Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO₃^?/CO₂ as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO₃^?/CO₂. Addition of exogenous HCO₃^? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.     

Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site

In maize chloroplasts, the ratio of HCO₃⁻ (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO₃⁻ to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO₃⁻ and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO₃⁻ binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO₃⁻ with a dissociation constant near 80 micromolar. The location of the HCO₃⁻ binding site, and the low-affinity atrazine binding site, is not known.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263)

We have measured the exchange of ¹⁸O between CO₂ and H₂O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of ¹⁸O from CO₂ in the fluid outside the cells provides a method to study CO₂ and HCO₃⁻ kinetics in suspensions of algae that contain carbonic anhydrase since ¹⁸O loss to H₂O is catalyzed inside the cells but not in the external fluid. Low-CO₂ cells of Chlorella vulgaris (grown with air) were added to a solution containing ¹⁸O enriched CO₂ and HCO₃⁻ with 2 to 15 millimolar total inorganic carbon. The observed depletion of ¹⁸O from CO₂ was biphasic and the resulting ¹⁸C content of CO₂ was much less than the ¹⁸O content of HCO₃⁻ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO₃⁻ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10⁻⁴ s⁻¹ due to our experimental errors. The Fick's law rate constant for entry of CO₂ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO₂ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO₂ entry into the cell or both. The CO₂ hydration activity inside the cells was 160 times the uncatalyzed CO₂ hydration rate.     

Utilization of Inorganic Carbon by Ulva lactuca

Thalli discs of the marine macroalga Ulva lactuca were given inorganic carbon in the form of HCO₃⁻, and the progression of photosynthetic O₂ evolution was followed and compared with predicted O₂ evolution as based on calculated external formation of CO₂ (extracellular carbonic anhydrase was not present in this species) and its carboxylation (according to the K_m(CO₂) of ribulose-1,5-bisphosphate carboxylase/oxygenase), at two different pHs, assuming a photosynthetic quotient of 1. The K_m(inorganic carbon) was some 2.5 times lower at pH 5.6 than at the natural seawater pH of 8.2, whereas V_max was similar under the two conditions, indicating that the unnaturally low pH per se had no adverse effect on U. lactuca's photosynthetic performance. These results, therefore, could be evaluated with regard to differential CO₂ and HCO₃⁻ utilization. The photosynthetic performance observed at the lower pH largely followed that predicted, with a slight discrepancy probably reflecting a minor diffusion barrier to CO₂ uptake. At pH 8.2, however, dehydration rates were too slow to supply CO₂ for the measured photosynthetic response. Given the absence of external carbonic anhydrase activity, this finding supports the view that HCO₃⁻ transport provides higher than external concentrations of CO₂ at the ribulose-1,5-bisphosphate carboxylase/oxygenase site. Uptake of HCO₃⁻ by U. lactuca was further indicated by the effects of potential inhibitors at pH 8.2. The alleged band 3 membrane anion exchange protein inhibitor 4,4′-diisothiocyanostilbene-2,2′disulphonate reduced photosynthetic rates only when HCO₃⁻ (but not CO₂) could be the extracellular inorganic carbon form taken up. A similar, but less drastic, HCO₃⁻-competitive inhibition of photosynthesis was obtained with Kl and KNO₃. It is suggested that, under ambient conditions, HCO₃⁻ is transported into cells at defined sites either via facilitated diffusion or active uptake, and that such transport is the basis for elevated internal [CO₂] at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation.     

