Oxaziclomefone, a new herbicide, inhibits wall expansion in maize cell-cultures without affecting polysaccharide biosynthesis, xyloglucan transglycosylation, peroxidase action or apoplastic ascorbate oxidation

BACKGROUND AND AIMS: Oxaziclomefone (OAC), a new herbicide, inhibits cell expansion, especially in roots and cell-cultures of gramineous monocots. OAC does not affect turgor in cultured maize cells, and must therefore inhibit wall-loosening or promote wall-tightening. METHODS: The effects of OAC in living cultured maize cells on various biochemical processes thought to influence wall extension were studied. KEY RESULTS: OAC did not affect 14C-incorporation from D-[U-14C]glucose into the major sugar residues of the cell wall (cellulosic glucose, non-cellulosic glucose, arabinose, xylose, galactose, mannose or uronic acids). OAC had no effect on 14C-incorporation from trans-[U-14C]cinnamate into wall-bound ferulate or its oxidative coupling-products. OAC did not influence the secretion or in-vivo action of peroxidase or xyloglucan endotransglucosylase activities-proposed wall-tightening and -loosening activities, respectively. The herbicide did not affect the consumption of extracellular L-ascorbate, an apoplastic solute proposed to act as an antioxidant and/or to generate wall-loosening hydroxyl radicals. CONCLUSIONS: OAC decreased wall extensibility without influencing the synthesis or post-synthetic modification of major architectural wall components, or the redox environment of the apoplast. The possible value of OAC as a probe to explore aspects of primary cell wall physiology is discussed.     

Hardening of root cell walls: a growth inhibitory response to salinity stress

Primary roots of intact maize plants (Zea mays L.) grown for several days in nutrient solutions containing 100 mol m⁻³ NaCl and additional calcium, had relatively inhibited rates of elongation. Possible physical restraints underlying this salt induced inhibition were investigated. The inhibition did not involve reductions in osmotic potential gradients and turgor in the tip tissues responsible for root elongation growth. The apparent yield threshold pressure, which is related to capacity of cell walls to undergo loosening by stress relaxation, was estimated psychrometrically in excised root tips. Salinity increased yield threshold values. Comparative root extensibility values were obtained for intact plants by determining the initial (1 min) increase in root elongation rate induced by an 0.1 MPa osmotic jump. Comparative extensibility was significantly reduced in the salinized root tips. Salinity did not reduce capacities for water efflux and associated elastic contraction in root tip tissues of intact plants exposed to hypertonic mannitol. We conclude that cell wall hardening in the elongating root tips is an important component of root growth inhibition induced by long-term salinization.     

Salinity Stress Inhibits Bean Leaf Expansion by Reducing Turgor, Not Wall Extensibility

Treatment of bean (Phaseolus vulgaris L.) seedlings with low levels of salinity (50 or 100 millimolar NaCl) decreased the rate of light-induced leaf cell expansion in the primary leaves over a 3 day period. This decrease could be due to a reduction in one or both of the primary cellular growth parameters: wall extensibility and cell turgor. Wall extensibility was assessed by the Instron technique. Salinity did not decrease extensibility and caused small increases relative to the controls after 72 hours. On the other hand, 50 millimolar NaCl caused a significant reduction in leaf bulk turgor at 24 hours; adaptive decreases in leaf osmotic potential (osmotic adjustment) were more than compensated by parallel decreases in the xylem tension potential and the leaf apoplastic solute potential, resulting in a decreased leaf water potential. It is concluded that in bean seedlings, mild salinity initially affects leaf growth rate by a decrease in turgor rather than by a reduction in wall extensibility. Moreover, longterm salinization (10 days) resulted in an apparent mechanical adjustment, i.e. an increase in wall extensibility, which may help counteract reductions in turgor and maintain leaf growth rates.     

Analysis of the dynamic and steady-state responses of growth rate and turgor pressure to changes in cell parameters

The physical analysis of plant cell enlargment is extended to show the dependence of turgor pressure and growth rate under steady-state conditions on the parameters which govern cell wall extension and water transport in growing cells and tissues, and to show the dynamic responses of turgor and growth rate to instantaneous changes in one of these parameters. The analysis is based on the fact that growth requires simultaneous water uptake and irreversible wall expansion. It shows that when a growing cell is perturbed from its steady-state growth rate, it will approach the steady-state rate with exponential kinetics. The half-time of the transient adjustment depends on the biophysical parameters governing both water transport and irreversible wall expansion. When wall extensibility is small compared to hydraulic conductance, the growth rate is controlled by the yielding properties of the cell wall, while the half-time for changes in growth rate is controlled by the water transport parameters. The reverse situation occurs when hydraulic conductance is lower than wall extensibility. The analysis also shows explicitly that turgor pressure is tightly coupled with growth rate when growth is controlled by both water transport and wall yielding parameters.     

