Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon

The extent and occurrence of water stress-induced “patchy” CO₂ uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [¹⁴C]CO₂ before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to −1.1 megapascals during a 4-day period did result in heterogenous CO₂ assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO₂ assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO₂] in stressed bean leaves. When extent of heterogenous CO₂ uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to −1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO₂ assimilation patterns when stressed to a leaf water potential of −2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : I. General Characteristics

Xerosicyos danguyi H. Humb. (Cucurbitaceae) is an unusual leaf succulent endemic to Madagascar. Under well-watered conditions the plant exhibited Crassulacean acid metabolism (CAM), as characterized by large diurnal changes in titratable acidity, predominantly nighttime stomatal opening and CO₂ uptake, and high δ¹³C values. When plants were exposed to water stress for a minimum of a month, they shifted to a mode of carbon metabolism previously labeled CAM-idling. Under this mode of metabolism, the plants exhibited reduced stomatal opening, reduced CO₂ uptake, dampened diurnal fluctuations in titratable acidity, and no apparent changes in the δ¹³C values. Additionally, investigations showed that the stress hormones 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) and abscisic acid increased as much as 6-fold in the water-stressed plants. The results are discussed in relation to physiological significance and evolution of the CAM-idling mode of metabolism.     

Crassulacean Acid Metabolism in the Succulent C(4) Dicot, Portulaca oleracea L Under Natural Environmental Conditions

Crassulacean acid metabolism (CAM) was examined under natural environmental conditions in the succulent C₄ dicot Portulaca oleracea L. Two groups of plants were monitored; one was watered daily (well watered), while the other received water once every 3 to 4 weeks to produce a ψ of −8 bars (drought stressed). Gas exchange, transpiration rate, and titratable acidity were measured for 24-hour periods during the growing season. CAM activity was greatest in drought-stressed plants during late August which had 13 hour days and day/night temperatures of 35/15°C. Under these conditions net CO₂ uptake occurred slowly throughout the night. Diurnal fluctuations of titratable acidity took place in both leaves and stems with amplitudes of 17 and 47 microequivalents per gram fresh weight, respectively. Transpiration data indicated greater opening of stomata during the night than the day. CAM was less pronounced in drought-stressed P. oleracea plants in July and September; neither dark CO₂ uptake nor positive carbon balance occurred during the July measurements. In contrast, well-watered plants appeared to rely on C₄ photosynthesis throughout the season, although some acid fluctuations occurred in stems of these plants during September.     

Water Deficit and Associated Changes in Some Photosynthetic Parameters in Leaves of ;Valencia' Orange (Citrus sinensis [L.] Osbeck)

Photosynthetic CO₂ assimilation, transpiration, ribulose-1,5-bisphosphate carboxylase (RuBPCase), and soluble protein were reduced in leaves of water-deficit (stress) `Valencia' orange (Citrus sinensis [L.] Osbeck). Maximum photosynthetic CO₂ assimilation and transpiration, which occurred before midday for both control and stressed plants, was 58 and 40%, respectively, for the stress (−2.0 megapascals leaf water potential) as compared to the control (−0.6 megapascals leaf water potential). As water deficit became more severe in the afternoon, with water potential of −3.1 megapascals for the stressed leaves vs. −1.1 megapascals for control leaves, stressed-leaf transpiration declined and photosynthetic CO₂ assimilation rapidly dropped to zero. Water deficit decreased both activation and total activity of RuBPCase. Activation of the enzyme was about 62% (of fully activated enzyme in vitro) for the stress, compared to 80% for the control. Water deficit reduced RuBPCase initial activity by 40% and HCO₃⁻/Mg²⁺-saturated activity by 22%. However, RuBPCase for both stressed and control leaves were similar in K_cat (25 moles CO₂ per mole enzyme per second) and K_m for CO₂ (18.9 micromolar). Concentrations of RuBPCase and soluble protein of stressed leaves averaged 80 and 85%, respectively, of control leaves. Thus, reductions in activation and concentration of RuBPCase in Valencia orange leaves contributed to reductions in enzyme activities during water-deficit periods. Declines in leaf photosynthesis, soluble protein, and RuBPCase activation and concentration due to water deficit were, however, recoverable at 5 days after rewatering.     

