Low conductances for CO2 diffusion from stomata to the sites of carboxylation in leaves of woody species

Concurrent measurements of leaf gas exchange and on-line ¹³C discrimination were used to evaluate the CO₂ conductance to diffusion from the stomatal cavity to the sites of carboxylation within the chloroplast (internal conductance; g_i). When photon irradiance was varied it appeared that g_i and/or the discrimination accompanying carboxylation also varied. Despite this problem, g_i, was estimated for leaves of peach (Prunus persica), grapefruit (Citrus paradisi), lemon (C. limon) and macadamia (Macadamia integrifolia) at saturating photon irradiance. Estimates for leaves of C. paradisi, C. limon and M. integrifolia were considerably lower than those previously reported for well-nourished herbaceous plants and ranged from 1.1 to2.2μmol CO₂ m^?2 s^?1 Pa^?1, whilst P. persica had a mean value of 3.5 μmol CO₂ m^?2 s^?1 Pa^?1. At an ambient CO₂ partial pressure of 33Pa, estimates of chloroplastic partial pressure of CO₂ (C_c) using measurements of CO₂ assimilation rate (A) and calculated values of g_i, and of partial pressure of CO₂ in the stomatal cavity (C_st) were as low as 11.2 Pa for C. limon and as high as 17.8Pa for peach. In vivo maximum rubisco activities (V_max) were also determined from estimates of C_c. This calculation showed that for a given leaf nitrogen concentration (area basis) C. paradisi and C. limon leaves had a lower V_max than P. persica, with C. paradisi and C. limon estimated to have only 10% of leaf nitrogen present as rubisco. Therefore, low CO₂ assimilation rates despite high leaf nitrogen concentrations in leaves of the evergreen species examined were explained not only by a low C_c but also by a relatively low proportion of leaf nitrogen being used for photosynthesis. We also show that simple one-dimensional equations describing the relationship between leaf internal conductance from stomatal cavities to the sites of carboxylation and carbon isotope discrimination (Δ) can lead to errors in the estimate of g_i. Potential effects of heterogeneity in stomatal aperture on carbon isotope discrimination may be particularly important and may lead to a dependence of g_i upon CO₂ assimilation rate. It is shown that for any concurrent measurement of A and Δ, the estimate of C_c is an overestimate of the correct photosynthetic capacity-weighted value, but this error is probably less than 1.0 Pa.     

CO2 assimilation, respiration and chlorophyll fluorescence in peach leaves infected by Taphrina deformans

Photosynthesis, respiration and chlorophyll fluorescence parameters were determined in peach ( Prunus persica L. cv. Dixired) leaves naturally infected by Taphrina deformans (Berk.) Tul. and in healthy leaves (controls), in two successive springs. A drastic decrease in net photosynthesis and an evident increase in respiration in curled leaves were noted. The instantaneous PSII fluorescence yield, with no (F₀) and with (F₀) quenching component, and steady state fluorescence yield (under actinic light, F_s) were essentially unchanged. Maximal fluorescence in dark-adapted (F_m) and illuminated (F'_m) leaves and the corresponding variable fluorescence (F_v and F_v) clearly decreased. The indicators of PSII quantum yield (F_v/F_m) in dark-adapted leaves, and the potential PSII excitation capture efficiency (F'_v/F'_m) and the quantum yield of PSII (q_p [F'_v/F'_m]) in the light were also significantly lower in curled leaves. Decreasing tendencies were also noted for the PSII photochemical yield (photochemical quenching, q_p) and in the energy status of the chloroplast (non-photochemical quenching, q_N, and Stern-Vollmer value, NPQ) although the differences were not always significant. In curled leaves the main alteration documented is the imbalance between the drastic inhibition of CO₂ fixation and the moderate decrease in photochemical reactions (i.e. F_v/F_m and ΔF/F'_m), indicating changes in the energy flux.     

