pH Dependence of Photosynthesis and Photorespiration in Soybean Leaf Cells

The kinetics of "4C fixation, and inorganic C (Cm.Org) accumulation, have been followed in isolated pea mesophyli protoplasts. NaH'4CO3 was supplied to the protoplasts in media the pH of which was varied between 7 and 8.When "4CO2 fixation was plotted against the calculated concentration of free CO2 in the media, the apparent Km for CO2 was observed to rise as external pH increased. The V,,,. did not alter significantly. Similarly, when Ci,iorg uptake, either in the Ught or in the dark, was plotted against external CO2 concentration the slope of the curves was steeper at higher external pH.Investigation of the time course of uptake showed that internal C,org concentration rose throughout the experimental period, and that in the light it surpassed the external Cinorg concentration after about 3 minutes.Irradiation of protoplasts previously taking up 'C,llrg 2Abbreviations: Ci.,g, inorganic C; Evans Blue, 6,6'-[(3,3'-dimethyl-4,4'-biphenylene)bis(azo)]-bis(4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid)tetrasodium salt; DMO, 5,5-dimethyloxazoladine-2,4-dione.Poincelot (20) and Champigny and Bismuth (6) have deduced that spinach chloroplast envelopes are capable of transferring HCO3 at a rate which is highly significant for the provision of Ci..,g to the chloroplast stroma. This work has been challenged by Heldt (13) who maintains that Cig uptake proceeds via diffusion of CO2 across the chloroplast envelope. The work to be reported here has been carried out with isolated pea mesophyll protoplasts. The isolated protoplast system has the following advantages:Where the object of investigation is a membrane process, it is desirable that the membrane should be directly exposed to the solute species in question, without intervening compartments such as the cell walls and intercellular spaces.The bicarbonate transfer mechanism, if it exists, may be sited in the plasmalemma. Investigation of isolated chloroplasts, therefore, would not reveal it (see 28) but it should be detectable in protoplasts.A suspension of mesophyll protoplasts is a relatively uniform population, and allows precise control of factors at the membrane surface such as pH, concentration of solutes, etc.In the present investigation we have attempted to collect data which could contribute to a decision as to whether HCO3 can cross the protoplast membranes as an anion; if so, whether it crosses by diffusion or with the aid of a transport mechanism; and whether HCO3 transport capacity contributes toward limiting the rate of photosynthesis. MATERIALS AND METHODSThe experimental plant was Pisum sativum L. var. "Dan," and conditions of growth and the procedure for isolating protoplasts were as described in earlier papers (9, 10). Protoplasts were suspended in a medium made up of 0.55 M mannitol, 1 mim KNO3, 0.2 mm KH2PO4, 0.5 mm CaCl2, 3 ms MgSO4 (pH 5.8).Accumulation of acid-stable and acid-labile '4C during incubation of the protoplasts with 14Clo0g was followed by means of a filtering centrifugation technique similar to that described earlier (...

Section: Discussionmentioning

confidence: 76%

Evidence for Mediated HCO₃⁻ Transport in Isolated Pea Mesophyll Protoplasts

Volokita

Reinhold²

1981

“…As a result algae have C4-like photosynthetic characteristics such as very low r (CO2) and r (DIC) values (5, 18), r shows little or no sensitivity to 02 and temperature (5,8,18), there is little apparent 02 inhibition of photosynthesis (8,18) and photorespiration is minimal or absent (6,18). The intervention of a HCO3 transport mechanism, presumably located at the plasmalemma, of Coccochloris peniocystis and Anabaena variabilis has a profound effect on the kinetics of whole-cell photosynthesis (3,8) (25) indicated that K1/2 (CO2), r (CO2) and the magnitude of 02 inhibition of photosynthesis decreased with increasing pH. However, in these experiments CO2 equilibration between the aqueous and gaseous phases in the reaction vials was not taken into account (23).…”

Section: Resultsmentioning

confidence: 90%

Photosynthesis and Inorganic Carbon Transport in Isolated Asparagus Mesophyll Cells