Minor Physiological Response to Elevated CO(2) by the CAM Plant Agave vilmoriniana

One-year-old plants of the CAM leaf succulent Agave vilmoriniana Berger were grown outdoors at Riverside, California. Potted plants were acclimated to CO₂-enrichment (about 750 microliters per liter) by growth for 2 weeks in an open-top polyethylene chamber. Control plants were grown nearby where the ambient CO₂ concentration was about 370 microliters per liter. When the plants were well watered, CO₂-induced differences in stomatal conductances and CO₂ assimilation rates over the entire 24-hour period were not large. There was a large nocturnal acidification in both CO₂ treatments and insignificant differences in leaf chlorophyll content. Well watered plants maintained water potentials of −0.3 to −0.4 megapascals. When other plants were allowed to dry to water potentials of −1.2 to −1.7 megapascals, stomatal conductances and CO₂ uptake rates were reduced in magnitude, with the biggest difference in Phase IV photosynthesis. The minor nocturnal response to CO₂ by this species is interpreted to indicate saturated, or nearly saturated, phosphoenolpyruvate carboxylase activity at current atmospheric CO₂ concentrations. CO₂-enhanced diurnal activity of ribulose bisphosphate carboxylase activity remains a possibility.     

Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO₂ uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.

The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days.

     

Induction of Acid Metabolism in Portulacaria afra

Portulacaria afra, a succulent plant, shifts from a predominantly C₃ mode of gas exchange to a typical Crassulacean acid metabolism type CO₂ uptake in response to water or NaCl stress. Control plants in the absence of water stress assimilated CO₂ during the light (about 7-8 mg CO₂ dm⁻² hr⁻¹), transpiration (about 1.5 g dm⁻² hr⁻¹) was predominantly during the day, stomates were open during the day, and there was little diurnal organic acid fluctuation. Stressed plants showed only dark CO₂ uptake and dark water loss, nocturnal stomatal opening, and an increased diurnal fluctuation of titratable acidity. Within 2 weeks after rewatering, stressed plants returned to the control acid fluctuation levels indicating that the response to stress was reversible.     

Water-Stress-Induced Ethylene Production in Wheat : A Fact or Artifact?

Effects of water stress on ethylene evolution from excised leaf segments and intact plants of wheat (Triticum aestivum L. cv Katepwa) were studied. Excised leaf segments of 8 day or 6 week old plants were dried until they lost 8% of their fresh weight (water potential about −2.3 megapascals). These and nondried control leaf segments (water potential about −1.0 megapascal) were sealed in glass tubes, and their ethylene production rates were compared by head space analysis via gas-chromatography. The dried leaves of both ages produced significantly more ethylene than the corresponding controls. However, when 6 week old intact plants were water-stressed by withholding water supply, and their ethylene production measured using a continuousflow system, no increase in ethylene was deteceted despite a drop in water potential to −2.9 megapascals over 6 days. Even the leaf segments excised from plants that had been subjected to water stress for 2, 4, or 6 days produced no more ethylene (in sealed tubes) than the leaves from well-watered plants. In fact, the ethylene production by these segments decreased with the increase in the severity of stress experienced by the plants. The results show that the commonly reported overproduction of ethylene by excised leaves subjected to rapid drying represents an artifact, which has little relevance to the water stress responses of intact wheat plants.     

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

Effects of Various Levels of CO(2) on the Induction of Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

In response to water stress, Portulacaria afra (L.) Jacq. (Portulacaceae) shifts its photosynthetic carbon metabolism from the Calvin-Benson cycle for CO₂ fixation (C₃) photosynthesis or Crassulacean acid metabolism (CAM)-cycling, during which organic acids fluctuate with a C₃-type of gas exchange, to CAM. During the CAM induction, various attributes of CAM appear, such as stomatal closure during the day, increase in diurnal fluctuation of organic acids, and an increase in phosphoenolpyruvate carboxylase activity. It was hypothesized that stomatal closure due to water stress may induce changes in internal CO₂ concentration and that these changes in CO₂ could be a factor in CAM induction. Experiments were conducted to test this hypothesis. Well-watered plants and plants from which water was withheld starting at the beginning of the experiment were subjected to low (40 ppm), normal (ca. 330 ppm), and high (950 ppm) CO₂ during the day with normal concentrations of CO₂ during the night for 16 days. In water-stressed and in well-watered plants, CAM induction as ascertained by fluctuation of total titratable acidity, fluctuation of malic acid, stomatal conductance, CO₂ uptake, and phosphoenolpyruvate carboxylase activity, remained unaffected by low, normal, or high CO₂ treatments. In well-watered plants, however, both low and high ambient concentrations of CO₂ tended to reduce organic acid concentrations, low concentrations of CO₂ reducing the organic acids more than high CO₂. It was concluded that exposing the plants to the CO₂ concentrations mentioned had no effect on inducing or reducing the induction of CAM and that the effect of water stress on CAM induction is probably mediated by its effects on biochemical components of leaf metabolism.     