CO2 uptake and transport in leaf mesophyll cells

Abstract The acquisition of inorganic carbon for photosynthetic assimilation by leaf mesophyll cells and chloroplasts is discussed with particular reference to membrane permeation of CO₂ and HCO^?₃. Experimental evidence indicates that at the apoplast pH normally experienced by leaf mesophyll cells (pH 6–7) CO₂ is the principal species of inorganic carbon taken up. Uptake of HCO^?₃ may also occur under certain circumstances (i.e. pH 8.5), but its contribution to the net flux of inorganic carbon is small and HCO^?₃ uptake does not function as a CO₂-concentrating mechanism. Similarly, CO₂ rather than HCO^?₃ appears to be the species of inorganic carbon which permeates the chloroplast envelope. In contrast to many C₃ aquatic plants and C₄ plants, C₃ terrestrial plants lack specialized mechanisms for the acquisition and transport of inorganic carbon from the intercellular environment to the site of photosynthetic carboxylation, but rely upon the diffusive uptake of CO₂.     

Theoretical reconsiderations when estimating the mesophyll conductance to CO2 diffusion in leaves of C3 plants by analysis of combined gas exchange and chlorophyll fluorescence measurements

Existing methods to estimate the mesophyll conductance to CO₂ diffusion ( g _m) are often based on combined gas exchange and chlorophyll fluorescence measurements. However, estimations of average g _m by these methods are often unreliable either because the range of usable data is too narrow or because the estimations are very sensitive to measurement errors. We describe three method variants to estimate g _m, for which a wider range of data are usable. They use curve-fitting techniques, which minimise the sum of squared model deviations from the data for A (CO₂ assimilation rate) or for J (linear electron transport rate). Like the existing approaches, they are all based on common physiological principles assuming that electron transport limits A . The proposed variants were far less sensitive than the existing approaches to 'measurement noise' either created randomly in the generated data set or inevitably existing in real data sets. Yet, the estimates of g _m from the three variants differed by approximately 15%. Moreover, for each variant, a stoichiometric uncertainty in linear electron transport-limited photosynthesis can cause another 15% difference. Any estimation of g _m using gas exchange and chlorophyll fluorescence measurements should be considered with caution, especially when g _m is high.     

Salinity effects on CO2 assimilation and diffusive conductance of cowpea leaves

The effect of NaCl salinity at concentrations of 43–173 mM in nutrient solution on net gas exchange of attached cowpea [Vigna unguiculata (L.) Walp cv. California Black-eye No. 5 (CB5)] leaves was investigated under both greenhouse and growth chamber conditions.
There was a marked decrease in leaf conductance to water vapor after exposure to low salinity levels and a slighter decrease when salinity levels were higher. The decrease in net assimilation was much more gradual throughout the entire salinity range. The altered responses of net assimilation and leaf conductance to salinity were more evident at a high light intensity. A decrease in intercellular partial CO₂ pressure [p(CO₂)] was found at the low and intermediate salinity levels but not at the high level. These findings suggest that CO, assimilation was mainly controlled by stomatal conductance and the fixation of CO, might have been increased due to stimulated biochemical activity or to higher chlorophyll concentration per unit leaf area. A decrease in assimilation was already found one day after salinization and pro-ceeded up to 4 days when it was inhibited by 50% at 43 mM NaCl and up to 85% at 173 mM. The decrease in transpiration was larger than the decrease in net assimila-tion, and both were attributed to osmotic stress. Partial recovery was found thereaf-ter and new steady-state rates, in the range of 55 to 100% of the control, were then obtained for salinity levels between 43 and 130 mM. Inhibition of net CO, assimila-tion at this stage was attributed partly to a specific sodium effect and partly to plant water status. A linear relationship between leaf sodium content and net photosynthe-sis was also evident at this stage. Net CO, assimilation recovered more completely than transpiration when salt stress was removed, but at 173 mM NaCl recovery was neglible.     

Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol⁻¹ CO₂) and double ambient (720 μmol mol⁻¹ CO₂) carbon dioxide (CO₂) mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density) and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO₂ concentration on the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf variation. In general, a doubling of the atmospheric CO₂ concentration enhanced plant growth and significantly increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated CO₂ concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate. However, no significant differences were found for the maximum rate of carboxylation ( V _cmax) and the light saturated rate of electron transport ( J _max) between the control and elevated CO₂ treatment.     

Effect of salinity on growth, ion content and CO2 assimilation rate in lemon varieties on different rootstocks

Citrus rootstocks as well as lemon scions differ in their ability to restrict sodium and chloride ions and in their sensitivity to saline stress. To determine the behaviour of different rootstock-scion combinations, 3 lemon cultivars on 3 different rootstocks were grown in containers in a greenhouse and irrigated with 5, 25 and 50 m M NaCl. Growth of the plants and foliar contents of sodium and chloride as well as physiological parameters including transpiration rate, gas exchange, stomatal conductance and chlorophyll content were evaluated. Shoot length of the plants on sour orange and on C. volkameriana showed a greater reduction with salinity than those on C. macrophylla . Accumulation of salt in the leaves was also scion dependent, cv. 'Eureka' having higher concentrations of sodium and chloride than the others. Assimilation rate of CO₂ and stomatal conductance were greatly reduced by salinity in the leaves of Verna and Eureka on sour orange. Gas exchange in the leaves was highly correlated with chloride and sodium contents in all lemon-rootstock combinations. C. macrophylla showed a higher resistance to salinity than C. volkameriana and sour orange. Inferences on the mechanisms of action of salt on lemon trees are discussed.     

Parameterization of the CO2 and H2O gas exchange of several temperate deciduous broad-leaved trees at the leaf scale considering seasonal changes

A combined model to simulate CO₂ and H₂O gas exchange at the leaf scale was parameterized using data obtained from in situ leaf‐scale observations of diurnal and seasonal changes in the CO₂ and H₂O gas exchange of four temperate deciduous broad‐leaved trees using a porometric method. The model consists of a Ball et al. type stomatal conductance submodel [Ball, Woodrow & Berry, pp. 221–224 in Progress in Photosynthesis Research (ed. I. Biggins), Martinus‐Nijhoff Publishers, Dordrecht, The Netherlands, 1987] and a Farquhar et al. type biochemical submodel of photosynthesis (Farquhar, von Caemmerer & Berry, Planta 149, 78–90, 1980). In these submodels, several parameters were optimized for each tree species as representative of the quantitative characteristics related to gas exchange. The results show that the seasonal physiological changes of V_cmax25 in the biochemical model of photosynthesis should be used to estimate the long‐term CO₂ gas exchange. For R_d25 in the biochemical model of photosynthesis and m in the Ball et al. type stomatal conductance model, the difference should be counted during the leaf expansion period.     

The use of low [CO2] to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive saplings

