FLOW CYTOMETRIC ANALYSIS OF CYTOSOLIC PH OF MESOPHYLL CELL PROTOPLASTS FROM THE CRABGRASS DIGITARIA-SANGUINALIS

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is a key enzyme of photosynthesis in C-4 plants; it is specifically localized in the cyt osol of mesophyll cells and is regulated by a phosphorylation/dephosph orylation process. The light-dependent phosphorylation of PEPC is trig gered by an increase in the cytosolic pH (pHc) of mesophyll cell proto plasts, An epifluorescence and confocal microscopy analysis showed tha t the specific pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfl uorescein, acetoxymethyl ester (BCECF-AM), when used at low concentrat ion, was essentially localized in the protoplast cytosol, By the niger icin null-point method and now cytometry, the pHc of freshly isolated protoplasts was estimated to be 6.4. To observe the full activity of P EPC kinase and maximal phosphorylation of PEPC in vivo, such protoplas t suspensions must first be treated with a permeant weak base. The pre sent report shows that 20 mM NH4Cl raised the final pHc to 7.4, This m ethod can be useful for estimating rapid changes of pHc in plant cells . (C) 1996 Wiley-Liss, Inc.