Abstract
Mesophyll cells and bundle sheath strands were isolated rapidly from leaves of the C(4) species Digitaria pentzii Stent. (slenderstem digitgrass) by a chopping and differential filtration technique. Rates of CO(2) fixation in the light by mesophyll and bundle sheath cells without added exogenous substrates were 6.3 and 54.2 micromoles of CO(2) per milligram of chlorophyll per hour, respectively. The addition of pyruvate or phosphoenolpyruvate to the mesophyll cells increased the rates to 15.2 and 824.6 micromoles of CO(2) per milligram of chlorophyll per hour, respectively. The addition of ribose 5-phosphate increased the rate for bundle sheath cells to 106.8 micromoles of CO(2) per milligram of chlorophyll per hour. These rates are comparable to those reported for cells isolated by other methods. The K(m)(HCO(3) (-)) for mesophyll cells was 0.9 mm; for bundle sheath cells it was 1.3 mm at low, and 40 mm at higher HCO(3) (-) concentrations. After 2 hours of photosynthesis by mesophyll cells in (14)CO(2) and phosphoenolpyruvate, 88% of the incorporated (14)C was found in organic acids and 0.8% in carbohydrates; for bundle sheath cells incubated in ribose 5-phosphate and ATP, more than 58% of incorporated (14)C was found in carbohydrates, mainly starch, and 32% in organic acids. These findings, together with the stimulation of CO(2) fixation by phosphoenolpyruvate for mesophyll cells and by ribose 5-phosphate plus ATP for bundle sheath cells, and the location of phosphoenolpyruvate and ribulose bisphosphate carboxylases in mesophyll and bundle sheath cells, respectively, are in accord with the scheme of C(4) photosynthesis which places the Calvin cycle in the bundle sheath and C(4) acid formation in mesophyll cells.Starch and reducing sugars were present in both mesophyll and bundle sheath cells following a period of photosynthesis by whole leaves. However, when isolated cells were exposed to (14)CO(2) in the light, even with appropriate exogenous substrates, only bundle sheath cells accumulated appreciable amounts of labeled carbohydrates. Incubation of mesophyll cells in the light with ATP and either pyruvate and inorganic phosphate, or phosphoenolpyruvate, or 3-phosphoglycerate resulted in large increases in total carbohydrates. The 3-phosphoglycerate treatment produced the greatest increase. These results could not be explained on the basis of increased CO(2) fixation. They suggest that mesophyll cells are able to metabolize exogenously supplied 3-carbon compounds to carbohydrates, despite the apparent inability of these cells to utilize CO(2) for this purpose, and support the view that in the whole leaf 3-phosphoglycerate is transported from bundle sheath to mesophyll cells, where it is reduced to carbohydrate.Sucrose and sucrose-phosphate synthetases and invertase were localized mainly in bundle sheath cells. ADP-Glucose starch synthetase and amylase were present mainly in bundle sheath cells whereas starch phosphorylase was present mainly in mesophyll cells.