Growth Physics in Nitella: a Method for Continuous in Vivo Analysis of Extensibility Based on a Micro-manometer Technique for Turgor Pressure

The view that the plant cell grows by the yielding of the cell wall to turgor pressure can be expressed in the equation: rate = cell extensibility × turgor. All growth rate responses can in principle be resolved into changes in the 2 latter variables. Extensibility will relate primarily to the yielding properties of the cell wall, turgor primarily to solute uptake or production. Use of this simple relationship in vivo requires that at least 2 of the 3 variables be measured in a growing cell. Extensibility is not amenable to direct measurement. Data on rate and turgor for single Nitella cells can, however, be continuously gathered to permit calculation of extensibility (rate/turgor). Rate is accurately obtained from measurements on time-lapse film. Turgor is estimated in the same cell, to within 0.1 atm or less, by measurement of the ability of the cell to compress gas trapped in the closed end of a capillary the open end of which is in the cell vacuole. The method is independent of osmotic equilibrium. It operates continuously for several days, over a several fold increase in cell length, and has response time of less than one minute. Rapid changes in turgor brought on by changes in tonicity of the medium, show that extensibility, as defined above, is not constant but has a value of zero unless the cell has about 80% of normal turgor. Because elastic changes are small, extensibility relates to growth. Over long periods of treatment in a variety of osmotica the threshold value for extensibility and growth is seen to fall to lower values to permit resumption of growth at reduced turgor. A brief period of rapid growth (5× normal) follows the return to normal turgor. All variables then become normal and the cycle can be repeated. The cell remains essentially at osmotic equilibrium, even while growing at 5× the normal rate. The method has potential for detailed in vivo analyses of “wall softening.”     

Mechanism for increased leaf growth in elevated CO2

The effect of exposure to elevated CO₂ on the processes of leafcell production and leaf cell expansion was studied using primaryleaves of Phaseolus vulgaris L. Cell division and expansionwere separated temporally by exposing seedlings to dim red lightfor 10 d (when leaf cell division was completed) followed byexposure to bright white light for 14 d (when leaf growth wasentirely dependent on cell expansion). When plants were exposedto elevated CO₂ during the phase of cell expansion, epidermalcell size and leaf area development were stimulated. Three piecesof evidence suggest that this occurred as a result of increasedcell wall loosening and extensibility, (i) cell wall extensibility(WEx, measured as tensiometric extension using an Instron) wassignificantly increased, (ii) cell wall yield turgor (V, MPa)was reduced and (iii) xyloglucan endotransglycosylase (XET)enzyme activity was significantly increased. When plants wereexposed to elevated CO₂ during the phase of cell division, thenumber of epidermal cells was increased whilst final cell sizewas significantly reduced and this was associated with reducedfinal leaf area, WEx and XET activity. When plants were exposedto elevated CO₂ during both phases of cell division and expansion,leaf area development was not affected. For this treatment,however, the number of epidermal cells was increased, but cellexpansion was inhibited, despite exposure to elevated CO₂ duringthe expansion phase. Assessments were also made of the spatialpatterns of WEx across the expanding leaf lamina and the datasuggest that exposure to elevated CO₂ during the phase of leafexpansion may lead to enhanced extensibility particularly atbasal leaf margins which may result in altered leaf shape. The data show that both cell production and expansion were stimulatedby elevated CO₂, but that leaf growth was only enhanced by exposureto elevated CO₂ in the cell expansion phase of leaf development.Increased leaf cell expansion is, therefore, an important mechanismfor enhanced leaf growth in elevated CO₂, whilst the importanceof increased leaf cell production in elevated CO₂ remains tobe elucidated. Key words: Phaseolus vulgaris L., dwarf beans, elevated CO², biophysics of cell expansion, xyloglucan endotransglycosylase, XET, water relations     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

Reduced V-ATPase activity in the trans-Golgi network causes oxylipin-dependent hypocotyl growth Inhibition in Arabidopsis

