Open AccessFlow cytometric analysis of cytosolic pH of mesophyll cell protoplasts from the crabgrass Digitaria sanguinalis
Author(s) -
GiglioliGuivarc'h Nathalie,
Pierre JeanNoël,
Vidal Jean,
Brown Spencer
Publication year - 1996
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19960301)23:3<241::aid-cyto7>3.0.co;2-l
Subject(s) - protoplast , phosphoenolpyruvate carboxylase , cytosol , nigericin , c4 photosynthesis , biology , digitaria sanguinalis , flow cytometry , biochemistry , photosystem ii , dephosphorylation , phosphorylation , biophysics , microbiology and biotechnology , enzyme , photosynthesis , botany , phosphatase , membrane , weed
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is a key enzyme of photosynthesis in C 4 plants; it is specifically localized in the cytosol of mesophyll cells and is regulated by a phosphorylation/dephosphorylation process. The light‐dependent phosphorylation of PEPC is triggered by an increase in the cytosolic pH (pHc) of mesophyll cell protoplasts. An epifluorescence and confocal microscopy analysis showed that the specific pH probe 2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, acetoxymethyl ester (BCECF‐AM), when used at low concentration, was essentially localized in the protoplast cytosol. By the nigericin null‐point method and flow cytometry, the pHc of freshly isolated protoplasts was estimated to be 6.4. To observe the full activity of PEPC kinase and maximal phosphorylation of PEPC in vivo, such protoplast suspensions must first be treated with a permeant weak base. The present report shows that 20 mM NH 4 Cl raised the final pHc to 7.4. This method can be useful for estimating rapid changes of pHc in plant cells. © 1996 Wiley‐Liss, Inc.