than were stomata of birch. Water use efficiency of Gmelina seedlings was comparatively high, even when VPD was high and the stomata were fully open.     

Assimilate transport in phloem sets conditions for leaf gas exchange

Carbon uptake and transpiration in plant leaves occurs through stomata that open and close. Stomatal action is usually considered a response to environmental driving factors. Here we show that leaf gas exchange is more strongly related to whole tree level transport of assimilates than previously thought, and that transport of assimilates is a restriction of stomatal opening comparable with hydraulic limitation. Assimilate transport in the phloem requires that osmotic pressure at phloem loading sites in leaves exceeds the drop in hydrostatic pressure that is due to transpiration. Assimilate transport thus competes with transpiration for water. Excess sugar loading, however, may block the assimilate transport because of viscosity build‐up in phloem sap. Therefore, for given conditions, there is a stomatal opening that maximizes phloem transport if we assume that sugar loading is proportional to photosynthetic rate. Here we show that such opening produces the observed behaviour of leaf gas exchange. Our approach connects stomatal regulation directly with sink activity, plant structure and soil water availability as they all influence assimilate transport. It produces similar behaviour as the optimal stomatal control approach, but does not require determination of marginal cost of water parameter.     

Xylem tension affects growth-induced water potential and daily elongation of maize leaves

Diurnal rates of leaf elongation vary in maize (Zea mays L.) and are characterized by a decline each afternoon. The cause of the afternoon decline was investigated. When the atmospheric environment was held constant in a controlled environment, and water and nutrients were adequately supplied to the soil or the roots in solution, the decline persisted and indicated that the cause was internal. Inside the plants, xylem fluxes of water and solutes were essentially constant during the day. However, the forces moving these components changed. Tensions rose in the xylem, and gradients of growth-induced water potentials decreased in the surrounding growing tissues of the leaf. These potentials, measured with isopiestic thermocouple psychrometry, changed because the roots became less conductive to water as the day progressed. The increased tensions were reversed by applying pressure to the soil/root system, which rehydrated the leaf. Afternoon elongation immediately recovered to rapid morning rates. The rapid morning rates did not respond to soil/root pressurization. It was concluded that increased xylem tension in the afternoon diminished the gradients in growth-induced water potential and thus inhibited elongation. Because increased tensions cause a similar but larger inhibition of elongation if maize dehydrates, these hydraulics are crucial for shaping the growth-induced water potential and thus the rates of leaf elongation in maize over the entire spectrum of water availability.     

Variations among woody plants in stomatal conductance and photosynthesis during and after drought

Acer saccharum, Fraxinus americana, Juglans nigra, Acer rubrum, Cornus amomum, and Ulmus americana seedlings were subjected to a soil drying cycle and then rewatered. At frequent intervals during the drying cycle and following rewatering, determinations were made of equilibrium photosynthesis rates, leaf conductances and leaf water potentials. As the drying cycle progressed, leaf water potentials decreased, stomata closed, and rates of transpiration and photosynthesis were reduced. Stomata of the two Acer species initially were more sensitive to water stress than were those of the other species. At low leaf water potentials, stomata of Juglans and Cornus were more open than those of the other species. Photosynthesis of Acer saccharum, Fraxinus and Juglans was significantly reduced by plant water stress, while photosynthetic water use efficiency of Cornus and Juglans was most unfavourable. Photosynthesis/leaf conductance ratios in water stressed leaves were higher in Fraxinus than in the other species. Immediately after rewatering, only limited stomatal opening occurred in Acer saccharum and Cornus with recovery of stomatal opening most protracted in Fraxinus and Ulmus. There was extended reduction of photosynthesis of all species as a result of the soil drying treatment. This effect was most significant in Acer saccharum and Juglans. Survival of plants on moist and dry sites is discussed in relation to stomatal control of transpiration and metabolic responses to water stress. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and the International Shade Tree Conference. The cooperation of the Wisconsin Department of Natural Resources is acknowledged. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and the International Shade Tree Conference. The cooperation of the Wisconsin Department of Natural Resources is acknowledged.     

The plant secondary metabolite citral alters water status and prevents seed formation in Arabidopsis thaliana

Based on previous results, which showed that the secondary metabolite citral causes disturbances to plant water status, the present study is focused on demonstrating and detailing these effects on the water‐related parameters of Arabidopsis thaliana adult plants, and their impact on plant fitness. Clear evidence of effects on water status and fitness were observed: plants treated with 1200 and 2400 μm citral showed decreased RWC, reduced Ψ_s, increased Ψ_w and reduced stomatal opening, even 7 days after the beginning of the experiment. Plant protection signals, such as leaf rolling or increased anthocyanin content, were also detected in these plants. In contrast, 14 days after beginning the treatment, treated plants showed signs of citral‐related damage. Moreover, the reproductive success of treated plants was critically compromised, with prematurely withered flowers and no silique or seed development. This effect of citral on fitness of adult plants suggests a promising application of this natural compound in weed management by reducing the weed seed bank in the soil.     