Multiple anion effects on photosystem II in chloroplast membranes

We investigated the activity of several anions at various sites on photosystem II, in particular those associated with the Cl^- effect (anion binding-site I) and the HCO₃ ^- effect (anion binding-site II). Chlorophyll a fluorescence changes were used to monitor partial photosystem II reactions either in the oxygen-evolving mechanism or involving endogenous quinone electron acceptors. We find that anions such as NO₃ ^-, HCO₃ ^-, HCO₂ ^-, F^-, NO₂ ^-, and acetate can, depending on conditions, bind to either anion binding-site I, anion binding-site II, or both sites simultaneously. The anions N₃ ^- and Au(CN)₂ ^- are exceptions. In their presence, oxygen-consumption reactions are enhanced. The results demonstrate that an exclusive site or mode of action of an anion on photosystem II cannot be determined by measuring the Hill reaction alone. Anion interactions with photosystem II are shown to be very complex and, therefore, caution is advisable in interpreting related experiments. Carbonic anhydrase associated with photosystem II was also investigated as a possible target for some anion effects. In Cl^--depleted thylakoids, NO₃ ^-, stimulated both electron transport and carbonic anhydrase activity at low concentrations, while higher concentrations inhibited both. However, carbonic anhydrase was more sensitive to inhibition by NO₃ ^- than was electron flow. Possible interpretations are discussed; the electron transport and carbonic anhydrase activity appear not to be functionally linked.Abbreviations ABSI Anion binding-site(s) I associated with the oxygen-evolving mechanism - ABSII Anion binding-site(s) II, which controls quinone-related reactions on the electron-acceptor side of photosystem II - OAc^- Acetate - Chl Chlorophyll - DCMU 3—(3,4-dichlorophenyl)-1,1-dimethyl urea - DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - Mes 2-[N-morpholino]ethanesulphonic acid - Mops 3-[N-morpholino]propanesulphonic acid - Tes N-Tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid - Tricine N-Tris[hydroxymethyl]methylglycine     

Absence of a Formate-Induced Release of Bicarbonate from Photosystem II

Formate has been proposed to inhibit electron flow in photosystem II by replacing endogenous bound bicarbonate on the reaction center complex. A mass spectrometer was used to measure directly the CO₂/HCO₃⁻ released when maize thylakoids, showing normal rates of electron flow, were treated with formate. Although the formate inhibited electron flow by 95%, no release (displacement) of CO₂/HCO₃⁻ was detected. This is consistent with the concept that membrane-bound HCO₃⁻ is not a requirement for normal rates of electron flow through photosystem II. Moreover, formate and other monovalent anions do not inhibit electron flow by removing bound HCO₃⁻ but by binding to empty sites. The “bicarbonate effect” is a reversal, by high concentrations of exogenous bicarbonate, of anion inhibition of photosystem II.     

Evidence for HCO(3) Transport by the Blue-Green Alga (Cyanobacterium) Coccochloris peniocystis

The possibility of HCO₃⁻ transport in the blue-green alga (cyanobacterium) Coccochloris peniocystis has been investigated. Coccochloris photosynthesized most rapidly in the pH range 8 to 10, where most of the inorganic C exists as HCO₃⁻. If photosynthesis used only CO₂ from the external solution the rate of photosynthesis would be limited by the rate of HCO₃⁻ dehydration to CO₂. Observed rates of photosynthesis at alkaline pH were as much as 48-fold higher than could be supported by spontaneous dehydration of HCO₃⁻ in the external solution. Assays for extracellular carbonic anhydrase were negative. The evidence strongly suggests that HCO₃⁻ was a direct C source for photosynthesis.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

A Model for HCO(3) Accumulation and Photosynthesis in the Cyanobacterium Synechococcus sp: Theoretical Predictions and Experimental Observations

A simple model based on HCO₃⁻ transport has been developed to relate photosynthesis and inorganic carbon fluxes for the marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1). Predicted relationships between inorganic carbon transport, CO₂ fixation, internal carbonic anhydrase activity, and leakage of CO₂ out of the cell, allow comparisons to be made with experimentally obtained data. Measurements of inorganic carbon fluxes and internal inorganic carbon pool sizes in these cells were made by monitoring time-courses of CO₂ changes (using a mass spectrometer) during light/dark transients. At just saturating CO₂ conditions, total inorganic carbon transport did not exceed net CO₂ fixation by more than 30%. This indicates CO₂ leakage similar to that estimated for C₄ plants.

