Wheat Leaf Phosphoenolpyruvate Carboxylase Activity

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-NO3leaves) or 40 millimolar KNO3 (high-NO3leaves) 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 NO3-. In contrast, the lower rate of increase recorded for low-NO3leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO3effect increased linearly with the level of NO3uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO3leaves, illuminated high-NO3leaves. 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, 32p 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-NO3conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO3-, an interpretation of the role of NO3as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO3-, 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 NO3modulates 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. A decrease in the rate of sucrose synthesis in illuminated wheat leaves upon transition from the low-NO3to the highNO3state has recently been reported (30). This effect is observed within minutes of feeding NO3to detached nitrogen-deficient leaves. The decrease in sucrose synthesis is linearly correlated with the rate ofNO3uptake and assimilation. The diversion of '4C-labeled photosynthetic carbon away from carbohydrate synthesis toward that oforganic acids and amino acids provided evidence that, in the presence of NO3-, the regulation of sucrose synthesis is modified to allow partitioning of C between the NO3assimilation and carbohydrate biosynthesis pathways in wheat leaves (8). Measurements of the level of intermediary metabolites and the changes of the fructose-2,6-bisphosphate concentration have revealed that the increased demand for carbon skeletons created by high rates of NO3assimilation is associated with (a) a concomitant decrease in phosphoenolpyruvate contents showing that PEPcase' (EC 4.1.1.31) and/or pyruvate kinase (EC 2.7.1.40) are activated, and (b) a decrease of SPS (EC 2.4.1.14) activity, which restricts the rate of sucrose synthesis (our unpublished data). Thus, it appears that in wheat leaves, light and NO3might regulate the activity of two enzymes that are involved in primary (PEPcase) and secondary (SPS) carbon metabolism in an inverse fashion. The results reported in the present paper are consistent with this hypothesis. We provide evidence that PEPcase is activated and SPS is deactivated when low-NO3wheat leaves are challenged by high-NO3nutrition in the light. PEPcase has been described as the protein whose changes are most affected by N availability in leaves (26); however, the regulation of PEPcase in C3 plants is not well understood. This is in marked contrast to the situation for PEPcase activity in C4 species and CAM plants, in which the light effect on PEPcase activity has been described repeatedly and thoroughly studied (2). The mechanism of the light modulation of the C4 and CAM plant PEPcase is considered to involve an interaction among the two effectors (glucose-6-phosphate and malate), phosphorylation, and aggregation/disaggregation of the enzyme-protein (13, 15, 20, 21, 27). Although it is generally sensitive to the same effectors, the PEPcase of C3 plants is thought to need less "tight" regulation than the C4 and CAM enzyme (18). Because the evidence for in vivo leaf PEPcase activation was first obtained from a C3 plant, the work reported here has focused on wheat leaf PEPcase with the objective of establish'Abbreviations: PEPcase, phosphoenolpyruvate carboxylase; SPS, sucrose phosphate synthase, 150, concentration of malate for 50% inhibition of the PEP case activity. 1476 www.plantphysiol. org on May 1, 2019 Published by Downloaded from Copyright © 1991 American Society of Plant Biologists. All rights reserved. WHEAT LEAF PEPcase: MODULATION BY LIGHT AND NO3ing and characterizing the dependency of PEPcase activity on light and nitrate. Assuming that the mechanism of the activation of C3 plant PEPcase might be similar to that of the C4 and CAM plant enzymes, the approach undertaken to elucidate the underlying mechanisms in this study was as follows: (a) comparison of the immunological properties; (b) sensitivity of PEPcase to the effectors malate and glucose-6-P (32); (c) sensitivity to okadaic acid (an inhibitor of phosphoprotein phosphatase activity) (9); and (d) sensitivity to cycloheximide (an inhibitor of protein synthesis) in crude extracts from lowNO3and high-NO3wheat leaves in the light as well as from low-NOleaves sampled in the dark (control). In addition, treatment of the leaves with 32Pi was performed in order to determine whether or not the wheat leaf PEPcase was modulated by reversible phosphorylation, as is the PEPcase from C4 and CAM plants, and SPS. The results presented here provide evidence for modulation of PEPcase by light and NO3in mature wheat leaves and establish that the covalent modification of the enzyme is due to reversible phosphorylation. An interpretation of the interaction ofNO3with the light effect, based on the comparison of the responses of wheat leaf PEPcase and SPS, is proposed. MATERIALS AND METHODS