Leaf and root growth dynamics in Eucalyptus globulus seedlings grown in drying soil

Summary Seedlings of Eucalyptus globulus growing in soil columns were subjected to a 24 day soil drying treatment. Water and solute potentials of both young expanding and fully expanded leaves declined under reduced soil water availability, while slightly higher turgor was sustained by the fully expanded leaves. Although leaf area of unwatered seedlings was smaller, the corresponding leaf dry weight was quite similar to that of well-watered seedlings. Soon after rewatering, leaf area of plants experiencing water shortage was comparable to that of well-watered plants. It seems that a difference in wall properties between juvenile and mature leaves allows for an effective pattern of water use by eucalypt plants growing in drying soil. Some stomatal opening is sustained and therefore, presumably, some carbon may be fixed, keeping the carbon balance of the whole plant positive, and allowing a continuous cell division despite the limited water supply. The highest root density of both well-watered and unwatered plants was found in the upper soil layers. However, root growth of unwatered seedlings was gradually increased in the deeper soil layers, where thicker root apices and higher soil water depletion rates per unit root length were recorded. As a consequence, root absorbing surface area was as large in unwatered plants as in well-watered plants.     

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.     

Plant morphological and biochemical responses to field water deficits: I. Responses of glutathione reductase activity and paraquat sensitivity

The effects of water deficits on plant morphology and biochemistry were analyzed in two photoperiodic strains of field-grown cotton (Gossypium hirsutum L.). Plants grown under dryland conditions exhibited a 40 to 85% decrease in leaf number, leaf area index, leaf size, plant height, and total weight per plant. Gross photosynthesis decreased from 0.81 to 0.47 milligram CO₂ fixed per meter per second and the average midday water, osmotic, and turgor potentials decreased to −2.1, −2.4, and 0.3 megapascals, respectively.

There was a progressive increase in glutathione reductase activity and in the cellular antioxidant system in the leaves of stressed plants compared to the irrigated controls. The stress-induced increases in enzyme activity occurred at all canopy positions analyzed.

Irrigation of the dryland plots following severe water stress resulted in a 50% increase in leaf area per gram fresh weight in newly expanded leaves of both strains over the leaves which had expanded under the dryland conditions. Paraquat resistance (a relative measure of the cellular antioxidant system) decreased in the strain T25 following irrigation. Glutathione reductase activities remained elevated in the T25 and T185 leaves which were expanded fully prior to irrigation and in the leaves which expanded following the irrigation treatment.

     

Analysis of Stomatal and Nonstomatal Components in the Environmental Control of CO(2) Exchange in Leaves of Welwitschia mirabilis

In well-watered plants of Welwitschia mirabilis, grown in the glass-house under high irradiance conditions, net CO₂ assimilation was almost exclusively observed during the daytime. The plants exhibited a very low potential for Crassulacean acid metabolism, which usually resulted in reduced rates of net CO₂ loss for several hours during the night. In leaves exposed to the diurnal changes in temperature and humidity typical of the natural habitats, CO₂ assimilation rates in the light were markedly depressed under conditions resembling those occurring during midday, when leaf temperatures and the leaf-air vapor pressure differences were high (36°C and 50 millibars bar⁻¹, respectively). Studies on the relationship between CO₂ assimilation rate and intercellular CO₂ partial pressure at various temperatures and humidities showed that this decrease in CO₂ assimilation was largely due to stomatal closure. The increase in the limitation of photosynthesis by CO₂ diffusion, which is associated with the strong decline in stomatal conductance in Welwitschia exposed to midday conditions, may significantly contribute to the higher ¹³C content of Welwitschia compared to the majority of C₃ species.     

