Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 mM Mg2+ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg2+ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre‐incubation of crude extracts with ATP increased Mg2+ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg2+ 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 Mg2+ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO2 exchange. However, in contrast to the rhythm in CO2 exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post‐translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm.

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca2+, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C3 leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C3 protoplasts than in C4 protoplasts or leaf tissue.

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg2+ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO 3 - ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO 3 - . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.

SummaryPhosphoenolpyruvate 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.

Maize seedlings were grown in pots either with or without preconditioned seeds of the parasitic weed, Striga hermonthica. After between 4 and 8 weeks, net photosynthesis in the leaves of maize plants infected with Striga decreased compared to leaves of uninfected control plants, The activities of four enzymes of photosynthetic metabolism were, however, little affected by infection. A pulse-chase experiment using (CO2)-C-14 showed that C-4 acids were the main early products of assimilation even when the rate of photosynthesis was much decreased by infection, but more radioactivity appeared in glycine and serine than in leaves of healthy maize plants, Leaves of infected maize required longer to reach a steady rate of photosynthesis upon enclosure in a leaf chamber than leaves of uninfected plants after similar treatment.Electron microscopy of transverse sections of the leaves of infected maize indicated that the cell walls in the bundle sheath and vascular tissue were less robust than in leaves of healthy plants, The results suggest that infection with Striga causes an increase in the permeability of cell walls in the bundle sheath, leakage of CO2 from the bundle sheath cells and decreased effectiveness of C-4 photosynthesis in host leaves.

The parasitic angiosperm Striga hermonthica is native to Africa where it parasitizes maize and can reduce crop yield to zero. Pulse-chase experiments showed that carbon was assimilated in control and infected plants mainly by the C4 pathway but the distribution of 14C among photosynthetic intermediates indicated an increase in photorespiratory metabolism in leaves of infected plants. Measurements with an infra-red gas analyser showed that maize infected with Striga hermonthica had decreased rates of photosynthesis. All the leaves were affected. The decrease often began before parasitic shoots had emerged above the soil and the extent of the decrease was not correlated with the number of parasitic shoots that eventually emerged. The decrease in photosynthesis was not due to a reduction in activity of four major photosynthetic enzymes. Glutathione reductase activity was increased as a result of infection. Microscopic studies of fresh sections showed abnormalities in bundle sheath cells of leaves from infected plants. Electron micrographs showed that the bundle sheath and vascular cells of infected plants had thinner walls and were not as rounded in outline as in control plants. Immunofluorescent labelling of RuBisCO showed it to be localised in the bundle sheath cells in leaf tissue of both control and infected plants, but decreased autofluorescence indicated that the walls of the bundle sheath cells were thirmer in leaves of infected plants. The effects of extracts of leaves on cells in culture provided no evidence for the presence of a toxin, but suggested a change in growth hormone content caused by infection. Differences in the content of soluble carbohydrate, starch and α-amino nitrogen showed that Striga infection changed host resource partitioning.