Separation of fatty acids or methyl esters including positional and geometric isomers by alumina argentation thin-layer chromatography.

This paper describes novel and rapid thin-layer chromatography procedures for the analysis of fatty acids and methyl esters using silver-impregnated alumina sheets. These techniques are known in most laboratories, and the equipment is readily available. The fatty acid method allows a separation of petroselinic (C18:1 delta 6c), oleic (C18:1 delta 9c), elaidic (C18:1 delta 9t), erucic (C22:1 delta 13c), and brassidic acids (C22:1 delta 13t), and the methyl ester method gives an excellent resolution with respect to the number, configuration, and position of the unsaturated centers. Sufficient separation for the subsequent ozonolysis and chromatographic quantification of isomeric C18 and C22 fatty acid methyl esters is obtained with both methods.

Effects of water stress on the gas exchange, the activities of some enzymes of carbon and nitrogen metabolism, and on the pool sizes of some organic acids in maize leaves.

Water-stressed maize (Zea mays L.) leaves showed a large decrease in leaf conductance during photosynthesis. Net CO2 uptake and evaporation declined fast at mild stress (ψ=-0.6 to -1.0 MPa) and slower at more severe stress (ψ=-1.0 to -1.2 MPa), whereas the CO2 concentration in the intercellular spaces (Ci) did not drop to the CO2 compensation point. The activities of the enzymes of photosynthetic carbon metabolism tested in this study dropped by approx. 30% at ψ=-1.2 MPa. Glutamine synthetase activity was unaffected by water stress, whereas the activity of nitrate reductase was almost completely inhibited. The decline of enzyme activities in relation to ψ was correlated with a concomitant decrease in the content of total soluble protein of the stressed leaves. The total leaf pools of malate, pyruvate and oxaloacetate decreased almost linearly in relation to ψ, thus obviously contradicting the almost constant Ci. In comparison to the controls (ψ=0.6 MPa) the content of citrate and isocitrate increaed markedly at ψ=-0.9 MPa and decreased again at ψ=-1.2 MPa.

The activity of nitrate reductase and the pool sizes of some amino acids and some sugars in water-stressed maize leaves.

The activity of nitrate reductase and the pool sizes of some amino acids and some sugars were measured in relation to the leaf water potential (ψ) of maize leaves. The activity of nitrate reductase was severely inhibited in water-stressed maize leaves. This was not due to substrate shortage or the presence of an inhibitor at reduced leaf water potential. While the typical proteinogenic amino acids valine, tyrosine, leucine and isoleucine were almost undetectable in the leaves of the control plants, their concentrations markedly increased with declining ψ, thus indicating protein degradation. The concentrations of serine, glycine and glutamate increased upon water stress, their total amount in severely stressed leaves ranging 5- to 6-fold higher than the total amount of valine, tyrosine, leucine and isoleucine at this stage of water deficit. The pool sizes of glucose, fructose and sucrose decreased in relation to decreasing ψ. The total amount of organic solutes remained almost constant at least up to a ψ of approx.-1.0 MPa and then dropped to about 50% when ψ reached -1.25 MPa.

(15)N and inhibitor studies on the photorespiratory nitrogen cycle in maize leaves.

A combination of inhibitor and (15)N studies were used to investigate the photorespiratory nitrogen cycle in maize, a C4 plant. Inhibitors used included isonicotinyl hydrazide which blocks the conversion of glycine to serine, methionine sulfoximine an inhibitor of GS and azaserine an inhibitor of GOGAT. Results from levels of ammonia and amino acids and the distribution of (15)N into NH3, serine, glutamine and glutamate indicated that the photorespiratory N-cycle occurs in this C4 plant, but the rate of flux through this pathway is low as compared with that in C3 plants.

Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower, bean and maize to water stress.

Gas exchange and fluorescence measurements of attached leaves of water stressed bean, sunflower and maize plants were carried out at two light intensities (250 µmol quanta m(-2)s(-1) and 850 µmol quanta m(-2)s(-1)). Besides the restriction of transpiration and CO2 uptake, the dissipation of excess light energy was clearly reflected in the light and dark reactions of photosynthesis under stress conditions. Bean and maize plants preferentially use non-photochemical quenching for light energy dissipation. In sunflower plants, excess light energy gave rise to photochemical quenching. Autoradiography of leaves after photosynthesis in (14)CO2 demonstrated the occurrence of leaf patchiness in sunflower and maize but not in bean. The contribution of CO2 recycling within the leaves to energy dissipation was investigated by studies in 2.5% oxygen to suppress photorespiration. The participation of different energy dissipating mechanisms to quanta comsumption on agriculturally relevant species is discussed.

