Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Salinity Stiffens the Epidermal Cell Walls of Salt-Stressed Maize Leaves: Is the Epidermis Growth-Restricting?

As a result of salt (NaCl)-stress, sensitive varieties of maize (Zea mays L.) respond with a strong inhibition of organ growth. The reduction of leaf elongation investigated here has several causes, including a modification of the mechanical properties of the cell wall. Among the various tissues that form the leaf, the epidermis plays a special role in controlling organ growth, because it is thought to form a rigid outer leaf coat that can restrict elongation by interacting with the inner cell layers. This study was designed to determine whether growth-related changes in the leaf epidermis and its cell wall correspond to the overall reduction in cell expansion of maize leaves during an osmotic stress-phase induced by salt treatment. Two different maize varieties contrasting in their degree of salt resistance (i.e., the hybrids Lector vs. SR03) were compared in order to identify physiological features contributing to resistance towards salinity. Wall loosening-related parameters, such as the capacity of the epidermal cell wall to expand, β-expansin abundance and apoplastic pH values, were analysed. Our data demonstrate that, in the salt-tolerant maize hybrid which maintained leaf growth under salinity, the epidermal cell wall was more extensible under salt stress. This was associated with a shift of the epidermal apoplastic pH into a range more favourable for acid growth. The more sensitive hybrid that displayed a pronounced leaf growth-reduction was shown to have stiffer epidermal cell walls under stress. This may be attributable to the reduced abundance of cell wall-loosening β-expansin proteins following a high salinity-treatment in the nutrient solution (100 mM NaCl, 8 days). This study clearly documents that salt stress impairs epidermal wall-loosening in growth-reduced maize leaves.     

Rapid and reversible modifications of extension capacity of cell walls in elongating maize leaf tissues responding to root addition and removal of NaCl

A creep extensiometer technique was used to provide direct evidence that short (20 min) and long-term (3d) exposures of roots to growth inhibitory levels of salinity (100mol m^-3 NaCl) induce reductions in the irreversible extension capacity of cell walls in the leaf elongation zone of intact maize seedlings (Zea mays L.). The long-term inhibition of cell wall extension capacity was reversed within 20 min of salt withdrawal from the root medium. Inhibited elongation of leaf epidermal tissues was also reversed after salt removal. The salt-induced changes in wall extension capacity were detected using in vivo and in vitro assays (shortly after localized freeze/thaw treatment of the basal elongation zone). The rapid reversal of the inhibition of wall extensibility and leaf growth after salt removal from root medium of long-term salinized plants, suggested that neither deficiencies in growth essential mineral nutrients nor toxic effects of NaCl on plasmamembrane viability were directly involved in the inhibition of leaf growth. There was consistent agreement between the scale, direction and timing of salinity-induced changes in leaf elongation growth and wall extension capacity. Rapid metabolically regulated changes in the physical properties of growing cell walls, caused by osmotic (or other) effects, appear to be a factor regulating maize leaf growth responses to root salinization.     

Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium ± NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls.     

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.     

Do pH changes in the leaf apoplast contribute to rapid inhibition of leaf elongation rate by water stress? Comparison of stress responses induced by polyethylene glycol and down‐regulation of root hydraulic conductivity

We have dissected the influences of apoplastic pH and cell turgor on short-term responses of leaf growth to plant water status, by using a combination of a double-barrelled pH-selective microelectrodes and a cell pressure probe. These techniques were used, together with continuous measurements of leaf elongation rate (LER), in the (hidden) elongating zone of the leaves of intact maize plants while exposing roots to various treatments. Polyethylene glycol (PEG) reduced water availability to roots, while acid load and anoxia decreased root hydraulic conductivity. During the first 30 min, acid load and anoxia induced moderate reductions in leaf growth and turgor, with no effect on leaf apoplastic pH. PEG stopped leaf growth, while turgor was only partially reduced. Rapid alkalinization of the apoplast, from pH 4.9 ± 0.3 to pH 5.8 ± 0.2 within 30 min, may have participated to this rapid growth reduction. After 60 min, leaf growth inhibition correlated well with turgor reduction across all treatments, supporting a growth limitation by hydraulics. We conclude that apoplastic alkalinization may transiently impair the control of leaf growth by cell turgor upon abrupt water stress, whereas direct hydraulic control of growth predominates under moderate conditions and after a 30-60 min delay following imposition of water stress.     

