Effects of Water and Turgor Potential on Malate Efflux from Leaf Slices of Kalanchoë daigremontiana

Malate efflux from leaf cells of the Crassulacean acid metabolism plant Kalanchoë daigremontiana Hamet et Perrier was studied using leaf slices submerged in experimental solutions. Leaves were harvested at the end of the dark phase and therefore contained high malate levels. Water potentials of solutions were varied between 0 and −5 bar using mannitol (a slowly permeating solute) and ethylene glycol (a rapidly permeating solute), respectively. Mannitol solutions of water potentials down to −5 bar considerably reduced malate efflux. The slowly permeating solute mannitol reduces both water potential and turgor potential of the cells. The water potential of a mannitol solution of −5 bar is just above plasmolyzing concentration. Malate efflux in ethylene glycol at −5 bar was only slightly smaller than at 0 bar, and much higher than in mannitol at −5 bar. Tissues in rapidly permeating ethylene glycol would have turgor potentials similar to tissues in 0.1 mm CaSO₄. The results demonstrate that malate efflux depends on turgor potential rather than on water potential of the cells.     

Chloroplast Response to Low Leaf Water Potentials: III. Differing Inhibition of Electron Transport and Photophosphorylation

Cyclic and noncyclic photophosphorylation and electron transport by photosystem 1, photosystem 2, and from water to methyl viologen (“whole chain”) were studied in chloroplasts isolated from sunflower (Helianthus annus L. var Russian Mammoth) leaves that had been desiccated to varying degrees. Electron transport showed considerable inhibition at leaf water potentials of −9 bars when the chloroplasts were exposed to an uncoupler in vitro, and it continued to decline in activity as leaf water potentials decreased. Electron transport by photosystem 2 and coupled electron transport by photosystem 1 and the whole chain were unaffected at leaf water potentials of −10 to −11 bars but became progressively inhibited between leaf water potentials of −11 and −17 bars. A low, stable activity remained at leaf water potentials below −17 bars. In contrast, both types of photophosphorylation were unaffected by leaf water potentials of −10 to −11 bars, but then ultimately became zero at leaf water potentials of −17 bars. Although the chloroplasts isolated from the desiccated leaves were coupled at leaf water potentials of −11 to −12 bars, they became progressively uncoupled as leaf water potentials decreased to −17 bars. Abscisic acid and ribonuclease had no effect on chloroplast photophosphorylation. The results are generally consistent with the idea that chloroplast activity begins to decrease at the same leaf water potentials that cause stomatal closure in sunflower leaves and that chloroplast electron transport begins to limit photosynthesis at leaf water potentials below about −11 bars. However, it suggests that, during severe desiccation, the limitation may shift from electron transport to photophosphorylation.     

Growth and Nodulation Responses of Rhizobium meliloti to Water Stress Induced by Permeating and Nonpermeating Solutes

Isolates of Rhizobium meliloti, representing antigenically distinct indigenous serogroups 31 and 17, were grown in yeast extract-mannitol broth (YEM) containing NaCl or polyethylene glycol (PEG) to provide external water potentials ranging from −0.15 to −1.5 MPa. Several differences were found between representatives of the two groups in their abilities to adapt to water stress induced by the nonpermeating solute PEG. At potentials below −0.5 MPa, strain 31 had a lower specific growth rate than strain 17 and an irregular cell morphology. In contrast, neither growth nor cell morphology of either strain was affected significantly over the same range of water potentials created by a permeating solute, NaCl. Despite the superior growth of strain 17 at the low water potentials imposed by PEG, upshock of water-stressed cells (−1.0 MPa; PEG) into normal YEM (−0.15 MPa) resulted in a faster recovery of growth by strain 31 than by strain 17. Different responses of the two strains to a water potential increase were also revealed in nodulation studies. Strain 31 required significantly fewer days to nodulate alfalfa than strain 17 did when the strains were transferred from YEM with PEG at −1.0 MPa onto the roots of alfalfa seedlings in plant growth medium (−0.1 MPa). The addition of supplemental calcium (0.1 mM) to growth medium with PEG (−1.0 MPa) reduced the differences between strains in their responses to water stress. The severe growth restriction and morphological abnormalities shown by strain 31 were corrected, and the prolonged recovery time shown by water-stressed cells (−1.0 MPa; PEG) of strain 17 upon transfer to normal YEM was shortened. The latter strain also nodulated earlier and more rapidly after growth in PEG medium at −1.0 MPa in the presence of supplemental calcium ions. These results indicate that the efficacy of osmoregulation can vary among strains of the same species and that the mechanism of osmoregulation may differ depending on the nature of the water stress.     

