Miyake and Wada (8) and Sweeney et al. (10) reported that the nitrogen enrichment between nitrate and phytoplankton was null.The purpose of the experiment presented in this study was to investigate the source of the discrepant results described above by studying the effect of time during the growing season and external N03 concentration on isotopic fractionation associated with N03 uptake.The results given in this paper suggest that the apparent discrepancy of the previous reports are not surprising. It will be shown in this paper that the observed isotope effect for NO3 uptake varies systematically in response to environmental conditions which have an impact on the relative rates of the relevant reactions. (15N/14N) of the increment of product which appears in an infinitely short time at time t, and R, the isotope ratio of the substrate at the same time. MATERIALS ANDThe isotope enrichment factor is:(p/ff= (ap/8 -1) X 1,000When the quantity of substrate is infinite compared to the quantity of product (6), the isotopic ratio of the product Rp is constant.

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The For many years, extensive research has been devoted to the regulation of the synthesis of sucrose, the exported form of photoassimilate in most leaves (7,11,17,43). This has provided the basic elements for the description of the fine control network that regulates sucrose synthesis and photosynthetic C partitioning between the cytosol (sucrose synthesis) and the chloroplasts (starch synthesis). In leaves, NO3-assimilation takes place in the same compartments, i.e. cytosol (NO3-reduction to NO2-) and chloroplasts (reduction of NO2-to NH4' and assimilation of the latter into glutamate) as sucrose and starch synthesis, respectively. The reduction of N02-to NH4+ uses photochemically generated reducing power, as does the reduction of CO2 to carbohydrate, and is considered to be an important sink for the products of photosynthetic electron flow (31). In consideration of the common requirements of NO3-and CO2 metabolism in terms of reducing power, ATP, and carbon skeletons, important questions have been raised concerning the impact of NO3-on photosynthesis and the partitioning of photosynthetic products to amino acids at the expense of carbohydrate synthesis (44).The effect of nitrate on the rate of sucrose synthesis was studied with mature leaves detached from wheat seedlings (Triticum aestivum) (49). A linear inverse relationship was shown between the rate of sucrose synthesis and the rate of uptake and assimilation of NO3-, with no effect on insoluble carbohydrate synthesis. The effect of NO3-on sucrose synthesis appeared within the first hour after the leaves were fed NO3-. It slowed the rate of synthesis without altering the sucrose storage capacity of leaves. The rate of CO2 fixation was only slightly reduced (5).The diversion of 14C-labeled photosynthetic carbon away from carbohydrate synthesis toward organic acid and amino acid synthesis in leaves fed with NO3-provided evidence that sucrose synthesis is regulated at the level of partitioning of C between the two pathways in the leaves of higher plants (5). The increased carbon flux to amino acids is always associated with a decrease in the PEP' content and the activation of PEPcase in the leaves. The activation is not impaired by cycloheximide (48). The increased demand for carbon skeletons created by high rates of NO3-or NH4' assimilation was thus met by the diversion of fixed carbon to the anapleurotic pathway. Concomitantly, the rate of sucrose synthesis was restricted by a decrease in SPS activity. These results are in agreement with the suggestions that (a) PEPcase is the protein whose activity is most affected by N availability in leaves (45) and (b) SPS activity is a major component that controls the flux of carbon into sucrose (29,43

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.

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH1 and NADP-IDH2 (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH1 while NADP-IDH2 represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH2 was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH1, and as a consequence NADP-IDH2 does not result from a post-translational modification of NADP-IDH1. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH1 is restricted to the cytosol and NADP-IDH2 to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH2 was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.