Decreased reactive oxygen species concentration in the elongation zone contributes to the reduction in maize leaf growth under salinity

Reactive oxygen species (ROS) in the apoplast of cells in the growing zone of grass leaves are required for elongation growth. This work evaluates whether salinity-induced reductions in leaf elongation are related to altered ROS production. Studies were performed in actively growing segments (SEZ) obtained from leaf three of 14-d-old maize (Zea mays L.) seedlings gradually salinized to 150 mM NaCl. Salinity reduced elongation rates and the length of the leaf growth zone. When SEZ obtained from the elongation zone of salinized plants (SEZs) were incubated in 100 mM NaCl, the concentration where growth inhibition was approximately 50%, O2*- production, measured as NBT formazan staining, was lower in these than in similar segments obtained from control plants. The NaCl effect was salt-specific, and not osmotic, as incubation in 200 mM sorbitol did not reduce formazan staining intensity. SEZs elongation rates were higher in 200 mM sorbitol than in 100 mM NaCl, but the difference could be cancelled by scavenging or inhibiting O2*- production with 10 mM MgCl2 or 200 microM diphenylene iodonium, respectively. The actual ROS believed to stimulate growth is *OH, a product of O2*- metabolism in the apoplast. SEZ(s) elongation in 100 mM NaCl was stimulated by a *OH-generating medium. Fusicoccin, an ATPase stimulant, and acetate buffer pH 4, could also enhance elongation in these segments, although both failed to increase ROS activity. These results show that decreased ROS production contributes to the salinity-associated reduction in grass leaf elongation, acting through a mechanism not associated with pH changes.     

Inhibitory effects of water deficit on maize leaf elongation

The growth rate of maize leaves has been investigated for plants grown in pots in controlled conditions and subjected to slow drying over a period of days. The elongation of leaves experiencing water deficit was inhibited primaily during the afternoon. No decrease in the turgor of the growing cells could be detected at that time. Solute concentration in the growing cells increased in tissue experiencing water deficit, but this was shown to occur after the growth rate had fallen. Calculations of the rate of solute accumulation necessary to maintain these concentrations indicated that the rate was less in slowly growing than in rapidly growing cells. The growing tissue of well-watered leaves excreted protons into the apoplastic space, but this acidification decreased in tissue exposed to water deficit. The pH of the apoplastic space correlated with the growth rate of the tissue. In vitro acidification of isolated, frozen-thawed tissue, maintained under constant tension, increased wall extensibility. The results suggest that one role of proton excretion may be to promote wall-loosening events necessary for cell enlargement, and that inhibition of this process may have reduced growth rate in leaves exposed to water deficit.     

Spatial and temporal quantitative analysis of cell division and elongation rate in growing wheat leaves under saline conditions

Leaf growth in grasses is determined by the cell division and elongation rates, with the duration of cell elongation being one of the processes that is the most sensitive to salinity. Our objective was to investigate the distribution profiles of cell production, cell length and the duration of cell elongation in the growing zone of the wheat leaf during the steady growth phase. Plants were grown in loamy soil with or without 120 mmol/L NaCl in a growth chamber, and harvested at day 3 after leaf 4 emerged. Results show that the elongation rate of leaf 4 was reduced by 120 mmol/L NaCl during the steady growth phase. The distribution profile of the lengths of abaxial epidermal cells of leaf 4 during the steady growth stage shows a sigmoidal pattern along the leaf axis for both treatments. Although salinity did not affect or even increased the length of the epidermal cells in some locations in the growth zone compared to the control treatment, the final length of the epidermal cells was reduced by 14% at 120 mmol/L NaCl. Thus, we concluded that the observed reduction in the leaf elongation rate derived in part from the reduced cell division rate and either the shortened cell elongation zone or shortened duration of cell elongation. This suggests that more attention should be paid to the effects of salinity on those properties of cell production and the period of cell maturation that are related to the properties of cell wall.     