Water Transfer in an Alfalfa/Maize Association : Survival of Maize during Drought

We investigated the possibility of interspecific water transfer in an alfalfa (Medicago sativa L.) and maize (Zea mays L.) association. An alfalfa plant was grown through two vertically stacked plastic tubes. A 5 centimeter air gap between tubes was bridged by alfalfa roots. Five-week old maize plants with roots confined to the top tube were not watered, while associated alfalfa roots had free access to water in the bottom tube (the −/+ treatment). Additional treatments included: top and bottom tubes watered (+/+), top and bottom tubes droughted (−/−), and top tube droughted after removal of alfalfa root bridges and routine removal of alfalfa tillers (−^*). Predawn leaf water potential of maize in the −/+ treatment fell to −1.5 megapascals 13 days after the start of drought; thereafter, predawn and midday potentials were maintained near −1.9 megapascals. Leaf water potentials of maize in the −/− and −^* treatments declined steadily; all plants in these treatments were completely desiccated before day 50. High levels of tritium activity were detected in water extracted from both alfalfa and maize leaves after ³H₂O was injected into the bottom −/+ tube at day 70 or later. Maize in the −/+ treatment was able to survive an otherwise lethal period of drought by utilizing water lost by alfalfa roots.     

Assimilation, Distribution, and Root Exudation of C by Ponderosa Pine Seedlings under Induced Water Stress

The effect of specific levels of induced water stress on the root exudation of ¹⁴C from 9-month-old and 12-month-old ponderosa pine (Pinus ponderosa Laws.) seedlings was examined. Polyethylene glycol (PEG-4000) was used to decrease root solution water potentials by 0, −1.9, −2.6, −5.5, −9.6 and −11.9 bars in either aerated 0.25X Hoagland's nutrient solution or aerated distilled water.     

Characteristics of cultured tomato cells after prolonged exposure to medium containing polyethylene glycol

Cell lines of tomato (Lycopersicon esculentum Mill., cv. VFNT-Cherry) have been isolated, which are capable of growing in media containing polyethylene glycol (PEG) 6000 with water potentials as low as −15 or −22 bar. After prolonged exposure to media containing PEG, these cell populations have reduced abilities to grow in the absence of PEG. Upon resuspension in PEG-free medium, the cells swell and begin to release metabolites, including protein. Measurement by plasmometry of the osmotic potential of cells selected in medium with −22 bar water potential indicates that they maintain, at the end of the growth cycle, an osmotic potential of approximately −26 bar. This is compared to an osmotic potential of −9 bar for nonselected cells in medium without PEG, having an initial water potential of −4 bar. Thus, considerable osmotic adjustment occurs as a result of exposure to external low water potential. The results also indicate that PEG does not contribute significantly to osmotic adjustment of the cells.     

Effects of the Ionic Composition and Water Potential of Aqueous Solution on the Activity and Survival of Orrina phyllobia

The activity and survival of Orrina phyllobia fourth-stage juveniles (J4) were examined in aqueous solutions representing 96 combinations of eight predominant soil solution ions at total concentrations of 100, 200, and 1,000 meq/liter. Various water potentials were imposed by the addition of mannitol or polyethylene glycol to ionic solutions. Nematode longevity increased as water potential was decreased. Longevity was approximately doubled at a water potential of -23 × 10⁵ Pa and more than tripled at -60 × 10⁵ Pa. No combination oflons at 200 meq/liter was lethal after a 6-day exposure. Several ion combinations significantly increased longevity at -10 and -23 × 10⁵ Pa. Single cation Na⁺ solutions consistently inhibited activity and more than doubled nematode longevity.     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