Fluorescence Quenching and Gas Exchange in a Water Stressed C(3) Plant, Digitalis lanata

A leaf cuvette has been adapted for use with a pulse-modulation fluorometer and an open gas exchange system. Leaf water potential (ψ) was decreased by withholding watering from Digitalis lanata EHRH. plants. At different stages of water deficiency the photochemical (q_Q) and nonphotochemical (q_E) fluorescence quenching was determined during the transition between darkness and light-induced steady state photosynthesis of the attached leaves. In addition, the steady state CO₂ and H₂O gas exchange was recorded. Following a decrease of leaf water potential with increasing water deficiency, the transition of photochemical quenching was almost unaffected, whereas nonphotochemical quenching increased. This is indicative of an enhanced thylakoid membrane energization during the transition and is interpreted as a partial inhibition of either the ATP generating or the ATP consuming reaction sequences. Complete reversion of the stress induced changes was achieved within 6 hours after rewatering. In contrast to the variations during transition, the final steady state values of q_Q and q_E remained unchanged over the entire stress range from −0.7 to −2.5 megapascals. From these results we conclude that, once established, electron transport via photosystem II and the transmembrane proton gradient remain unaffected by water stress. These data are indicative of a protective mechanism against photoinhibition during stress, when net CO₂ uptake is limited.     

Protoplast volume:water potential relationship and bound water fraction in spinach leaves

Methods used to estimate the (nonosmotic) bound water fraction (BWF) (i.e. apoplast water) of spinach (Spinacia oleracea L.) leaves were evaluated. Studies using three different methods of pressure/volume (P/V) curve construction all resulted in a similar calculation of BWF; approximately 40%. The theoretically derived BWF, and the water potential (Ψ_w)/relative water content relationship established from P/V curves were used to establish the relationship between protoplast (i.e. symplast) volume and Ψ_w. Another method of establishing the protoplast volume/Ψ_w relationship in spinach leaves was compared with the results from P/V curve experiments. This second technique involved the vacuum infiltration of solutions at a range of osmotic potentials into discs cut from spinach leaves. These solutions contained radioactively labeled H₂O and sorbitol. This dual label infiltration technique allowed for simultaneous measurement of the total and apoplast volumes in leaf tissue; the difference yielded the protoplast volume. The dual label infiltration experiments and the P/V curve constructions both showed that below −1 megapascals, protoplast volume decreases sharply with decreasing water potential; with 50% reduction in protoplast volume occurring at −1.8 megapascals leaf water potential.     

Facultative CAM photosynthesis (crassulacean acid metabolism) in four species of <Emphasis Type="Italic">Calandrinia</Emphasis>, ephemeral succulents of arid Australia

Crassulacean acid metabolism (CAM) was demonstrated in four small endemic Australian terrestrial succulents from the genus Calandrinia (Montiaceae) viz. C. creethiae, C. pentavalvis, C. quadrivalvis and C. reticulata. CAM was substantiated by measurements of CO₂ gas-exchange and nocturnal acidification. In all species, the expression of CAM was overwhelmingly facultative in that nocturnal H⁺ accumulation was greatest in droughted plants and zero, or close to zero, in plants that were well-watered, including plants that had been droughted and were subsequently rewatered, i.e. the inducible component was proven to be reversible. Gas-exchange measurements complemented the determinations of acidity. In all species, net CO₂ uptake was restricted to the light in well-watered plants, and cessation of watering was followed by a progressive reduction of CO₂ uptake in the light and a reduction in nocturnal CO₂ efflux. In C. creethiae, C. pentavalvis and C. reticulata net CO₂ assimilation was eventually observed in the dark, whereas in C. quadrivalvis nocturnal CO₂ exchange approached the compensation point but did not transition to net CO₂ gain. Following rewatering, all species returned to their original well-watered CO₂ exchange pattern of net CO₂ uptake restricted solely to the light. In addition to facultative CAM, C. quadrivalvis and C. reticulata exhibited an extremely small constitutive CAM component as demonstrated by the nocturnal accumulation in well-watered plants of small amounts of acidity and by the curved pattern of the nocturnal course of CO₂ efflux. It is suggested that low-level CAM and facultative CAM are more common within the Australian succulent flora, and perhaps the world succulent flora, than has been previously assumed.     

Osmotic Response of Sugar Beet Source Leaves at CO(2) Compensation Point

As sugar beet source leaves lowered the CO₂ concentration to compensation point in a closed atmosphere, leaf thickness and relative water content decreased. Leaf water potential declined rapidly from −0.5 to −1.4 megapascals. At 340 microliters CO₂ per liter, water potential and sucrose, glucose, and fructose contents were steady in photosynthesizing source leaves. Within 90 minutes after leaves were exposed to a CO₂ concentration at the compensation point, leaf sucrose content declined to 60% of the preteatment level, rapidly in the first 30 minutes and then more slowly. During the subsequent 200 minutes, sucrose content increased to 180% of pretreatment level. Glucose and fructose remained unchanged during the treatment. Degradation of starch was sufficient to account for the additional sucrose that accumulated. Labeled carbon lost from starch appeared in sucrose and several other compounds that likely contributed to the recovery in leaf water content.     