For this leakage rate, the model predicts the cell would need a conductance to CO₂ of around 10⁻⁵ centimeters per second. This is similar to estimates made for the same cells using inorganic carbon pool sizes and CO₂ efflux measurements. The model predicts that carbonic anhydrase is necessary internally to allow a sufficiently fast rate of CO₂ production to prevent a large accumulation of HCO₃⁻. Intact cells show light stimulated carbonic anhydrase activity when assayed using ¹⁸O-labeled CO₂ techniques. This is also supported by low but detectable levels of carbonic anhydrase activity in cell extracts, sufficient to meet the requirements of the model.

     

Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and ¹⁸O contents of extracellular CO₂ and HCO₃⁻ in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring ¹⁸O exchange between CO₂ and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO₂ hydration rate in different samples of cells that were grown on bubbled air (low-CO₂ conditions). This activity was only weakly inhibited by ethoxzolamide with an I₅₀ near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO₂-starved cells there is considerable generation of CO₂ from intracellular stores, a factor that can cause errors in measurement of net CO₂ uptake unless accounted for. It was demonstrated that use of ¹³C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO₂ readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO₃⁻ was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO₃⁻ from Synechococcus.     

CO2 permeability of the rat erythrocyte membrane and its inhibition

We have studied the CO₂ permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs ¹⁸O-labelled CO₂. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO₃⁻ permeability. For normal rat erythrocytes, we find at 37 °C a CO₂ permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10⁻³cm/s. We studied whether the rat erythrocyte membrane possesses protein CO₂ channels similar to the human red cell membrane by applying the potential CO₂ channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO₂ permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO₂ channels that are responsible for at least 50% of its CO₂ permeability.     

Light-Induced Proton Release by the Cyanobacterium Anabaena variabilis: Dependence on CO(2) and Na

Light-induced acidification by the cyanobacterium Anabaena variabilis is biphasic (a fast phase I and slow phase II) and shown to be sodium-dependent with an optimum concentration of 40 to 60 millimolar Na⁺. Cells grown under low CO₂ concentrations at pH 9 (i.e. mainly HCO₃⁻ present in the medium) exhibited the slow phase II of proton efflux only, while cells grown under low CO₂ concentrations at pH 6.3 (i.e. CO₂ and HCO₃⁻ present) exhibited both phases. Light-induced proton release of phase I was dependent on inorganic carbon available in the bathing medium with an apparent K_m for CO₂ of 20 to 70 micromolar. As was concluded from the CO₂ dependence of acidification measured at different pH of the bathing medium, bicarbonate inhibited phase-I acidification noncompetetively. Acidification was inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Apparently, acidification of phase I is due to a light-dependent uptake of CO₂ being converted to HCO₃⁻ by a carbonic anhydrase-like function of the HCO₃⁻-transport system (M Volokita, D Zenvirth, A Kaplan, L Reinhold 1984 Plant Physiol 76: 599-602) before or during entering the cell, thus releasing one proton per CO₂ converted to HCO₃⁻.     

The effects of carbonic anhydrase inhibitors formate,bicarbonate, acetazolamide,and imidazole on photosystem II in maize chloroplasts

Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N₃^?, I^?, OA_c^?, and Cl^? is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor Q. This inhibitory effect of low levels of HCO₃^? along with the known enhancement by HCO₃^? of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the “bicarbonate-effect” associated with PS II in chloroplasts.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

Inorganic Carbon Uptake during Photosynthesis : I. A Theoretical Analysis Using the Isotopic Disequilibrium Technique