Endosperm cell division in maize kernels cultured at three levels of water potential

The influence of osmoticum treatments on early kernel development of maize (Zea mays L.) was studied using an in vitro culture method. Kernels with subtending cob sections were placed in culture at 5 days after pollination. Sucrose (0.29, 0.44, or 0.58 molar) and sorbitol (0, 0.15, or 0.29 molar) were used to obtain six media with water potentials of −1.1, −1.6, or −2.0 megapascals. Kernel water potential declined in correspondence with the water potential of the medium; however, fresh weight growth was not significantly inhibited from 5 to 12 days after pollination. In stress treatments with media water potentials of −1.6 or −2.0 megapascals, endosperm tissue accumulated water and solutes from 10 and 12 days after pollination at a rate similar to or greater than that of the control (−1.1 megapascals). In contrast, endosperm cell division was inhibited in all treatments relative to control. At 10 days after pollination, endosperm sucrose concentration was greater in two of the −2.0 megapascal treatments with 0.44 or 0.58 molar media sucrose compared to control kernels cultured in 0.29 molar sucrose at −1.1 megapascals. Significant increases in abscisic acid content per gram of fresh weight were detected in two −2.0 megapascal treatments (0.29 molar sucrose plus 0.29 molar sorbitol and 0.58 molar sucrose) at 10 days after pollination. We conclude that in cultured maize kernels, endosperm cell division was more responsive than fresh weight accumulation to low water potential treatments. Data were consistent with mechanisms involving abscisic acid or lowered tissue water potential, or an interaction of the two factors.     

Kinetics of Adaptation to Osmotic Stress in Lentil (Lens culinaris Med.) Roots

When intact roots of lentil (Lens culinaris Med.) are subjected to severe osmotic stress by treatment with a solution of low water potential, they immediately begin to shrink. Within 10 to 15 minutes, shrinkage ceases, and within 20 minutes, the roots resume growth. The time lag between application of osmoticum and resumption of growth varies from about 10 to 30 minutes over the range of external water potentials of −2 to −12.4 bars. For external water potentials as low as −8.7 bars the new steady rate of growth in the presence of osmoticum is approximately equal to that prevailing before application of osmoticum. For external water potentials between −8.7 and −13 bars growth resumes, but the new rate is less than that prior to addition of osmoticum. Measurements of changes in the internal solute content during adaptation show that the solute content of the root increases but that the magnitude of the increase is, by itself, insufficient to account for the resumption of rapid growth.     

Does Water Deficit Stress Promote Ethylene Synthesis by Intact Plants?

The effect of plant water deficit on ethylene production by intact plants was tested in three species, beans (Phaseolus vulgaris L.), cotton (Gossypium hirsutum L.) and miniature rose (Rosa hybrida L., cv Bluesette). Compressed air was passed through glass, plant-containing cuvettes, ethylene collected on chilled columns, and subsequently assayed by gas chromatography. The usual result was that low water potential did not promote ethylene production. When plants were subjected to cessation of irrigation, ethylene production decreased on a per plant or dry weight basis of calculation. No significant promotion of ethylene production above control levels was detected when water deficit-treated bean or cotton plants were rewatered. The one exception to this was for cotton subjected to a range of water deficits, plants subjected to deficits of −1.4 to −1.6 MPa exhibited a transient increase of ethylene production of 40 to 50% above control levels at 24 or 48 hours. Ethylene was collected from intact leaves while plants developed a water deficit stress of −2.9 megapascals after rewatering, and no significant promotion of ethylene production was detected. The shoots of fruited, flowering cotton plants produced less ethylene when subjected to cessation of irrigation. In contrast, the ability of bench drying of detached leaves to increase ethylene production several-fold was verified for both beans and cotton. The data indicate that detached leaves react differently to rapid drying than intact plants react to drying of the soil with regard to ethylene production. This result suggests the need for additional attention to ethylene as a complicating factor in experiments employing excised plant parts and the need to verify the relevance of shock stresses in model systems.     

