Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

Abstract

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C(4) plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O(2) evolution by chloroplasts isolated from protoplasts.Chloroplasts were isolated from protoplasts by several passages of the protoplasts through a 20-micrometer nylon mesh. Mesophyll chloroplasts were judged approximately 90 to 95% intact and bundle sheath chloroplasts 80 to 90% intact based on retention of chloroplast marker enzymes and the ferricyanide test for intactness. It was necessary to include 10 millimolar MgCl(2) in media for osmotically shocking the chloroplasts in order to obtain maximum and linear rates of ferricyanide-dependent O(2) evolution.Chloroplasts isolated from mesophyll protoplast preparations had low rates of light-dependent O(2) evolution in the presence of 10 millimolar NaHCO(3) (0.13 micromoles per milligram chlorophyll per minute) in comparison to bundle sheath chloroplasts (1 to 2.5 micromoles per milligram chlorophyll per minute). The mesophyll chloroplasts catalyze high rates of 3-phosphoglycerate-dependent O(2) evolution (2 to 4 micromoles per milligram chlorophyll per minute). Orthophosphate but not phosphoenolpyruvate inhibited the 3-phosphoglycerate-dependent O(2) evolution by the mesophyll chloroplasts. Rates of O(2) evolution were much higher with mesophyll than with bundle sheath chloroplasts in the presence of pyruvate plus oxaloacetate. The results are discussed in relation to the proposed function of these chloroplasts during C(4) photosynthesis.