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans.

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air.

Mass spectrometric measurements of (16)O2 and (18)O2 isotopes were used to compare the rates of gross O2 evolution (E0), O2 uptake (U0) and net O2 evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO2 (CO2-grown) and by cells grown in 5% CO2 and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 µmol photons m(-2) s(-1)), pH=7.0 and 28°C, U0 equalled E0 at the DIC compensation point which was 10µM DIC for CO2-grown and zero for air-grown cells. Both E0 and U0 were strongly dependent on DIC and reached DIC saturation at 480 µM and 70 µM for CO2-grown and air-grown algae respectively. U0 increased from DIC compensation to DIC saturation. The U0 values were about 40 (CO2-grown), 165 (air-adapted) and 60 µmol O2 mg Chl(-1) h(-1) (air-grown). Above DIC compensation the U0/E0 ratios of air-adapted and air-grown algae were always higher than those of CO2-grown cells. These differences in O2 exchange between CO2- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O2 uptake measured 5 min after darkening were considerably lower than the rates of O2 uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U0 is discussed and the energy requirement of the inducable CO2/HCO3 (-) concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.

Effect of Photon Fluence Rate on Oxygen Evolution and Uptake by Chlamydomonas reinhardtii Suspensions Grown in Ambient and CO(2)-Enriched Air.

A closed system consisting of an assimilation chamber furnished with a membrane inlet from the liquid phase connected to a mass spectrometer was used to measure O(2) evolution and uptake by Chlamydomonas reinhardtii cells grown in ambient (0.034% CO(2)) or CO(2)-enriched (5% CO(2)) air. At pH = 6.9, 28 degrees C and concentrations of dissolved inorganic carbon (DIC) saturating for photosynthesis, O(2) uptake in the light (U(o)) equaled O(2) production (E(o)) at the light compensation point (15 micromoles photons per square meter per second). E(o) and U(o) increased with increasing photon fluence rate (PFR) but were not rate saturated at 600 micromoles photons per square meter per second, while net O(2) exchange reached a saturation level near 500 micromoles photons per square meter per second which was nearly the same for both, CO(2)-grown and air-grown cells. Comparison of the U(o)/E(o) ratios between air-grown and CO(2)-grown C. reinhardtii showed higher values for air-grown cells at light intensities higher than light compensation. For both, air-grown and CO(2)-grown algae the rates of mitochondrial O(2) uptake in the dark measured immediately before and 5 minutes after illumination were much lower than U(o) at PFR saturating for net photosynthesis. We conclude that noncyclic electron flow from water to NADP(+) and pseudocyclic electron flow via photosystem I to O(2) both significantly contribute to O(2) exchange in the light. In contrast, mitochondrial respiration and photosynthetic carbon oxidation cycle are regarded as minor O(2) consuming reactions in the light in both, air-grown and CO(2)-grown cells. It is suggested that the "extra" O(2) uptake by air-grown algae provides ATP required for the energy dependent CO(2)/HCO(3) (-) concentrating mechanism known to be present in these cells.

Ammonia assimilation by Aspergillus nidulans: [15N]ammonia study.

15N kinetic labelling studies were done on liquid cultures of wild-type Aspergillus nidulans. The labelling pattern of major amino acids under 'steady state' conditions suggests that glutamate and glutamine-amide are the early products of ammonia assimilation in A. nidulans. In the presence of phosphinothricin, an inhibitor or glutamine synthetase, 15N labelling of glutamate, alanine and aspartate was maintained whereas the labelling of glutamine was low. This pattern of labelling is consistent with ammonia assimilation into glutamate via the glutamate dehydrogenase pathway. In the presence of azaserine, an inhibitor of glutamate synthase, glutamate was initially more highly labelled than any other amino acid, whereas its concentration declined. Isotope also accumulated in glutamine. Observations with these two inhibitors suggest that ammonia assimilation can occur concurrently via the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways in low-ammonia-grown A. nidulans. From a simple model it was estimated that about half of the glutamate was synthesized via the glutamate dehydrogenase pathway; the other half was formed from glutamine via the glutamate synthase pathway. The transfer coefficients of nine other amino acids were also determined.

