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THE PLANT CELL, Vol 8, Issue 4 573-586, Copyright © 1996 by American Society of Plant Biologists
The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis
N. Giglioli-Guivarc'h, J. N. Pierre, S. Brown, R. Chollet, J. Vidal and P. Gadal
Institut de Biotechnologie des Plantes, Centre National de la Recherche Scientifique, UA D1128, Universite de Paris-Sud, Batiment 630, 91405 Orsay Cedex, France
Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from
C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and
shown to display the structural, functional, and regulatory properties
typical of a C4 PEPC. In situ increases in the apparent phosphorylation
state of the enzyme and the activity of its Ca2+-independent protein-serine
kinase were induced by light plus NH4Cl or methylamine. The
photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a
substitute for the weak base in these experiments. The early effects of
light plus the weak base or 3-PGA treatment were alkalinization of
protoplast cytosolic pH, shown by fluorescence cytometry, and calcium
mobilization from vacuoles, as suggested by the use of the calcium channel
blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the
apparent phosphorylation state of PEPC also were blocked in situ by the
electron transport and ATP synthesis inhibitors DCMU and gramicidin,
respectively, the calcium/calmodulin antagonists W7, W5, and compound
48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These
results suggest that the production of ATP and/or NADPH by the illuminated
mesophyll chloroplast is required for the activation of the transduction
pathway, which presumably includes an upstream Ca2+-dependent protein
kinase and a cytosolic protein synthesis event. The collective data support
the view that the C4 PEPC light transduction pathway is contained entirely
within the mesophyll cell and imply cross-talk between the mesophyll and
bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.
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