Stomatal behaviour and leaf water potential of chilled and water-stressed Solanum melongena, as influenced by growth history

Abstract Leaf diffusion resistance and leaf water potential of intact Solanum melongena plants were measured during a period of chilling at 6 °C. Two pretreatments, consisting of a period of water stress or a foliar spraying of abscisic acid (ABA), were imposed upon the plants prior to chilling. The control plants did not receive a pretreatment. In addition to intact plant studies, stomatal responses to water loss and exogenous abscisic acid were investigated using excised leaves, and the influence of the pretreatment observed. Chilled, control plants wilted slowly and maintained open stomata despite a decline in leaf water potential to –2.2 MPa after 2 d of chilling. In contrast plants that had been water stressed or had been sprayed with abscisic acid, prior to chilling, did not wilt and maintained a higher leaf water potential and a greater leaf diffusion resistance. In plants that had not received a pretreatment, abscisic acid caused stomatal closure at 35 °C, but at 6°C it did not influence stomatal aperture. The two pretreatments greatly increased stomatal sensitivity to both exogenous ABA and water stress, at both temperatures. Stomatal response to water loss from excised leaves was greatly reduced at 6°C. These results are discussed in relation to low temperature effects on stomata and the influence of preconditioning upon plant water relations.     

Stomatal conductance and transpiration of the two faces of Acacia phyllodes

Summary The daily course of stomatal conductance and transpiration was monitored on each separate face of vertical phyllodes of various acacias. The selected phyllodes had a north-south orientation so that one side faced eastwards and the other westwards. The principal measurements were made on Acacia longifolia and A. melanoxylon in Portugal in late summer and autumn, and additional measurements were made on A. ligulata and A. melanoxylon in Australia. In Portugal, irrespective of soil moisture status, conductance showed on early morning maximum with a subsequent gradual decline and sometimes a subsidiary peak in the late afternoon. Maximum conductances appeared to be a function of soil moisture status, whereas the decline in conductance in the late morning and afternoon was correlated with changes in phyllode-to-air vapour pressure deficits rather than changes in phyllode water status. The relationship of transpiration to phyllode water potential did not appear to be influenced by soil moisture status, although transpiration was less in drier soils and in the afternoons, this latter factor contributing to a marked hysteresis in the relationship. The opposing faces of the phyllodes exhibited a high degree of synchrony, showing parallel stomatal opening and closing, despite their large differences in irradiance. Stomatal conductance tended to be higher on the eastern faces in the morning and lower in the afternoon. In A. longifolia the daily average of relative conductance was much the same for both faces, but in A. melanoxylon that of the eastern face was higher and was retained even when the normal orientation of the phyllodes was reversed by turning them through 180°. Synchrony must be achieved by the stomata of both sides responding to common environmental or endogenous signals which are perceived by both surfaces with equal sensitivity.     

Senescence in oat leaf segments under hypobaric conditions

The physiological effects of storing plants under hypobaric conditions were studied using oat ( Avena sativa L. cv. Victory) leaf segments as a test system. The segments from seven day old plants were floated on water and stored in darkness at 12°C, 1.6 kPa or at 25°C, 6 kPa. Low temperature or hypobaric conditions delayed senescence, whereas the combination arrested the syndrome at an early stage. One of the effects of low pressure was to force the stomata open. The hormones abscisic acid and kinetin, which affect the stomatal aperture and also senescence, did not show any effect in hypobarically stored plant material. The stomata were forced open in darkness when the pressure was lower than 77 kPa and opening time was 8 h. The senescence syndrome in hypobarically stored segments developed similar to those treated with kinetin at 101 kPa.     

Stomatal responses to vapour pressure deficit are regulated by high speed gene expression in angiosperms

Plants dynamically regulate water use by the movement of stomata on the surface of leaves. Stomatal responses to changes in vapour pressure deficit (VPD) are the principal regulator of daytime transpiration and water use efficiency in land plants. In angiosperms, stomatal responses to VPD appear to be regulated by the phytohormone abscisic acid (ABA), yet the origin of this ABA is controversial. After a 20 min exposure of plants, from three diverse angiosperm species, to a doubling in VPD, stomata closed, foliar ABA levels increased and the expression of the gene encoding the key, rate‐limiting carotenoid cleavage enzyme (9‐cis‐epoxycarotenoid dioxygenase, NCED) in the ABA biosynthetic pathway was significantly up‐regulated. The NCED gene was the only gene in the ABA biosynthetic pathway to be up‐regulated over the short time scale corresponding to the response of stomata. The closure of stomata and rapid increase in foliar ABA levels could not be explained by the release of ABA from internal stores in the leaf or the hydrolysis of the conjugate ABA‐glucose ester. These results implicate an extremely rapid de novo biosynthesis of ABA, mediated by a single gene, as the means by which angiosperm stomata respond to natural changes in VPD.     

Changes in plant–soil hydraulic pressure gradients of soybean in response to soil drying

Plant gas‐exchange response to drying soil in many instances tracks a common pattern when expressed as a function of fraction of transpirable soil water (FTSW). There is little decrease in gas exchange until FTSW decreases to a value in the range of about 0.3–0.45, then with further drying gas exchange declines approximately linearly. This unique pattern is hypothesised to reflect mainly changes in the water potential gradient between bulk soil and plant. The primary objective was to directly document the basis of this response by measuring the hydrostatic pressure gradient required in the soil to maintain leaf xylem at zero potential with decreasing FTSW. Pots in which soybean (Glycine max) plants were grown were placed in a pressure chamber and the pressure adjusted to maintain zero water potential in a leaf petiolule. These results showed a small, relatively constant hydrostatic pressure had to be applied to the soil to maintain zero leaf xylem water potential until FTSW decreased to approximately 0.3–0.45. Thereafter, the required hydrostatic pressure gradient increased as FTSW continued to decrease. Hydraulic conductance was calculated to be relatively stable early in the drying cycle, and then decrease as the soil dried to comparatively high FTSW of 0.5–0.7.