In this study it has been shown that increased diffusional resistances caused by salt stress may be fully overcome by exposing attached leaves to very low [CO₂] (～ 50 µmol mol^?1), and, thus a non‐destructive‐in vivo method to correctly estimate photosynthetic capacity in stressed plants is reported. Diffusional (i.e. stomatal conductance, g_s, and mesophyll conductance to CO₂, g_m) and biochemical limitations to photosynthesis (A) were measured in two 1‐year‐old Greek olive cultivars (Chalkidikis and Kerkiras) subjected to salt stress by adding 200 mm NaCl to the irrigation water. Two sets of A–C_i curves were measured. A first set of standard A–C_i curves (i.e. without pre‐conditioning plants at low [CO₂]), were generated for salt‐stressed plants. A second set of A–C_i curves were measured, on both control and salt‐stressed plants, after pre‐conditioning leaves at [CO₂] of ～ 50 µmol mol^?1 for about 1.5 h to force stomatal opening. This forced stomata to be wide open, and g_s increased to similar values in control and salt‐stressed plants of both cultivars. After g_s had approached the maximum value, the A–C_i response was again measured. The analysis of the photosynthetic capacity of the salt‐stressed plants based on the standard A–C_i curves, showed low values of the J_max (maximum rate of electron transport) to V_cmax (RuBP‐saturated rate of Rubisco) ratio (1.06), that would implicate a reduced rate of RuBP regeneration, and, thus, a metabolic impairment. However, the analysis of the A–C_i curves made on pre‐conditioned leaves, showed that the estimates of the photosynthetic capacity parameters were much higher than in the standard A–C_i responses. Moreover, these values were similar in magnitude to the average values reported by Wullschleger (Journal of Experimental Botany 44, 907–920, 1993) in a survey of 109 C₃ species. These findings clearly indicates that: (1) salt stress did affect g_s and g_m but not the biochemical capacity to assimilate CO₂ and therefore, in these conditions, the sum of the diffusional resistances set the limit to photosynthesis rates; (2) there was a linear relationship (r² = 0.68) between g_m and g_s, and, thus, changes of g_m can be as fast as those of g_s; (3) the estimates of photosynthetic capacity based on A–C_i curves made without removing diffusional limitations are artificially low and lead to incorrect interpretations of the actual limitations of photosynthesis; and (4) the analysis of the photosynthetic properties in terms of stomatal and non‐stomatal limitations should be replaced by the analysis of diffusional and non‐diffusional limitations of photosynthesis. Finally, the C₃ photosynthesis model parameterization using in vitro‐measured and in vivo‐measured kinetics parameters was compared. Applying the in vivo‐measured Rubisco kinetics parameters resulted in a better parameterization of the photosynthesis model.     

The influence of leaf thickness on the CO2 transfer conductance and leaf stable carbon isotope ratio for some evergreen tree species in Japanese warm-temperate forests

1. The influence of leaf thickness on internal conductance for CO₂ transfer from substomatal cavity to chloroplast stroma ( g _i) and carbon isotope ratio (δ¹³C) of leaf dry matter was investigated for some evergreen tree species from Japanese temperate forests. g _i was estimated based on the combined measurements of gas exchange and concurrent carbon isotope discrimination.
2. Leaves with thicker mesophyll tended to have larger leaf dry mass per area (LMA), larger surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area ( S _mes) and smaller volume ratio of intercellular spaces to the whole mesophyll (mesophyll porosity).
3. g _i of these leaves was correlated positively to S _mes but negatively to mesophyll porosity. The variation in g _i among these species would be therefore primarily determined by variation of the conductance in liquid phase rather than that in gas phase.
4. δ¹³C was positively correlated to mesophyll thickness and leaf nitrogen content on an area basis. However, g _i values did not correlate to δ¹³C. These results suggest that difference in δ¹³C among the species was not caused by the variation in g _i, but mainly by the difference in long-term photosynthetic capacity.
5. Comparison of our results with those of previous studies showed that the correlation between leaf thickness and g _i differed depending on leaf functional types (evergreen, deciduous or annual). Differences in leaf properties among these functional types were discussed.     

Interaction between CO2 enrichment and salinity stress in the C4 non-halophyte Andropogon glomeratus (Walter) BSP

Abstract Increasing atmospheric CO₂ may result in alleviation of salinity stress in salt-sensitive plants. In order to assess the effect of enriched CO₂ on salinity stress in Andropogon glomeratus, a C₄ non-halophyte found in the higher regions of salt marshes, plants were grown at 350, 500, and 650 cm³ m^?3 CO₂ with 0 or 100 mol m^?3 NaCl watering treatments. Increases in leaf area and biomass with increasing CO₂ were measured in salt-stressed plants, while decreases in these same parameters were measured in non-salt-stressed plants. Tillering increased substantially with increasing CO₂ in salt-stressed plants, resulting in the increased biomass. Six weeks following initiation of treatments, there was no difference in photosynthesis on a leaf area basis with increasing CO₂ in salt-stressed plants, although short-term increases probably occurred. Stomatal conductance decreased with increasing CO₂ in salt-stressed plants, resulting in higher water-use efficiency, and may have improved the diurnal water status of the plants. Concentrations of Na⁺ and Cl^? were higher in salt stressed-plants while the converse was found for K ⁺. There were no differences in leaf ion content between CO₂ treatments in the salt-stressed plants. Decreases in photosynthesis in salt-stressed plants occurred primarily as a result of decreased internal (non-stomatal) conductance.     

