Higher plant phosphoenolpyruvate carboxylase kinase is regulated at the level of translatable mRNA in response to light or a circadian rhythm

Abstract

Summary Phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to light in C3 and C4 plants and to a circadian oscillator in CAM plants. Increases in phosphoenolpyruvate carboxylase kinase activity require protein synthesis. This requirement has been analysed by quantifying translatable mRNA for this protein kinase using in vitro translation of isolated RNA followed by direct assay of kinase activity. In leaves of the CAM plant Bryophyllum (Kalanchoë) fedtschenkoi, in normal diurnal conditions, kinase mRNA was 20‐fold more abundant at night than in the day. In constant environmental conditions (continuous darkness, CO2‐free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. The appearance of kinase mRNA and activity was blocked by cordycepin and puromycin. In maize and barley, kinase mRNA increased in response to light. For all three plants, the phosphoenolpyruvate carboxylase kinase activity generated during in vitro translation was Ca2+‐independent. These results demonstrate that phosphoenolpyruvate carboxylase kinase activity is regulated at the level of translatable mRNA in C3, C4 and CAM plants.

Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO2 Fixation in Crassulacean Acid Metabolism

“…Leaf extracts were prepared and desalted as described by Hartwell et al (1996). The activity of PEPc was assayed and its apparent K i for l-malate estimated as described by Nimmo et al (1984).…”

“…Following the method of Hartwell et al (1996), RNA was isolated and translated in vitro using a rabbit reticulocyte lysate, and a sample of the translation products was as-sayed for PEPc kinase activity. The PEPc was isolated by immunoprecipitation, resolved by SDS gel electrophoresis, and the incorporation of 32 P into PEPc was quantified by phosphor imaging.…”

“…The PEPc was isolated by immunoprecipitation, resolved by SDS gel electrophoresis, and the incorporation of 32 P into PEPc was quantified by phosphor imaging. These values were corrected to take into account any differences in the efficiency of translation between the different samples, as estimated by the incorporation of [ 35 S]Met into protein (Hartwell et al, 1996). The values are therefore equivalent to data from northern blot analysis corrected for RNA loading.…”

“…Recently, Hartwell et al (1996) used a novel approach in which the products of in vitro translation of leaf RNA were assayed directly for PEPc kinase activity to demonstrate that the activity of PEPc kinase reflects changes in the level of its translatable mRNA. Thus, levels of kinase mRNA were approximately 20-fold higher at night than during the daytime in leaves of the CAM plant Kalanchoë (Bryophyllum) fedtschenkoi (Hartwell et al, 1996).…”

“…While the levels of PEPc kinase mRNA in C 3 and C 4 plants appear to respond to photosynthesis and, thus, light-dark transitions, in CAM plants a circadian oscillator controls the levels of kinase activity and translatable mRNA under constant environmental conditions (Carter et al, 1991;Hartwell et al, 1996). This results in a circadian rhythm in the phosphorylation state of PEPc (Nimmo et al, 1987), which plays an important role in generating the endogenous rhythms of CO 2 exchange in CAM plants first described by Wilkins (1959).…”