Espie¹,

Colman²

1982

The possibility of HC03-transport into isolated leaf mesophyOl cells of Asparagus sprengeri Regel has been investigated. Measurement of the inorganic carbon pool in these ceOls over an external pH range 6.2 to 8.0, using the silicone-fluid filtration technique, indicated that the pool was larger than predicted by passive "4CO2 distribution, suggesting that HC03-as wel as CO2 crosses the plasmalemma. Intracelular pH values, calculated from the distribution of 14CO2 between the cells and the medium, were found to be higher (except at pH 8.0) than those previously determined by 5,5-dimethyll2-14Cloxazolidine-2,4dione distribution. It is suggested that the inorganic carbon accumulated above predicted concentrations may be bound to proteins and membranes and thus may not represent inorganic carbon actively accumulated by the cells, inasmuch as in a closed system at constant CO2 concentration, the photosynthetic rates at pH 7.0 and 8.0 were 5 to 8 times lower than the maximum rate which could be supported by CO2 arising from the spontaneous dehydration of HC03-. Furthermore, CO2 compensation points of the cells in liquid media at 21% 02 at pH 7.0 and 8.0, and the K1/2 CO2 (CO2 concentration supporting the half maximal rate of 02 evolution) at 2% 02 at pH 7.0 and 8.0 are not consistent with HCO3-transport. These results indicate that the principal inorganic carbon species crossing the plasmalemma in these cells is CO2.is substantially higher in an alkaline medium compared to that in an acidic medium under the condition of constant CO2 concentration. Other workers (3,9,15,(19)(20)(21) have used more exacting criteria to establish active HCO3 transport, which in algal cells has the following characteristics: that the observed rate of photosynthesis, in alkaline medium, is substantially greater than the rate of photosynthesis which could be supported solely by the spontaneous dehydration of HCO3 to C02, within a closed system (19, 21), and there is an accumulation of a large, acid-labile, inorganic carbon pool within the cell which occurs against concentration and pH gradients (3,9,15,20).In the present study, we have applied the criteria established for HCO3 transport in algae, to test for the presence of inorganic carbon transport in isolated mesophyll cells ofAsparagus sprengeri Regel. These cells can be obtained quickly and in large quantities by a simple mechanical isolation technique (10), thus eliminating the potential hazards associated with enzymic isolation (11,22) and the deleterious effects of osmotic stress on photosynthesis (10,22). Asparagus mesophyll cells can maintain high rates of photosynthesis for a prolonged period of time (10) and their robust nature permits vigorous stirring of the reaction media and reduction of the surface boundary layer without significant damage to cellular integrity or loss of photosynthetic activity. MATERIALS AND METHODSThe chemical species of inorganic carbon which crosses the plasmalemma of the photesynthetic cells of higher plants is not known with certainty, but h...

“…After 60 min incubation in the presence of 33 nm SO2, the decrease of the pH of the cytoplasm was calculated from known buffering capacities (27,28) to be about 0.7 pH units. In the chloroplast stroma, such a decrease should inhibit photosynthesis by inhibiting fructose bisphosphatase and sedoheptulose bisphosphatase (4,6,7,30,33,38). At an SO2 concentration of 82 nm ( Fig.…”

mentioning

confidence: 99%

Flux of SO₂ into Leaf Cells and Cellular Acidification by SO₂

Pfanz¹,

Martinoia²,

Lange³

et al. 1987

ABSTRACIA comparison of fluxes of SO2 from the atmosphere into leaves with fluxes across biomembranes revealed that, apart from the cuticle, the main barrier to S02 entry into leaves are the stomates. S02 fluxes into leaves can be calculated with an accuracy sufficient for many purposes on the assumption that the intracellular S02 concentration is zero. SO2 entering green leaf cells is trapped in the cytoplasm. In the light, the products formed in its reaction with water are processed particularly in the chloroplasts. Flux of SO2 to the acidic central vacuole of leaf cells is insignificant. Intracellular acidification of barley mesophyll protoplasts by S02 was measured by the uptake of '4C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. The measured acidification was similar to the acidification calculated from known buffer capacities and the rate of SO2 influx when the H@/SO2 ratio was assumed to be 2. A comparison of photosynthesis inhibition by S02 with calculated acidification revealed different mechanisms of inhibition at low and at high concentrations of SO2. At very low concentrations, inhibition by S02 was even smaller than expected from calculated acidification. The data suggest that, ifacidification cannot be compensated by pH-stabilizing cellular mechanisms, it is a main factor of S02 toxicity at low SO2 levels. At high levels of SO2, anion toxicity and/or radical formation during oxidation of SO2 to sulfate may play a large role in inhibition.Diffusion of gases into a leaf is hindered by several barriers. Stomata can regulate entry of a gas into the interior of a leaf. Inside, aqueous phases of cellular systems are less important in restricting gas flux than lipid phases, if the solubility of a gas in an aqueous phase is high or if the gas can react with water in a reversible reaction to form ionic species which can contribute to and may actually dominate diffusional fluxes towards a sink. Both conditions are fulfilled by gases such as C02, which is the substrate of photosynthesis, and the air pollutants SO2 and NO2.When aqueous phases offer much less resistance to the diffusional flux of a gas or its reaction products than lipid phases, it may be sufficient for a flux analysis to know the resistance of biomembranes which have to be crossed by a gas on its way into a leaf cell or its organelles. In the previous publication (29) thereby releasing protons. Protons are trapped by alkali. As the pK, of sulfurous acid is about 1.8 and the pK2 about 7, the HS03-anion dominates below pH 7 and the S032-anion above pH 7. Thus, the products of the reaction of S02 with water will accumulate particularly in alkaline cell compartments. Release of protons in these compartments will decrease pH. The extent of the decrease is a function of the amount of SO2 trapped (i.e. of the flux of S02 into the leaf), the pH of the compartment and its buffering capacity. If the decrease in pH caused by influx of SO2 cannot be compensated by cellular mechanisms aimed at stabilizing intracellular pH, metabolism will be affected. For ...