Participation of Plant Hormones in Growth Resumption of Wheat Shoots Following Short-Term NaCl Treatment

The effects of sodium-chloride salinity on the leaf elongation rate, transpiration rate, cell sap osmolality, and phytohormone content in 7-day-old shoots of durum wheat (Triticum durum L.) were studied. Leaf growth was suppressed under the salinity stress and resumed 1 h after NaCl removal. The resumption of leaf growth coincided with a decrease in the transpiration rate due to the rapid ABA accumulation in the differentiation leaf zone. The increased IAA concentration in the growing leaf zone promoted the formation of the attraction signal. The authors concluded that the changes in phytohormonal status in wheat plants occurred already following short-term (up to 1 h) salinity and were directed to the maintenance of plant growth under these conditions.     

Apoplastic pH and growth in expanding leaves of Vicia faba under salinity

Salinity affects water availability in the soil and subsequently the plant uptake capacity. Upon exposure to salt stress, leaf growth in monocot plants has been shown to be reduced instantaneously, followed by a gradual acclimation. The growth reactions are caused by an initial water deficit and an accompanied osmotic effect, followed by an IAA-induced sequestration of protons into the apoplast that increases leaf growth again as explained by the acid growth theory. In this study, we investigated the dynamics of growth reactions and apoplastic pH in leaves of the dicot Vicia faba in the presence of NaCl during the initiation of salt stress. Concurrent changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein tetramethylrhodamine dextran. To elucidate the possible relation between the dynamics of leaf growth and apoplastic pH, results of the ratio imaging technique were combined with an in vivo growth analysis imaging approach. Leaf growth rate of V. faba was highest in the dusk and the early night phase; at this time a concomitant decrease of the apoplastic pH was observed. Under salinity, the apoplastic pH in leaves of V. faba increased with a simultaneous decrease of leaf growth towards increasing developmental stages, but with complex aberrations in the 24-h-leaf-growth pattern compared to control leaves. In conclusion, these results show that salt stress leads to an increase in apoplastic pH and to a declined leaf growth activity with complex 24-h-interactions of growth and pH in V. faba.     

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Submergence stimulates elongation of the leaves of Rumex palustris and under laboratory conditions the maximum final leaf length (of plants up to 7 weeks old) was obtained within a 9 day period. This elongation response, mainly determined by petiole elongation, depends on the availability of storage compounds and developmental stage of a leaf. A starch accumulating tap root and mature leaves and petioles were found to supply elongating leaves with substrates for polysaccharide synthesis in expanding cell walls. Changes in the composition of cell wall polysaccharides of elongated petioles suggest a substantial cell wall metabolism during cell extension. Reduced starch levels or removal of mature leaves caused a substantial limitation of submerged leaf growth. From the 5th leaf onward enough reserves were available to perform submerged leaf growth from early developmental stages. Very young petioles had a limited capacity to elongate. In slightly older petioles submergence resulted in the longest final leaf lengths and these values gradually decreased when submergence was started at more mature developmental stages. Submerged leaf growth is mainly a matter of petiole elongation in which cell elongation has a concurrent synthesis of xylem elements in the vascular tissue. Mature petioles still elongated (when submerged) by cell and tissue elongation only: the annular tracheary elements stretched enabling up to 70% petiole elongation.     

