Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO(3) (-) leaves) or 40 millimolar KNO(3) (high-NO(3) (-) leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO(3) (-). In contrast, the lower rate of increase recorded for low-NO(3) (-) leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO(3) (-) effect increased linearly with the level of NO(3) (-) uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO(3) (-) leaves, illuminated high-NO(3) (-) leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, (32)P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO(3) (-) conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO(3) (-), an interpretation of the role of NO(3) (-) as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO(3) (-), the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO(3) (-) modulates the relative protein kinase/protein phosphatase ratio to favor increased phosphorylation of both enzymes in order to redirect carbon flow away from sucrose synthesis and toward amino acid synthesis.

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO(3) (-) uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO(3) (-) leaves) or 40 mm NO(3) (-) solution (high-NO(3) (-) leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO(3) (-)-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO(3) (-) and high-NO(3) (-) leaves, indicating that NO(3) (-) had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO(3) (-) compared with low NO(3) (-) leaves showed evidence that NO(3) (-) enhanced the protein kinase activity. These results support the concept that NO(3) (-), or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).

Experiments were carried out with fully expanded leaves from three-week-old seedlings of wheat (Triticum aestivum L.) raised without NO 3 (sup-) . Nitrate was supplied to the leaves through the transpiration stream in the light. Uptake of NO 3 (sup-) was linear with NO 3 (sup-) concentrations from 0 to 80 mM in the solution. Net sucrose synthesis showed inverse relationships versus nitrate uptake, assimilation, and accumulation, with correlation coefficients close to 1. By contrast, no alteration in sucrose synthesis was observed when KCl was substituted for KNO3 in the uptake solution. Sucrose synthesis was not affected by nitrate in seedlings treated with tungstate which absorbed but did not reduce NO 3 (sup-) . After 10 h, the final amount of sucrose in the tissues was only slightly decreased in the presence of NO 3 (sup-) , indicating that the effect of NO 3 (sup-) did not result from an altered sucrose-storage capacity. Comparison of the carbon skeleton and energy reductant necessary for NO 3 (sup-) and CO2 assimilation is consistent with the hypothesis that the processes of NO 3 (sup-) assimilation and sucrose synthesis compete for photosynthetic energy and carbon.

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