A three-dimensional model of CO2 transport in airspaces and mesophyll cells of a silver birch leaf

A realistic numerical three‐dimensional (3D) model was constructed to study CO₂ transport inside a birch leaf. The model included chloroplasts, palisade and spongy mesophyll cells, airspaces, stomatal opening and the leaf boundary layer. Diffusion equations for CO₂ were solved for liquid(mesophyll) and gaseous(air) phases. Simulations were made in typical ambient field conditions varying stomatal opening, photosynthetic capacity and temperature. Doubled ambient CO₂ concentration was also considered. Changes in variables caused non‐linear effects in the total flux, especially when compared with the results of double CO₂ concentration. The reduction in stomatal opening size had a smaller effect on the total flux in doubled concentrations than ambient CO₂. The reduced photosynthetic capacity had a similar effect on the flux in both cases. The palisade and spongy mesophyll cells had unequal roles mainly due to the light absorption profile. Results from the 3D simulation were also compared to the classical one‐dimensional resistance approach. Liquid and gas phase resistances were estimated and found strongly variable according to changes in temperature and degree of stomatal opening. For the birch leaves modelled, intercellular airspace resistance was small (2% of the total resistance in saturating irradiance conditions at 25 °C at stomatal opening diameter of 4 µm) whereas the liquid phase resistance was significant (23% for mesophyll and chloroplasts in the same ‘base case’). The absorption of CO₂ into water at cell surfaces caused additional (strongly temperature dependent) resistance which accounted for 36% of the total resistance in the base case.     

RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.     

Limitation of net CO2 assimilation rate by internal resistances to CO2 transfer in the leaves of two tree species (Fagus sylvatica L. and Castanea sativa Mill.)

Using a combination of gas-exchange and chlorophyll fluorescence measurements, low apparent CO₂/O₂ specificity factors (1300 mol mol_?1) were estimated for the leaves of two deciduous tree species (Fagus sylvatica and Castanea sativa). These low values contrasted with those estimated for two herbaceous species and were ascribed to a drop in the CO₂ mole fraction between the intercellular airspace (C_i) and the catalytic site of Rubisco (C_c) due to internal resistances to CO₂ transfer. C_c. was calculated assuming a specificity of Rubisco value of 2560 mol mol^?1. The drop between C_i and C_c was used to calculate the internal conductance for CO₂ (g_i). A good correlation between mean values of net CO₂ assimilation rate (A) and g_i was observed within a set of data obtained using 13 woody plant species, including our own data. We report that the relative limitation of A, which can be ascribed to internal resistances to CO₂ transfer, was 24–30%. High internal resistances to CO₂ transfer may explain the low apparent maximal rates of carboxylation and electron transport of some woody plant species calculated from A/C_i curves.     

On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar–von Caemmerer–Berry leaf photosynthesis model