Light Energy Dissipation under Water Stress Conditions: Contribution of Reassimilation and Evidence for Additional Processes.

Using (14)CO(2) gas exchange and metabolite analyses, stomatal as well as total internal CO(2) uptake and evolution were estimated. Pulse modulated fluorescence was measured during induction and steady state of photosynthesis. Leaf water potential of Digitalis lanata EHRH. plants decreased to -2.5 megapascals after withholding irrigation. By osmotic adjustment, leaves remained turgid and fully exposed to irradiance even at severe water stress. Due to the stress-induced reduction of stomatal conductance, the stomatal CO(2) exchange was drastically reduced, whereas the total CO(2) uptake and evolution were less affected. Stomatal closure induced an increase in the reassimilation of internally evolved CO(2). This ;CO(2) recycling' consumes a significant amount of light energy in the form of ATP and reducing equivalents. As a consequence, the metabolic demand for light energy is only reduced by about 40%, whereas net photosynthesis is diminished by about 70% under severe stress conditions. By CO(2) recycling, carbon flux, enzymatic substrate turnover and consumption of light energy were maintained at high levels, which enabled the plant to recover rapidly after rewatering. In stressed D. lanata plants a variable fluorescence quenching mechanism, termed ;coefficient of actinic light quenching,' was observed. Besides water conservation, light energy dissipation is essential and involves regulated metabolic variations.

Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate.

By measuring (18)O exchange from doubly labeled CO(2) ((13)C(18)O(18)O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C(i)]) or air enriched with 5% CO(2) (high C(i)). Intact low C(i) protoplasts had a 10-fold higher carbonic anhydrase activity than did high C(i) protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of (18)O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C(i) cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C(i) than in high C(i) preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment.

A new approach to measure gross CO2 fluxes in leaves. Gross CO2 assimilation, photorespiration, and mitochondrial respiration in the light in tomato under drought stress.

We developed a new method using 13CO2 and mass spectrometry to elucidate the role of photorespiration as an alternative electron dissipating pathway under drought stress. This was achieved by experimentally distinguishing between the CO2 fluxes into and out of the leaf. The method allows us to determine the rates of gross CO2 assimilation and gross CO2 evolution in addition to net CO2 uptake by attached leaves during steady-state photosynthesis. Furthermore, a comparison between measurements under photorespiratory and non-photorespiratory conditions may give information about the contribution of photorespiration and mitochondrial respiration to the rate of gross CO2 evolution at photosynthetic steady state. In tomato (Lycopersicon esculentum Mill. cv Moneymaker) leaves, drought stress decreases the rates of net and gross CO2 uptake as well as CO2 release from photorespiration and mitochondrial respiration in the light. However, the ratio of photorespiratory CO2 evolution to gross CO2 assimilation rises with water deficit. Also the contribution of re-assimilation of (photo) respiratory CO2 to gross CO2 assimilation increases under drought.

The efficiency of water use in water stressed plants is increased due to ABA induced stomatal closure.

Gas exchange and abscisic acid content of Digitalis lanata EHRH. have been examined at different levels of plant water stress. Net photosynthesis, transpiration and conductance of attached leaves declined rapidly at first, then more slowly following the withholding of irrigation. The intercellular partial pressure of CO2 decreased slightly. The concentration of 2-cis(S)ABA increased about eight-fold in the leaves of non-irrigated plants as compared with well-watered controls. A close linear correlation was found between the ABA content of the leaves and their conductance on a leaf area basis. In contrast, the plot of net assimilation versus ABA concentration was curvilinear, leading to an increased efficiency of water use during stress. After rewatering, photosynthesis reached control values earlier than transpiration, leaf conductance and ABA content. From these data it is concluded that transpiration through the stomata is directly controlled by the ABA content, whereas net photosynthesis is influenced additionally by other factors.Possible reasons for the responses of photosynthesis and water use efficiency to different stress and ABA levels are discussed.

(16)O 2/ (18)O 2 analysis of oxygen exchange in Dunaliella tertiolecta. Evidence for the inhibition of mitochondrial respiration in the light.