Regulated cell expansion allows plants to adapt their morphogenesis to prevailing environmental conditions. Cell expansion is driven by turgor pressure created by osmotic water uptake and is restricted by the extensibility of the cell wall, which in turn is regulated by the synthesis, incorporation, and cross-linking of new cell wall components. The vacuolar H(+)-ATPase (V-ATPase) could provide a way to coordinately regulate turgor pressure and cell wall synthesis, as it energizes the secondary active transport of solutes across the tonoplast and also has an important function in the trans-Golgi network (TGN), which affects synthesis and trafficking of cell wall components. We have previously shown that det3, a mutant with reduced V-ATPase activity, has a severe defect in cell expansion. However, it was not clear if this is caused by a defect in turgor pressure or in cell wall synthesis. Here, we show that inhibition of the tonoplast-localized V-ATPase subunit isoform VHA-a3 does not impair cell expansion. By contrast, inhibition of the TGN-localized isoform VHA-a1 is sufficient to restrict cell expansion. Furthermore, we provide evidence that the reduced hypocotyl cell expansion in det3 is conditional and due to active, hormone-mediated growth inhibition caused by a cell wall defect.     

Estimation of growth parameters in salt-stressed maize: comparison of the pressure-block and applied-tension techniques

A custom-built pressure block was used to estimate the effective turgor (turgor pressure minus the yield threshold) and the cell wall extensibility of the growing zone of the third leaves of 8-d-old maize (Zea mays L.) seedlings. In response to cell wall loosening, pressure in the chamber increased rapidly and reached a maximum after approximately 60 min. Plants treated with 80 mol m^?3 NaCl for 4 h were compared to control plants. Pressure-block analysis revealed that salinity reduced effective turgor, but had no effect on cell wall extensibility. These results are qualitatively and quantitatively similar to those obtained with an applied-tension technique used previously in our laboratory. This study indicates that the pressure-block and applied-tension techniques, which use very different methodologies, estimate similar growth parameters.     

Inhibition of maize endosperm cell division and endoreduplication by exogenously applied abscisic acid

Abscisic acid (ABA) is thought to play a role in inhibiting or aborting kernel growth during water deficit. To test the responsiveness of early endosperm development to ABA concentrations, cylinders containing (±)ABA in a buffered agar medium were applied to the apical pericarp surface of kernels on intact, well‐watered maize ( Zea mays L. cv. Pioneer Brand 3925) plants from 5 to 11 days after pollination (DAP). Endosperm nuclei were analyzed by flow cytometry to assess effects on cell division and endoreduplication. ABA treatments of ≥ 100 µM substantially decreased endosperm cell numbers and fresh weight accumulation, but did not affect average cell size. ABA at ≥ 300 µM decreased the proportion of nuclei in the size classes ≥ 12C, indicating that the rate of transition to endoreduplication status was inhibited, and decreased the progressive advance from 12C to 24C to 48C, indicating that the rate of S‐phase cycling of endoreduplicating cells was inhibited. We conclude that cell division was more responsive to ABA concentrations than were endoreduplication or cell expansion growth.     

Dynamic Relation between Expansion and Cellular Turgor in Growing Grape (Vitis vinifera L.) Leaves

Measurements of the growth and water relations of expanding grape (Vitis vinifera L.) leaves have been used to determine the relationship between leaf expansion rate and leaf cell turgor. Direct measurement of turgor on the small (approximately 15 micrometer diameter) epidermal cells over the midvein of expanding grape leaves was made possible by improvements in the pressure probe technique. Leaf expansion rate and leaf water status were perturbed by environmentally induced changes in plant transpiration. After establishing a steady state growth rate, a step decrease in plant transpiration resulted in a rapid and large increase in leaf cell turgor (0.25 megapascal in 5 minutes), and leaf expansion rate. Subsequently, leaf expansion rate returned to the original steady state rate with no change in cell turgor. These results indicate that the expansion rate of leaves may not be strongly related to the turgor of the leaf cells, and that substantial control of leaf expansion rate, despite changes in turgor, may be part of normal plant function. It is suggested that a strictly physical interpretation of the parameters most commonly used to describe the relationship between turgor and growth in plant cells (cell wall extensibility and yield threshold) may be inappropriate when considering the process of plant cell expansion.     