The response of foliar gas exchange to exogenously applied ethylene

The responsiveness to ethylene of net photosynthesis and stomatal conductance to water vapor in intact plants was investigated in 13 herbaceous species representing seven plant families. Exposures were conducted in an open, whole-plant exposure system providing controlled levels of irradiance, air temperature, CO₂, relative humidity, and ethylene concentration. Net photosynthesis and stomatal conductance to water vapor in units of moles per square meter per second were measured on recently expanded leaves in control and ethylene-treated plants using a remotely operated single-leaf cuvette. The ethylene concentration was either 0 or 210 micromoles per cubic meter and was maintained for 4 hours. Species varied substantially in the response of their foliar gas exchange to ethylene. In 7 of the 13 species, net photosynthesis was inhibited statistically by 4 hours of ethylene exposure. As a function of the rate in control plants, the responses were most pronounced and statistically significant in Arachis hypogaea (−51.1%), Gossypium hirsutum (−31.7%), Glycine max (−24.8%), Cucurbita pepo (−20.4%), Phaseolus vulgaris (−18.4%), Setaria viridis (−17.5%), and Raphanus sativus (−4.4%). Whereas the responsiveness of net photosynthesis to ethylene among the 13 species showed no specific taxonomic associations, the responsiveness was positively correlated with the intrinsic rate of net photosynthesis. Stomatal conductance to water vapor after 4 hours of ethylene exposure declined statistically in 6 of the 13 species. As a function of control rates, the most marked and statistically significant responses of stomatal conductance were in Glycine max (−53.6%), Gossypium hirsutum (−51.2%), Arachis hypogaea (−42.7%), Phaseolus vulgaris (−38.6%), Raphanus sativus (−26.8%), and Solanum tuberosum (−23.4%). Although ethylene-induced changes in net photosynthesis and stomatal conductance were positively correlated, there were species-specific exceptions in which net photosynthesis declined after 4 hours of exposure without a concurrent change in stomatal conductance, stomatal conductance declined without a change in net photosynthesis, and the decline in stomatal conductance substantially exceeded the corresponding decline in net photosynthesis. Thus, the responsiveness to ethylene of net photosynthesis and stomatal conductance to water vapor were not consistently synchronous or equivalent among the 13 species. It is concluded that foliar gas exchange is responsive to exogenously applied ethylene in many plant species. The sensitivity of foliar gas exchange to ethylene may play a role in general plant response to environmental stress in which one of the physiological sites of action for endogenously produced stress ethylene in the leaf is the plant's photosynthetic capacity and/or stomatal conductance to water vapor.     

Effects of turgor potential on 1-aminocyclopropane-1-carboxylic acid uptake into the vacuolar compartment and ethylene production in tomato pericarp slices

While solute transport and ethylene production by plant tissue are sensitive to the osmotic concentration of the solution bathing the tissue, the influence of tissue water relations and specifically tissue turgor potential on the kinetics of 1-aminocyclopropane-1-carboxylic acid (ACC) uptake into the vacuolar compartment and ethylene production have not been examined. 1-Aminocyclopropane-1-carboxylic acid transport and ethylene production were examined in tomato (Lycopersicon esculentum Mill. cv. Liberty) pericarp slices incubated in solutions having a range of mannitol, polyethylene glycol 3350 and ethylene glycol concentrations known to affect tissue water relations. Tissue osmotic and turgor potentials were derived from osmolality measurements of cell saps recovered by freeze-thawing and corrected for the contribution of the free-space solution. When relatively nonpermeable (mannitol or polyethylene glycol 3350) osmotica were used, both ACC uptake and ethylene production were greatest at a solution osmolality of 230 milliosmolal where tissue turgor potential ranged between 120 and 140 kPa. At higher and lower turgor potentials, the high-affinity saturating component of ACC uptake and ethylene production were inhibited, and ACC efflux from the vacuolar compartment was increased. The inhibition of ACC uptake was evident as a decrease in V_max with no effect on K_m. Turgor potential changes caused by adjusting solution osmolality with mannitol or polyethylene glycol 3350 were accompanied by changes in the osmotic potential and water potential of the tissue. The effects of turgor potential vs the osmotic and water potentials of tomato pericarp slices were differentiated by comparing responses to nonpermeable osmotica and mixtures of nonpermeable and permeable osmotica. Ethylene glycol-mannitol mixtures had effects on the osmotic potential and water potential of the tissue similar to those of nonpermeable osmotica but had less effect on tissue turgor, ACC transport and ethylene production. Incubating tissue in solutions without nonpermeable osmotica osmotically shocked the tissue. Increasing solution osmolality with ethylene glycol in the absence of nonpermeable osmotica increased tissue turgor and ethylene production. The present study indicates that tissue turgor is an important factor affecting the kinetics of ACC uptake into the vacuolar compartment and ethylene production in tomato pericarp slices.     