A mass spectrometric (16)O2/(18)O2-isotope technique was used to analyse the rates of gross O2 evolution, net O2 evolution and gross O2 uptake in relation to photon fluence rate by Dunaliella tertiolecta adapted to 0.5, 1.0, 1.5, 2.0 and 2.5 M NaCl at 25°C and pH 7.0.At concentrations of dissolved inorganic carbon saturating for photosynthesis (200 µM) gross O2 evolution and net O2 evolution increased with increasing salinity as well as with photon fluence rate. Light compensation was also enhanced with increased salinities. Light saturation of net O2 evolution was reached at about 1000 µmol m(-2)s(-1) for all salt concentrations tested. Gross O2 uptake in the light was increased in relation to the NaCl concentration but it was decreased with increasing photon fluence rate for almost all salinities, although an enhanced flow of light generated electrons was simultaneously observed. In addition, a comparison between gross O2 uptake at 1000 µmol photons m(-2)s(-1), dark respiration before illumination and immediately after darkening of each experiment showed that gross O2 uptake in the light paralleled but was lower than mitochondrial O2 consumption in the dark.From these results it is suggested that O2 uptake by Dunaliella tertiolecta in the light is mainly influenced by mitochondrial O2 uptake. Therefore, it appears that the light dependent inhibition of gross O2 uptake is caused by a reduction in mitochondrial O2 consumption by light.

Effect of light intensity on ammonia assimilation in maize leaves.

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 µmol m(-2) s(-1) PAR during incubation with the leaf base in 2 mM (15)NH4Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. (15)N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO2 assimilation (8.3 and 17.4 µmol m(-1) s(-1) at 150 and 1350 µmol m(-2) s(-1) PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% (15)N. In the high light regime the amino acids contained up to 15% (15)N, whereas in low light (15)N enrichments were small (up to 6%). The kinetics of (15)N incorporation indicated that NH3 was firstly assimilated into glutamine and then into glutamate. After 15 min (15)N was also found in glycine, serine and alanine. At high light intensity nearly half of the (15)N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant (15)N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.

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 (psi) 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 CO(2) and H(2)O 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 CO(2) uptake is limited.

Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L.

Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO(2) and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, (15)NH(4)Cl was injected into the central pith of stem sections. The kinetics of (15)N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of (15)N derived from (15)NH(4)Cl was almost completely inhibited. Injections of amido-(15)N glutamine demonstrated a potential for (15)N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations.

Chloroplasts with high rates of photosynthetic O2 evolution (up to 120 µmol O2· (mg Chl)(-1)·h(-1) compared with 130 µmol O2· (mg Chl)(-1)·h(-1) of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO2 concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O2 uptake could be observed in the dark by high- and low-CO2 adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 µmol photons·m(-2)·s(-1) for high- and low-CO2 adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200-300 µM for low-CO2 adapted chloroplasts, whereas high-CO2 adapted chloroplasts were not saturated even at 700 µM DIC. The concentrations of DIC required to reach half-saturated rates of net O2 evolution (Km(DIC)) was 31.1 and 156 µM DIC for low- and high-CO2 adapted chloroplasts, respectively. These results demonstrate that the CO2 concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light.

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO(2) assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in (15)N-labeled NH(3), glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25 degrees C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The (15)N-enrichment of various amino acids derived from these (15)N-substrates were examined. The amido-N of glutamine was the first (15)N-labeled product in leaves incubated with (15)NH(4)Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly (15)N-labeled products during incubation with [(15)N]glycine. In contrast, aspartate and alanine were the first (15)N-labeled products when [(15)N] glutamate was used. These results indicate that NH(3) was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii.

Mass spectrometric measurements of dissolved free (13)CO(2) were used to monitor CO(2) uptake by air grown (low CO(2)) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the (13)CO(2) concentration in the medium to close to zero. Similar results were obtained with low CO(2) cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO(2) uptake revealed that the removal of CO(2) from the medium in the light was due to selective and active CO(2) transport rather than uptake of total dissolved inorganic carbon. In the light, low CO(2) cells and protoplasts incubated with carbonic anhydrase took up CO(2) at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO(2) uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO(2) cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO(2) compensation point. During the linear uptake of CO(2), low CO(2) cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O(2) evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O(2) evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO(2) uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO(2) had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O(2) evolution. These results show low CO(2) cells of Chlamydomonas are able to transport both CO(2) and HCO(3) (-) but CO(2) is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO(2) from the dehydration of HCO(3) (-).