Evidence for Mediated HCO3− Transport in Isolated Pea Mesophyll Protoplasts

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Abstract
The kinetics of 14C fixation and inorganic C (Cinorg) accumulation were followed in isolated pea mesophyll protoplasts. NaH14CO3 was supplied to the protoplasts in media the pH of which was varied between 7-8. When 14CO2 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 Vmax did not alter significantly. Similarly, when Cinorg 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 Cinorg concentration rose throughout the experimental period and that in the light it surpassed the external Cinorg concentration after .apprx. 3 min. Irradiation of protoplasts previously taking up 14Cinorg in the dark brought about a sharp increase in the rate of 14Cinorg accumulation which was sustained for at least 20 min. 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 Cinorg distribution were always higher than those calculated from DMO distribution, i.e., the internal Cinorg 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 of Cinorg accumualtion. Various possible interpretations of the results are considered. 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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