Rheological analysis of viscoelastic cell wall changes in maize coleoptiles as affected by auxin and osmotic stress

The effects of auxin and osmotic stress on elongation growth of maize (Zea mays L.) coleoptile segments are accompanied by characteristic changes in the extensibility of the growth-limiting cell walls. At full turgor auxin causes growth by an increase in wall extensibility (wall looseining). Growth can be stopped by an osmotically produced step-down in turgor of 0.45 MPa. Under these conditions auxin causes the accumulation of a potential for future wall extension which is released after restoration of full turgor. Turgor reduction causes a reversible decrease in wall extensibility (wall stiffening) both in the presence and absence of auxin. These changes in vivo are correlated with corresponding changes in the rheological properties of the cell walls in vitro which can be traced back to specific modifications in the shape of the hysteretic stress-strain relationship. The longitudinally load-bearing walls of the coleoptile demonstrate almost perfect viscoelasticity as documented by a nearly closed hysteresis loop. Auxin-mediated wall loosening causes an increase of loop width and thus affects primarily the amount of hysteresis in the isolated wall. In contrast, turgor reduction by osmotic stress reduces loop length and thus affects primarily the amount of viscoelastic wall extensibility. Pretreatment of segments with anoxia and H₂O₂ modify the hysteresis loop in agreement with the conclusion that the wall-stiffening reaction visualized under osmotic stress in vivo is an O₂-dependent process in which O₂ can be substituted by H₂O₂. Cycloheximide specifically inhibits auxin-mediated wall loosening without affecting wall stiffening, and this is mirrored in specific changes of the hysteresis loop. Corroborating a previous in vivo study (Hohl et al. 1995, Physiol. Plant. 94: 491–498) these results show that cell wall stiffening in vivo can also be demonstrated by Theological measurements with the isolated cell wall and that this process can be separated from cell wall loosening by specific changes in the shape of the hysteresis loop.     

Growth control by cell wall pectins

Plant cell growth is controlled by the balance between turgor pressure and the extensibility of the cell wall. Several distinct classes of wall polysaccharides and their interactions contribute to the architecture and the emergent features of the wall. As a result, remarkable tensile strength is achieved without relinquishing extensibility. The control of growth and development does not only require a precisely regulated biosynthesis of cell wall components, but also constant remodeling and modification after deposition of the polymers. This is especially evident given the fact that wall deposition and cell expansion are largely uncoupled. Pectins form a functionally and structurally diverse class of galacturonic acid-rich polysaccharides which can undergo abundant modification with a concomitant change in physicochemical properties. This review focuses on homogalacturonan demethylesterification catalyzed by the ubiquitous enzyme pectin methylesterase (PME) as a growth control module. Special attention is drawn to the recently discovered role of this process in primordial development in the shoot apical meristem.     

Growth control by cell wall pectins

Plant cell growth is controlled by the balance between turgor pressure and the extensibility of the cell wall. Several distinct classes of wall polysaccharides and their interactions contribute to the architecture and the emergent features of the wall. As a result, remarkable tensile strength is achieved without relinquishing extensibility. The control of growth and development does not only require a precisely regulated biosynthesis of cell wall components, but also constant remodeling and modification after deposition of the polymers. This is especially evident given the fact that wall deposition and cell expansion are largely uncoupled. Pectins form a functionally and structurally diverse class of galacturonic acid-rich polysaccharides which can undergo abundant modification with a concomitant change in physicochemical properties. This review focuses on homogalacturonan demethylesterification catalyzed by the ubiquitous enzyme pectin methylesterase (PME) as a growth control module. Special attention is drawn to the recently discovered role of this process in primordial development in the shoot apical meristem.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

Rapid Response of the Yield Threshold and Turgor Regulation during Adjustment of Root Growth to Water Stress in Zea mays

Responses of cortical cell turgor (P) following rapid changes in osmotic pressure ([pi]m) were measured throughout the elongation zone of maize (Zea mays L.) roots using a cell pressure probe and compared with simultaneously measured root elongation to evaluate: yield threshold (Y) (minimum P for growth), wall extensibility, growth-zone radial hydraulic conductivity (K), and turgor recovery rate. Small increases in [pi]m (0.1 MPa) temporarily decreased P and growth, which recovered fully in 5 to 10 min. Under stronger [pi]m (up to 0.6 MPa), elongation stopped for up to 30 min and then resumed at lower rates. Recoveries in P through solute accumulation and lowering of Y enabled growth under water stress. P recovery was as much as 0.3 MPa at [pi]m = 0.6 MPa, but recovery rate declined as water stress increased, suggesting turgor-sensitive solute transport into the growth zone. Under strong [pi]m, P did not recover in the basal part of the growth zone, in conjunction with a 30% shortening of the growth zone. Time courses showed Y beginning to decrease within several minutes after stress imposition, from about 0.65 MPa to a minimum of about 0.3 MPa in about 15 min. The data concerning Y were not confounded significantly by elastic shrinkage. K was high (1.3 x 10-10 m2 s-1 MPa-1), suggesting very small growth-induced water potential gradients.     

Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported thatthe cell walls of growing rye coleoptiles exhibit irreversible(plastic) extensibility in a rheological extension test. Basicallysimilar measurements with cell walls of maize coleoptiles hadpreviously shown that the apparent plastic extensibility determinedin this material is in reality due to the slowly reversible(viscoelastic) extensibility of the walls. A recent reinvestigationof this discrepancy showed that rye coleoptile walls also behaveas a perfectly viscoelastic material if precautions are takento prevent measuring artefacts. Similar results were obtainedwith cell walls from the growing zone of various other seedlingorgans (maize mesocotyl, maize root, cucumber hypocotyl). Itis concluded that plastic extensibility has not yet been convincinglydemonstrated by rheological tests that determine the intrinsicmaterial properties of cell walls. Reported changes in mechanicalmaterial properties of cell walls produced by growth-controllingfactors such as auxin or light may generally be attributed tochanges in viscoelasticity which are not directly related tothe chemo-rheological processes controlling wall extension ofgrowing cells. Key words: Cell wall extensibility, extension growth, plastic cell wall extensibility, viscoelastic cell wall extensibility     

Wall extensibility, wall pH and tissue osmolality: significance for auxin and ethylene-enhanced petiole growth in semi-aquatic plants

Abstract. Auxin and ethylene both enhance cell elongation in intact petioles of the semi-aquatic plants Regnellidium diphyllum and Nymphoides peltata. The authors now show that auxin but not ethylene increases the in vitro extensibility of cell walls. No response to ethylene occurs in auxin-depleted tissue. Neither hormone regulates cell expansion by direct control of internal osmolality (OS). During growth of segments, OS (and hence turgor) declines rapidly over the first 5–6 h with a net loss of osmotic solutes. Thereafter, an apparent threshold OS is maintained with net gains in osmostic solutes ( Nymphoides ) or further net losses ( Regnellidium ). Although wall extensibility determines initial rates of hormone-induced cell expansion, the primary control of wall loosening appears to differ in the two species. Nymphoides shows typical 'acid growth', and fusicoccin, auxin and ethylene (with auxin) all enhance proton secretion. In Regnellidium , neither low pH nor fusicoccin (FC) alters the rate of cell expansion, although proton secretion is stimulated by FC. Stress relaxation studies using low pH treatment of living or frozen-thawed segments show increases in the extensibility of walls in vitro for Nymphoides but not for Regnellidium. The authors propose that extensibility may be controlled by wall pH in Nymphoides but the availability of effective wall-loosening sites determines extensibility in Regnellidium.     

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.     

Primary events regulating stem growth at low water potentials

Cell enlargement is inhibited by inadequate water. As a first step toward understanding the mechanism, all the physical parameters affecting enlargement were monitored to identify those that changed first, particularly in coincidence with the inhibition. The osmotic potential, turgor, yield threshold turgor, growth-induced water potential, wall extensibility, and conductance to water were measured in the elongating region, and the water potential was measured in the xylem of stems of dark-grown soybean (Glycine max [L.] Merr.) seedlings. A stepdown in water potential was achieved around the roots by transplanting the seedlings to vermiculite of low water content, and each of the parameters was measured simultaneously in the same plants while intact or within a few minutes of being intact using a newly developed guillotine psychrometer. The gradient of decreasing water potential from the xylem to the enlarging cells (growth-induced water potential) was the first of the parameters to decrease to a growth-limiting level. The kinetics were the same as for the inhibition of growth. The decreased gradient was caused mostly by a decreased water potential of the xylem. This was followed after 5 to 10 hours by a similar decrease in cell wall extensibility and tissue conductance for water. Later, the growth-induced water potential recovered as a result of osmotic adjustment and a rise in the water potential of the xylem. Still later, moderate growth resumed at a rate apparently determined by the low wall extensibility and tissue conductance for water. The turgor did not change significantly during the experiment. These results indicate that the primary event during the growth inhibition was the change in the growth-induced water potential. Because the growth limitation subsequently shifted to the low wall extensibility and tissue conductance for water, the initial change in potential may have set in motion subsequent metabolic changes that altered the characteristics of the wall and cell membranes.