Acid-base regulation during nitrate assimilation in Hydrodictyon africanum

Abstract The acid-base balance during NO₃^? assimilation in Hydrodictyon africanum has been investigated during growth from (1) an analysis of the elemental composition of the cells, (2) the alkalinity of the ash and (3) the net H⁺ changes in the medium during growth. These investigations agree in showing that some 0.25 excess organic negative charges are generated per N assimilation from No₃^? as N-source and C02 as C-source; the excess OH^? (0.75 OH^? per NO₃^? assimilated) appears in the medium. Approximately half of the excess organic negative charge is attributable to cell wall uronates; the remainder is intracellular. All of the excess OH^? appearing in the medium must have crossed the plasmalemma (as net downhill H⁺ influx or OH^? efflux). Previous work has shown that the value of ψco is more negative than ψ_K⁺ during NO₃^? assimilation, suggesting that the active electrogenic H⁺ extrusion pump is still operative despite the net downhill H⁺ influx. The interpretation of this in terms of H⁺?NO₃^? symport which causes the entry of more H⁺ than is consumed in NO₃^? metabolism, with extrusion of the excess H⁺via the active, electrogenic H⁺ pump, was tested by measuring short-term H⁺ influx upon addition of NO^?₃. A net H⁺ influx occurs before NOa assimilation (as indicated by additional O₂ evolution in the light) has commenced, suggesting a mechanistic relation of H⁺ and NO₃^? influxes. This is consistent with the interpretation suggested above. Determinations of cytoplasmic pH showed no significant effect of NO₃^? assimilation, suggesting that cytoplasmic pH changes sufficient to change the ‘pH-regulating’ H⁺ fluxes are smaller than the errors in the determination of cytoplasmic pH.     

Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Measurement of Cytoplasmic pH by the DMO Technique in Hydrodictyon africanum

The 5, 5-dimethyl-[2-¹⁴C]oxazolidine-2, 4-dione (DMO) distributiontechnique for the measurement of intracellular pH has been appliedto giant cells of Hydrodictyon africanum. Significant metabolism of DMO was found in this alga; the free[DMO + DMO–] in subcellular samples is thus derived fromthe total label in cells equilibrated in [¹⁴C]DMO solutionsby measuring and subtracting the label in metabolic productsof DMO. A further problem arises from the observation that theDMO concentration in the vacuolar sap is always lower than thatpredicted by the transmembrane equilibration of undissociatedDMO from the bathing medium. This is interpreted in terms ofa finite permeability to the anion DMO–. Since the effectof P_DMO– on the DMO distribution is much smaller at thetonoplast (where the transmembrane electrical potential differenceis small) than at the plasmalemma, the values of cytoplasmicpH are computed assuming equilibration of undissociated DMOacross the tonoplast. At an external pH of 7.0 the cytoplasmic pH is about 7.4; decreaseor increase of external pH by 1 unit causes a decrease and anincrease in cytoplasmic p11 respectively of about 0.2 pH units.Determinations of _vo at pH 6, 7, and 8, together with an assumedconstant value of _cv, permit calculations of µ_H+ at theplasmalemma and tonoplast. The values are relatively independentof external pH in the range pH 6–8 at 21–25 and12–14 kJ mol^–1 respectively. The significance ofthese results for the regulation of intracellular pH, and forthe regulation and energising of the fluxes of ions, is discussed.