Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Abstract

Leaves of the C3 plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO2 concentrations varying from 1 to 20% in air. When they were transferred back into CO2-free air, CO2 was rapidly released. It originated from dissolved CO2 and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO2 from bicarbonate. The data were fitted to a model which accounts for the CO2/bicarbonate equilibrium in buffers with different CO2 concentrations and initial pH values. From this, pH values and CO2-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO2 concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO2 between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H+ per pH unit. The work shows that measurements of solubilized CO2 are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.