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.
We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C 4 phosphoenolpyruvate carboxylase (PEPC) by its Ca 2؉ -insensitive protein-serine/threonine kinase (PEPC kinase) in the C 4 mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 mol m ؊2 s ؊1 photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll-and bundle-sheathspecific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO 2 -exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.
1996. Rhythms in magnesium ion inhibition and hysteretic properties of nitrate reductase in the CAM planl Bryophyllum fedtschenkoi ' -Phys'w]. Planl. 98: 140-146, Nitrate reductase (NR, EC 1,6,6,1) was tested in crude extracts of leaves from Biyophvlltim fedtschenkoi plants growing nnder alternating light/darkness as well as in excised leaves kept in continuous liglit or darkness. In most extracts NR activity was inhibited 2O-8O'7r by 5 mM Mg"* A^lighl to darkness shifl (30 min darkness) during the first part of the pholoperiod gave an increase in the Mg"' inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variation,s. Strongest inhibition was found in extracts made during the latter part of the pholoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg"' inhibition, indicating that phosphorylation of NR is involved m regulation of NR in Crassulaeean acid metabolism (CAM) plants. In continuous light an increase in Mg'* inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR, A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg"* inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in COT exchange. However, in contrast to the rhythm in CO, exchange, NR rhythtiis were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that posltranslational modification of CAM NR is influenced by light/darkne,ss and by an endogenous rhythm.
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