Equations have been developed which quantitatively predict the theoretical time-course of photosynthetic ¹⁴C incorporation when CO₂ or HCO₃⁻ serves as the sole source of exogenous inorganic carbon taken up for fixation by cells during steady state photosynthesis. Comparison between the shape of theoretical (CO₂ or HCO₃⁻) and experimentally derived time-courses of ¹⁴C incorporation permits the identification of the major species of inorganic carbon which crosses the plasmalemma of photosynthetic cells and facilitates the detection of any combined contribution of CO₂ and HCO₃⁻ transport to the supply of intracellular inorganic carbon. The ability to discriminate between CO₂ or HCO₃⁻ uptake relies upon monitoring changes in the intracellular specific activity (by ¹⁴C fixation) which occur when the inorganic carbon, present in the suspending medium, is in a state of isotopic disequilibrium (JT Lehman 1978 J Phycol 14: 33-42). The presence of intracellular carbonic anhydrase or some other catalyst of the CO₂-HCO₃⁻ interconversion reaction is required for quantitatively accurate predictions. Analysis of equations describing the rate of ¹⁴C incorporation provides two methods by which any contribution of HCO₃⁻ ions to net photosynthetic carbon uptake can be estimated.     

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

The nature of the inorganic carbon (C_i) species actively taken up by cyanobacteria CO₂ or HCO₃⁻ has been investigated. The kinetics of CO₂ uptake, as well as that of HCO₃⁻ uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO₂ was higher than that for HCO₃⁻. Though the calculated V_max was the same in both cases, the maximum rate of uptake actually observed was higher when HCO₃⁻ was supplied. C_i uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO₂ was the species supplied. Observations of photosynthetic rate as a function of intracellular C_i level (following supply of CO₂ or HCO₃⁻ for 5 seconds) led to the inference that HCO₃⁻ is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.

On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C_i transport apparatus binds CO₂ and releases HCO₃⁻ at or near a membrane porter. The latter transports HCO₃⁻ ions to the cell interior.

     

Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity

In high inorganic carbon grown (1% CO₂ [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO₂ uptake and to reduce the final internal inorganic carbon (C_i) pool size reached. The relationship between CO₂ fixation rate and internal C_i concentration in high C_i grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High C_i grown cells readily took up CO₂ but had little or no capacity for HCO₃⁻ uptake. These cells appear to possess a CO₂ utilizing C_i pump that has a CA-like function associated with the transport step such that HCO₃⁻ is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low C_i grown cells possess both CO₂ uptake and HCO₃⁻ uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO₂ and HCO₃⁻ uptake (such as the C_i pump) may have been affected. The inhibitor had no apparent effect on internal CO₂/HCO₃⁻ equilibria (internal CA function) in low C_i grown cells.     

Intracellular pH Regulation during NO(3) Assimilation in Shoot and Roots of Ricinus communis

Ricinus communis L. was used to test the Dijkshoorn-Ben Zioni hypothesis that NO₃⁻ uptake by roots is regulated by NO₃⁻ assimilation in the shoot. The fate of the electronegative charge arising from total assimilated NO₃⁻ (and SO₄²⁻) was followed in its distribution between organic anion accumulation and HCO₃⁻ excretion into the nutrient solution. In plants adequately supplied with NO₃⁻, HCO₃⁻ excretion accounted for about 47% of the anion charge, reflecting an excess nutrient anion over cation uptake. In vivo nitrate reductase assays revealed that the roots represented the site of about 44% of the total NO₃⁻ reduction in the plants. To trace vascular transport of ionic and nitrogenous constituents within the plant, the composition of both xylem and phloem saps was thoroughly investigated. Detailed dry tissue and sap analyses revealed that only between 19 and 24% of the HCO₃⁻ excretion could be accounted for from oxidative decarboxylation of shoot-borne organic anions produced in the NO₃⁻ reduction process. The results obtained in this investigation may be interpreted as providing direct evidence for a minor importance of phloem transport of cation-organate for the regulation of intracellular pH and electroneutrality, thus practically eliminating the necessity for the Dijkshoorn-Ben Zioni recycling process.     

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria

The induction of a high-affinity state of the CO₂-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO₂ concentrations were resuspended in low-CO₂ buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO₂-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO₂ and HCO₃⁻ fluxes indicated that only the affinity of HCO₃⁻ transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO₂ uptake remained unchanged. Fast induction of high-affinity HCO₃⁻ transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO₃⁻ transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO₃⁻ transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.