Acid-induced wall loosening is confined to the accelerating region of the root growing zone

The aims of this study were to quantify developmental differences in acid growth along the root axis and to determine whether these differences were due to alterations in cell turgor or cell wall properties. The apoplast pH of maize roots growing in hydroponics was altered from pH 7.0 to pH 3.4 using 2 mol m^-3 citrate-phosphate buffer or unbuffered solutions. Whole root elongation rate rapidly increased and measurement of the local growth profile indicated that this increase in growth occurred in young cells in the accelerating zone (apical 0-4 mm) while more proximal growing cells were unaffected. Unbuffered solutions of identical pH produced qualitatively similar results. Single cell turgor pressures were unchanged between pH treatments both longitudinally and radially in the root tip. This suggests that the rapid acid-induced changes in growth rate were due to an increase in cell wall loosening. Single cell osmotic pressure and water potential were not significantly different between pH treatments. Acid pH caused net solute import at the root tip to increase 3- to 4-fold, which, coupled with the maintenance of turgor and osmotic pressure, indicated that solute import was not limiting expansion. Thus, acidic solutions cause an increase in growth in accelerating but not decelerating regions. It has been shown for the first time that acid growth in intact, growing roots is not due to differences in turgor, assigning these changes to cell wall properties. Possible cell wall biochemical alterations are discussed.     

Kinematics and Dynamics of Sorghum (Sorghum bicolor L.) Leaf Development at Various Na/Ca Salinities (I. Elongation Growth)

In many salt-sensitive species, elevated concentrations of Ca in the root growth media ameliorate part of the shoot growth reduction caused by NaCl stress. The physiological mechanisms by which Ca exerts protective effects on leaf growth are still not understood. Understanding growth inhibition caused by a stress necessitates locating the leaf expansion region and quantifying the profile of the growth reduction. This will enable comparisons and correlations with spatial gradients of probable physiologically inhibiting factors. In this work we applied the methods of growth kinematics to analyze the effects of elevated Ca concentrations on the spatial and temporal distributions of growth within the intercalary expanding region of salinized sorghum (Sorghum bicolor [L.] Moench, cv NK 265) leaves. NaCl (100 mM) caused a decrease in leaf elongation rate by shortening the leaf growing zone by 20%, as well as reducing the peak value of the longitudinal relative elemental growth rate (REG rate). Increasing the Ca concentrations from 1 to 10 mM restored the length of the growing zone of both emerged and unemerged salinized leaves and increased the peak value of the REG rate. The beneficial effects of supplemental Ca were, however, more pronounced in leaves after their appearance above the whorl of encircling older leaf sheaths. Elevated Ca then resulted in a peak value of REG rate higher than in the salinized leaves. The peak value of unemerged leaves was not increased, although it was maintained over a longer distance. The duration of elongation growth associated with a cell during its displacement from the leaf base was longer in salinized than control leaves, despite the fact that the elongation zone was shorter in salinity. Although partially restoring the length of the elongation region, supplemental Ca had no effect on the age of cessation of growth. Elongation of a tissue element, therefore, ceased when a cellular element reached a certain age and not a specific distance from the leaf base.     

Kinetics of Maize Leaf Elongation: I. INCREASED YIELD THRESHOLD LIMITS SHORT-TERM, STEADY-STATE ELONGATION RATES AFTER EXPOSURE TO SALINITY

The short-term responses of leaf elongation to salinity areinvestigated in this study. The kinetics of maize (Zea maysL.) leaf elongation were measured with Linear Variable DifferentialTransformers (LVDTs). After exposure to salinity (0 to 120 molm^–3 NaCl), leaf elongation rates (LER) declined rapidly.Within 4 h, LER had recovered and reached a new steady-statefor all salinity treatments. These rates were reduced by 10,20, and 60% of control rates by 40, 80 and 120 mol m^–3NaCl, respectively. Osmotic adjustment in the growing zone ofleaves was correlated with the recovery of LER after plant exposureto salinity. However, after 4 h of exposure, the osmolalityof the cell sap continued to increase without effect on steady-stateLER. Estimates of the apparent turgor in the growing zone indicatedthat turgor was no longer limiting LER of salt-stressed plantsafter 4 h. An in vivo technique was developed to apply a unidirectionalforce to intact growing leaves of maize to mimic increases inelongation force. Relative elongation rate (RER) were increasedby adding weights to the LVDT core to increase elongation force.Plots of RER as a function of elongation force gave estimatesof two growth coefficients: the yield threshold and the yieldingcoefficient, mL/(m + L), where m is the cell wall extensibilityand L is the hydraulic conductivity. RER as a function of elongationforce was determined immediately, 05, 4, and 21 h after plantswere salinized. Estimates of the growth coefficients indicatedthat the apparent yield threshold decreased immediately aftersalinization. However, when LER reached steady-state, the yieldthreshold of salt-stressed plants had increased above controlvalues and was the only limiting growth coefficient. There wereno significant effects of salinity on the yielding coefficients,cell wall extensibility or hydraulic conductivity. One of theadvantages of this in vivo technique over other methods is thatyield threshold, yielding coefficient, and cell wall extensibilitycan be determined without the confounding effects of woundingor osmotic stress. This technique may prove widely applicableto the study of other growth regulating factors. Key words: Salinity, leaf growth, Zea mays L     