Daily Changes in CO(2) and Water Vapor Exchange, Chlorophyll Fluorescence, and Leaf Water Relations in the Halophyte Mesembryanthemum crystallinum during the Induction of Crassulacean Acid Metabolism in Response to High NaCl Salinity

Simultaneous measurements of net CO₂ exchange, water vapor exchange, and leaf water relations were performed in Mesembryanthemum crystallinum during the development of crassulacean acid metabolism (CAM) in response to high NaCl salinity in the rooting medium. Determinations of chlorophyll a fluorescence were used to estimate relative changes in electron transport rate. Alterations in leaf mass per unit area, which—on a short-term basis—largely reflect changes in water content, were recorded continuously with a beta-gauge. Turgor pressure of mesophyll cells was determined with a pressure probe. As reported previously (K Winter, DJ von Willert [1972] Z Pflanzenphysiol 67: 166-170), recently expanded leaves of plants grown under nonsaline conditions showed gas-exchange characteristics of a C₃ plant. Although these plants were not exposed to any particular stress treatment, water content and turgor pressure regularly decreased toward the end of the 12 hour light periods and recovered during the following 12 hours of darkness. When the NaCl concentration of the rooting medium was raised to 400 millimolar, in increments of 100 millimolar given at the onset of the photoperiods for 4 consecutive days, leaf water content and turgor pressure decreased by as much as 30 and 60%, respectively, during the course of the photoperiods. These transient decreases probably triggered the induction of the biochemical machinery which is required for CAM to operate. After several days at 400 millimolar NaCl, when leaves showed features typical of CAM, overall turgor pressure and leaf mass per unit area had increased above the levels before onset of the salt treatment, and diurnal alterations in leaf water content were reduced. Net carbon gain during photoperiods and average intercellular CO₂ partial pressures at which net CO₂ uptake occurred, progressively decreased upon salinization. Reversible diurnal depressions in leaf conductance and net CO₂ uptake, with minima recorded in the middle of the photoperiods, preceded the occurrence of nocturnal net CO₂ uptake. During these reductions, intercellular CO₂ partial pressure and rates of photosynthetic electron transport decreased. With advancing age, leaves of plants grown under nonsaline conditions exhibited progressively greater diurnal reductions in turgor pressure and developed a low degree of CAM activity.     

Utilization of Ammonium as a Nitrogen Source : Effects of Ambient Acidity on Growth and Nitrogen Accumulation by Soybean

Dry matter accumulation of plants utilizing NH₄⁺ as the sole nitrogen source generally is less than that of plants receiving NO₃⁻ unless acidity of the root-zone is controlled at a pH of about 6.0. To test the hypothesis that the reduction in growth is a consequence of nitrogen stress within the plant in response to effects of increased acidity during uptake of NH₄⁺ by roots, nonnodulated soybean plants (Glycine max [L.] Merr. cv Ransom) were grown for 24 days in flowing nutrient culture containing 1.0 millimolar NH₄⁺ as the nitrogen source. Acidities of the culture solutions were controlled at pH 6.1, 5.1, and 4.1 ± 0.1 by automatic additions of 0.01 n H₂SO₄ or Ca(OH)₂. Plants were sampled at intervals of 3 to 4 days for determination of dry matter and nitrogen accumulation. Rates of NH₄⁺ uptake per gram root dry weight were calculated from these data. Net CO₂ exchange rates per unit leaf area were measured on attached leaves by infrared gas analysis. When acidity of the culture solution was increased from pH 6.1 to 5.1, dry matter and nitrogen accumulation were reduced by about 40% within 14 days. Net CO₂ exchange rates per unit leaf area, however, were not affected, and the decreased growth was associated with a reduction in rates of appearance and expansion of new leaves. The uptake rates of NH₄⁺ per gram root were about 25% lower throughout the 24 days at pH 5.1 than at 6.1. A further increase in solution acidity from pH 5.1 to 4.1 resulted in cessation of net dry matter production and appearance of new leaves within 10 days. Net CO₂ exchange rates per unit leaf area declined rapidly until all viable leaves had abscised by 18 days. Uptake rates of NH₄⁺, which were initially about 50% lower at pH 4.1 than at 6.1, continued to decline with time of exposure until net uptake ceased at 10 days. Since these responses also are characteristic of the sequence of responses that occur during onset and progression of a nitrogen stress, they corroborate our hypothesis.