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Articles

Evidence for Mediated HCO₃^– Transport in Isolated Pea Mesophyll Protoplasts ¹

Department of Botany, The Hebrew University of Jerusalem, Israel

The kinetics of ¹⁴C fixation, and inorganic C (C_inorg) accumulation, have been followed in isolated pea mesophyll protoplasts. NaH¹⁴CO₃ was supplied to the protoplasts in media the pH of which was varied between 7 and 8.

When ¹⁴CO₂ fixation was plotted against the calculated concentration of free CO₂ in the media, the apparent K_m for CO₂ was observed to rise as external pH increased. The V_max did not alter significantly. Similarly, when C_inorg uptake, either in the light 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_inorg concentration rose throughout the experimental period, and that in the light it surpassed the external C_inorg concentration after about 3 minutes. Irradiation of protoplasts previously taking up ¹⁴C_inorg in the dark brought about a sharp increase in the rate of ¹⁴C_inorg accumulation which was sustained for at least 20 minutes.

Estimates of internal pH based on the distribution of labeled 5,5-dimethyloxazoladine-2,4-dione (DMO) between protoplast and medium suggested that internal pH altered relatively little with change in external pH. The values for internal pH as calculated from C_inorg distribution were always higher than those calculated from DMO distribution, i.e. the internal C_inorg concentration was higher than would be predicted on the assumption of passive distribution in accordance with pH.

Addition of carbonic anhydrase to the external solution was without effect either on rate of ¹⁴CO₂ fixation or C_inorg accumulation.

Various possible interpretations of the results are considered. It is concluded that the most reasonable explanation, consistent with all the data, is that HCO₃^– ions can cross the protoplast membranes, and that their passage is mediated by a transfer mechanism.

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