Rapid and tissue-specific changes in ABA and in growth rate in response to salinity in barley leaves

The addition of 100 mM NaCl to the root medium of barley plantscaused the rapid cessation of elongation of the growing leafthree, followed by a sudden resumption of growth during thefollowing hour. The idea that resumption of growth is precededand mediated by rapid and tissue-specific changes in ABA concentrationand by changes in transpiration was tested. Leaf elongationvelocity was recorded continuously using linear variable displacementtransducers (LVDT), ABA was determined by immunoassay, and transpirationand stomatal conductivity were measured gravimetrically andby porometry, respectively. Within 10 min following additionof salt, ABA increased 6-fold in the distal portion of the leafelongation zone; in the proximal portion, ABA accumulated witha delay. In the portion of the growing blade that had emergedABA increased 3-fold and remained elevated during the following20 min. This preceded a decrease in transpiration and stomatalconductivity, which, in turn, coincided with growth resumption.Twenty hours following the addition of salt, the ABA concentrationshad returned to the level before stress. Leaf elongation velocitywas still reduced. It is concluded that NaCl causes a rapidincrease in ABA in the transpiring portion of the growing leaf.This leads to a decrease in transpiration. As a result, xylemwater potential is expected to rise. The moment that the waterpotential gradient between the xylem and the peripheral cellsin the growth zone favours water uptake again into the latter,leaf elongation resumes. The results suggest that ABA causesdifferent responses in different leaf regions, all aimed atpromoting the resumption of leaf growth. Key words: Abscisic acid, cell elongation, Hordeum vulgare, leaf growth, salinity, water relations.     

The influence of salt stress on ABA and auxin concentrations in two maize cultivars differing in salt resistance

The plant hormones abscisic acid (ABA) and auxin (IAA, IBA) play important roles in plant responses to environmental stresses such as salinity. Recent breeding improvements in terms of salt resistance of maize have lead to a genotype with improved growth under saline conditions. By comparing this salt-resistant hybrid with a sensitive hybrid, it was possible to show differences in hormone concentrations in expanding leaves and roots. In response to salinity, the salt-resistant maize significantly increased IBA concentrations in growing leaves and maintained IAA concentration in roots. These hormonal adaptations may help to establish favorable conditions for growth-promoting agents such as β-expansins and maintain growth of resistant maize hybrids under salt stress. Moreover, ABA concentrations significantly increased in resistant maize leaves under salt stress, which may contribute to acidifying the apoplast, which in turn is a prerequisite for growth.     

pH regulation in apoplastic and cytoplasmic cell compartments of leaves

 The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO₂, HCl or NH₃ differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO₂ either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO₂ shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO₂ was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO₂-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO₂ under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO₂ stress increased in the presence of oxygen with the length of previous exposure to high CO₂. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO₂ was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO₂, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO₂ stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m² leaf area)⁻¹ s⁻¹ or less. Proton export under acid stress into the vacuole was about 1200 nmol m⁻² s⁻¹. The kinetics of pH responses to the addition or withdrawal of CO₂ indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast. Received: 19 July 1999 / Accepted: 29 December 1999