Maximising Photosynthetic Activity and Cell Integrity in Isolated Bundle Sheath Cell Strands From C₄ Species

Abstract

Bundle sheath cells prepared from C₄ leaves have a variety of experimental applications because of their high permeability to metabolites. We determined the factors affecting the physical and metabolic integrity of Panicum miliaceum bundle sheath cell strands immediately following isolation and during subsequent storage. Cell integrity was monitored by determining chloroplast intactness (as ferricyanidedependent O₂ evolution) and photosynthetic activity (usually as HCO₃^-, 3-phosphoglycerate- or C₄ acid-dependent O₂ evolution). In freshly isolated cell preparations, at least 85-90% of the chloroplasts were apparently intact and there was no significant decline in this value during storage for up to 5 h. With the best extraction conditions, light-dependent O₂ evolution in response to adding HCO₃^- or C₄ acids ranged between 3 and 6 µmol min^-1 mg^-1 chlorophyll. Preillumination of leaves was critical for good isolated cell photosynthetic activity and the activity due to C₄ acids in particular was increased substantially by including EDTA. During storage for up to 5 h at 0°C as much as 80% of the capacity for HCO₃^-- or C₄ acid-dependent O₂ evolution was lost; this loss was very largely prevented by storing the cells with EDTA. Large kinetic lags (induction phase) also developed during storage of cells. Both the loss of photosynthetic activity and the kinetic lags were reduced or eliminated by including photosynthetic intermediates such as dihydroxyacetone phosphate or ribose 5-phosphate in the storage medium. Under the best conditions, bundle sheath cell preparations could be stored for at least 3 h with little apparent change in cell integrity or metabolic capacity.