Modulation of phosphoenolpyruvate carboxylase in vivo by Ca < sup > 2+ < /sup > in Amaranthus hypochondriacus, a NAD-ME type C < inf > 4 < /inf > plant: Possible involvement of Ca < sup > 2+ < /sup > in up-regulation of PEPC-protein kinase in vivo

Abstract

The properties of phosphoenolpyruvate carboxylase (PEPC) were studied, with respect to calcium (Ca2+), in leaves of Amaranthus hypochondriacus, a C4 plant. Experiments were conducted in vitro (by adding Ca 2+ during enzyme assay) or in vivo (by feeding Ca2+ to intact leaves through petiole). Inclusion of 10 μM Ca2+ during assay marginally increased ( < 30%) malate sensitivity of PEPC in extracts from dark-adapted leaves. The effect of Ca2+ was marginal on PEPC in extracts from illuminated leaves. Upon applying a low concentration of Ca 2+ to leaves, the PEPC activity in leaves increased by 1.5-fold, while inhibition by malate decreased markedly. The light activation of PEPC in Ca2+-fed leaves was slightly higher than in the absence of Ca 2+-ethyleneglycol-bis-(β-aminoethyl ether) N,N,N′, N′-tetra acetic acid (EGTA). To assess further the role of Ca 2+, 5 mM EGTA (Ca2+ chelator) was either added during the enzyme assay or fed to leaves through petiole. EGTA had no effect on PEPC, when added during enzyme assay. Upon feeding EGTA, the PEPC activity in the dark-adapted leaf extracts increased by 30%, and the effect on malate sensitivity was marginal. However, there was a decrease in PEPC activity in illuminated extracts, resulting in a marked decrease in the extent of light activation of PEPC. The extent of phosphorylation of PEPC was much higher in Ca2+ or Ca2+-EGTA-fed leaves than in the control, but EGTA decreased the light-induced phosphorylation. Our results suggest that optimal alone concentration of Ca2+ is essential for PEPC in leaves of A. hypochondriacus, particularly in vivo. We suggest that Ca2+ regulates PEPC, at an upstream level, such as transcription, by modulating PEPC-protein kinase, thus facilitating the light activation of PEPC. © 2005 Elsevier GmbH. All rights reserved.