Chloroplast response to low leaf water potentials: I. Role of turgor

The effect of decreases in turgor on chloroplast activity was studied by measuring the photochemical activity of intact sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves having low water potentials. Leaf turgor, calculated from leaf water potential and osmotic potential, was found to be affected by the dilution of cell contents by water in the cell walls, when osmotic potentials were measured with a thermocouple psychrometer. After the correction of measurements of leaf osmotic potential, both the thermocouple psychrometer and a pressure chamber indicated that turgor became zero in sunflower leaves at leaf water potentials of −10 bars. Since most of the loss in photochemical activity occurred at water potentials below −10 bars, it was concluded that turgor had little effect on the photochemical activity of the leaves.     

Differential Response in the Water Status of Immature and Mature Fronds of the Ostrich Fern (Matteuccia struthiopteris [L.] Todaro) to a Mild Water Stress

Experiments were conducted in growth chambers to examine the effect of a mild water stress (−200 kilopascals polyethylene glycol) on frond elongation and water status of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Measurements were taken for two days, starting one day after the application of polyethylene glycol. Total water potential in control (well-watered) plants was always significantly higher in immature fronds than in mature fronds. The osmotic potential in mature fronds was always significantly lower (about 800 kilopascals) than in immature fronds in both control and stressed plants. In immature fronds, the stress decreased elongation and total water and pressure potentials, while in mature fronds it increased total water and pressure potentials. The decreases in total and pressure potentials in immature fronds were approximately equal to the increases in mature fronds.     

Stomatal response to abscisic Acid is a function of current plant water status

We investigated, under laboratory and field conditions, the possibility that increasing abscisic acid (ABA) concentrations and decreasing water potentials can interact in their effects on stomata. One experiment was carried out with epidermal pieces of Commelina communis incubated in media with a variety of ABA and polyethylene glycol concentrations. In the media without ABA, incubation in solutions with water potentials between −0.3 and −1.5 megapascals had no significant effect on stomatal aperture. Conversely, the sensitivity of stomatal aperture to ABA was trebled in solutions at −1.5 megapascals compared with sensitivity at −0.3 megapascals. The effect of the change in sensitivity was more important than the absolute effect of ABA at the highest water potential. In a field experiment, sensitivity of maize stomatal conductance to the concentration of ABA in the xylem sap varied strongly with the time of the day. We consider that the most likely explanation for this is the influence of a change in leaf or epidermal water potential that accompanies an increase in irradiance and saturation deficit as the day progresses. These observations suggest that epidermal water relations may act as a modulator of the responses of stomata to ABA. We argue that such changes must be taken into account in studies or modeling of plant responses to drought stress.     

Increased endogenous abscisic Acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials

Roots of maize (Zea mays L.) seedlings continue to grow at low water potentials that cause complete inhibition of shoot growth. In this study, we have investigated the role of abscisic acid (ABA) in this differential growth sensitivity by manipulating endogenous ABA levels as an alternative to external applications of the hormone. An inhibitor of carotenoid biosynthesis (fluridone) and a mutant deficient in carotenoid biosynthesis (vp 5) were used to reduce the endogenous ABA content in the growing zones of the primary root and shoot at low water potentials. Experiments were performed on 30 to 60 hour old seedlings that were transplanted into vermiculite which had been preadjusted to water potentials of approximately −1.6 megapascals (roots) or −0.3 megapascals (shoots). Growth occurred in the dark at near-saturation humidity. Results of experiments using the inhibitor and mutant approaches were very similar. Reduced ABA content by either method was associated with inhibition of root elongation and promotion of shoot elongation at low water potentials, compared to untreated and wild-type seedlings at the same water potential. Elongation rates and ABA contents at high water potential were little affected. The inhibition of shoot elongation at low water potential was completely prevented in fluridone-treated seedlings during the first five hours after transplanting. The results indicate that ABA accumulation plays direct roles in both the maintenance of primary root elongation and the inhibition of shoot elongation at low water potentials.     

Stimulation of ethylene production in citrus leaf discs by mannitol

Wound ethylene formation induced in leaf tissue of citrus (Citrus sinensis [L.] Osbeck cv. “Washington Navel”) by excision was significantly stimulated by mannitol after a lag period of about 6 hours. The extent of stimulation was dependent upon the concentration of mannitol (10 to 100 millimolar). This increased ethylene production was not simply due to osmotic effect or water stress as other osmoticums tested failed to exert such an effect. The stimulatory effect of mannitol resulted from both the enhancement of 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. The effect on the latter step was particularly pronounced in aged discs. The use of labeled mannitol showed that it was taken up by the leaf discs, utilized for respiration, and metabolized to sucrose, but no radioactivity was detected in the ethylene.     

Chloroplast Response to Low Leaf Water Potentials: IV. Quantum Yield Is Reduced

Quantum yields were measured for CO₂ fixation by sunflower (Helianthus annuus L.) leaves having various water potentials and for dichlorophenolindophenol photoreduction by chloroplasts isolated from similar leaves having various water potentials. In red radiation, the quantum yield for CO₂ was 0.076 for an attached sunflower leaf at a water potential of −3 to −4 bars but was 0.020 for the same leaf at −15.3 bars. After recovery to a water potential of −5 bars, the quantum yield rose to 0.060. Soybean (Glycine max L. [Merr.]) leaves behaved similarly. Chloroplasts from a sunflower leaf with a water potential of −3.6 bars had a quantum yield for 4 equivalents of 0.079, but when tissue from the same leaf had a water potential of −14.8 bars, the quantum yield of the chloroplasts decreased to 0.028. The decrease could not be attributed to differences in rates of respiration by the leaves or the chlorophyll content or absorption spectrum of the leaves and chloroplasts.     

H Uptake and Release during Circadian Rhythmic Movements of Excised Samanea Motor Organs : Effects of Mannitol, Sorbitol, and External pH

We investigated H⁺ fluxes during circadian rhythmic movements of Samanea saman leaflets by monitoring the pH of a weakly buffered medium bathing extensor or flexor motor tissue excised at different times during 51 hours of darkness. Experiments were made in media of two different osmotic potentials: −0.3 megapascal (control medium) and −1.2 megapascals (control medium supplemented with 0.4 molar mannitol or sorbitol). Both extensor and flexor tissue took up H⁺ from the control medium at all times when the initial pH was 5.5. Rates of uptake by the extensor varied rhythmically in phase with the leaflet movement rhythm, whereas rates for the flexor were similar at all times. Addition of 0.4`molar mannitol (or sorbitol) to the medium magnified the amplitude of the rhythm in H⁺ uptake and release by extensor tissue and revealed a rhythm with flexor tissue. In the flexor, mannitol promoted H⁺ release (or reduced H⁺ uptake) at all times. We propose that mannitol reduces flexor cell turgor, and that low turgor activates the H⁺ pump. The magnitude and/or direction of pH changes varied with the initial pH of the medium. The pH values after 60 minutes converged to a narrow range, suggesting that cell wall pH might be regulated.