caa a22 4500 477101674 CHVBK 20180405111543.0 cr unu---uuuuu 170330e19960701xx s 000 0 eng 10.1007/BF00195741 doi (NATIONALLICENCE)springer-10.1007/BF00195741 In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phospho enol pyruvate-carboxylase kinase signal-transduction chain [Elektronische Daten] [Stephen Duff, Nathalie Giglioli-Guivarc'h, Jean-Noël Pierre, Jean Vidal, Shirley Condon, Raymond Chollet] Regulation of the light activation of C4 phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro 32P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH4Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca2+ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca2+ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C4 MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531-537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C4-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C4 mesophyll cytosol. Springer-Verlag, 1996 C4 photosynthesis nationallicence Digitaria nationallicence Mesophyll cells and protoplasts nationallicence Phospho enol pyruvate carboxylase nationallicence Protein-Ser/Thr kinase nationallicence BS : bundle-sheath nationallicence CAM : Crassulacean acid metabolism nationallicence DHAP : dihydroxyacetone phosphate nationallicence FPLC : fast-protein liquid chromatography nationallicence Glc6P : glucose 6-phosphate nationallicence I0.5 : 50% inhibition constant nationallicence MC : mesophyll cell(s) nationallicence MP : me-sophyll protoplast(s) nationallicence PEP : phosphoenolpyruvate nationallicence PEPC : PEP carboxylase nationallicence PEPC-PK : PEPC protein-Ser/Thr kinase nationallicence 2-PGA : 2-phosphoglycerate nationallicence 3-PGA : 3-phosphoglycerate nationallicence PPFD : photosynthetic photon flux density nationallicence Pyr : pyruvate nationallicence Ser : serine nationallicence Duff Stephen Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664, Lincoln, NE, USA aut Giglioli-Guivarc'h Nathalie Institut de Biotechnologie des Plantes, UA CNRS D-1128, Université de Paris-Sud, Bâtiment 630, F-91405, Orsay Cedex, France aut Pierre Jean-Noël Institut de Biotechnologie des Plantes, UA CNRS D-1128, Université de Paris-Sud, Bâtiment 630, F-91405, Orsay Cedex, France aut Vidal Jean Institut de Biotechnologie des Plantes, UA CNRS D-1128, Université de Paris-Sud, Bâtiment 630, F-91405, Orsay Cedex, France aut Condon Shirley Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664, Lincoln, NE, USA aut Chollet Raymond Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664, Lincoln, NE, USA aut Planta Springer-Verlag 199/3(1996-07-01), 467-474 0032-0935 199:3<467 1996 199 425 https://doi.org/10.1007/BF00195741 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00195741 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Duff Stephen Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664, Lincoln, NE, USA aut NATIONALLICENCE 700 1- Giglioli-Guivarc'h Nathalie Institut de Biotechnologie des Plantes, UA CNRS D-1128, Université de Paris-Sud, Bâtiment 630, F-91405, Orsay Cedex, France aut NATIONALLICENCE 700 1- Pierre Jean-Noël Institut de Biotechnologie des Plantes, UA CNRS D-1128, Université de Paris-Sud, Bâtiment 630, F-91405, Orsay Cedex, France aut NATIONALLICENCE 700 1- Vidal Jean Institut de Biotechnologie des Plantes, UA CNRS D-1128, Université de Paris-Sud, Bâtiment 630, F-91405, Orsay Cedex, France aut NATIONALLICENCE 700 1- Condon Shirley Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664, Lincoln, NE, USA aut NATIONALLICENCE 700 1- Chollet Raymond Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664, Lincoln, NE, USA aut NATIONALLICENCE 773 0- Planta Springer-Verlag 199/3(1996-07-01), 467-474 0032-0935 199:3<467 1996 199 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer