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In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

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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 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.

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Crassulacean acid metabolism


dihydroxyacetone phosphate


fast-protein liquid chromatography


glucose 6-phosphate

I0.5 :

50% inhibition constant


mesophyll cell(s)


me-sophyll protoplast(s)




PEP carboxylase


PEPC protein-Ser/Thr kinase






photosynthetic photon flux density






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Author information

Correspondence to Raymond Chollet.

Additional information

The authors thank Ms. Jill Myatt for her help with some of the MC preparations. This work was supported in part by grants INT-9115566 and MCB-9315928 from the U.S. National Science Foundation (to R.C.). S.M.G.D. was a recipient of an NSERC of Canada Post-Doctoral Fellowship. This paper is Journal Series No. 11 395 of the University of Nebraska Agricultural Research Division.

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Duff, S.M.G., Giglioli-Guivarc'h, N., Pierre, J. et al. In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain. Planta 199, 467–474 (1996). https://doi.org/10.1007/BF00195741

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Key words

  • C4 photosynthesis
  • Digitaria
  • Mesophyll cells and protoplasts
  • Phosphoenolpyruvate carboxylase
  • Protein-Ser/Thr kinase