Virtually all current estimates of the maximum carboxylation rate (V_cmax) of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and the maximum electron transport rate (J_max) for C₃ species implicitly assume an infinite CO₂ transfer conductance (g_i). And yet, most measurements in perennial plant species or in ageing or stressed leaves show that g_i imposes a significant limitation on photosynthesis. Herein, we demonstrate that many current parameterizations of the photosynthesis model of Farquhar, von Caemmerer & Berry (Planta 149, 78–90, 1980 ) based on the leaf intercellular CO₂ concentration (C_i) are incorrect for leaves where g_i limits photosynthesis. We show how conventional A–C_i curve (net CO₂ assimilation rate of a leaf –A_n– as a function of C_i) fitting methods which rely on a rectangular hyperbola model under the assumption of infinite g_i can significantly underestimate V_cmax for such leaves. Alternative parameterizations of the conventional method based on a single, apparent Michaelis–Menten constant for CO₂ evaluated at C_i[K_m(CO₂)_i] used for all C₃ plants are also not acceptable since the relationship between V_cmax and g_i is not conserved among species. We present an alternative A–C_i curve fitting method that accounts for g_i through a non‐rectangular hyperbola version of the model of Farquhar et al. (1980 ). Simulated and real examples are used to demonstrate how this new approach eliminates the errors of the conventional A–C_i curve fitting method and provides V_cmax estimates that are virtually insensitive to g_i. Finally, we show how the new A–C_i curve fitting method can be used to estimate the value of the kinetic constants of Rubisco in vivo is presented     

Photosynthesis and conductance of spring-wheat leaves: field response to continuous free-air atmospheric CO2 enrichment

Spring wheat was grown from emergence to grain maturity in two partial pressures of CO₂ (pCO₂): ambient air of nominally 37 Pa and air enriched with CO₂ to 55 Pa using a free-air CO₂ enrichment (FACE) apparatus. This experiment was the first of its kind to be conducted within a cereal field without the modifications or disturbance of microclimate and rooting environment that accompanied previous studies. It provided a unique opportunity to examine the hypothesis that continuous exposure of wheat to elevated pCO₂ will lead to acclimatory loss of photosynthetic capacity. The diurnal courses of photosynthesis and conductance for upper canopy leaves were followed throughout the development of the crop and compared to model-predicted rates of photosynthesis. The seasonal average of midday photosynthesis rates was 28% greater in plants exposed to elevated pCO₂ than in contols and the seasonal average of the daily integrals of photosynthesis was 21% greater in elevated pCO₂ than in ambient air. The mean conductance at midday was reduced by 36%. The observed enhancement of photosynthesis in elevated pCO₂ agreed closely with that predicted from a mechanistic biochemical model that assumed no acclimation of photosynthetic capacity. Measured values fell below predicted only in the flag leaves in the mid afternoon before the onset of grain-filling and over the whole diurnal course at the end of grain-filling. The loss of enhancement at this final stage was attributed to the earlier senescence of flag leaves in elevated pCO₂. In contrast to some controlled-environment and field-enclosure studies, this field-scale study of wheat using free-air CO₂ enrichment found little evidence of acclimatory loss of photosynthetic capacity with growth in elevated pCO₂ and a significant and substantial increase in leaf photosynthesis throughout the life of the crop.     

The mechanism of bicarbonate assimilation by the polar leaves of Potamogeton and Elodea. CO2 concentrations at the leaf surface

Abstract. Photosynthetic utilization of HCO, in leaves of Poiamogeton and Elodea occurs at the lower leaf side, with subsequent OH∼ release at the upper side. It is accompanied by transport of cations, in the present experiment K +, across the leaf. The resulting pH and K+ concentration changes near the leaf surface were recorded with miniature electrodes. From the pH and K+ concentration the concentrations of the different inorganic carbon species were calculated and compared with photosynthetic O, production. HCO⁻₃ utilization is accompanied by a drastic increase in the free CO₂ concentration near the lower epidermis. Experiments with CO₂− and HCO₃⁻free solutions showed an oscillating acidification near the lower epidermis and alkalinization near the upper epidermis. It is concluded that the acidification results from the activity of light-dependent H+ pumps. The finding that an increase in pH at the upper side always coincided with a decrease at the lower in these experiments shows that the H+ pumps and the OH− extruding mechanism are coupled although occurring in different cell layers. Previously we have suggested that the first step in the process of photosynthetic HCO₃⁻ utilization is external conversion of HCO₃⁻" by acidification caused by light-dependent H+ pumps. The present results strongly support this hypothesis. Two possible pathways for the accompanying K + transport are discussed. The model presented here explains the known inhibiting effects of buffers and high pH on photosynlhetic HCO₃⁻ utilization.