Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions

IT is generally considered that glutamate dehydrogenase (EC.1.4.1.3) is the enzyme that is chiefly responsible for the incorporation of nitrogen into the α-amino group of amino acids: We suggest that this is not the case in the leaves of higher plants.

Alternative route for nitrogen assimilation in higher plants.

Publisher Summary This chapter describes the preparation and extraction of nitrate reductase from higher plants. For preparation of nitrate reductase, chlorophyllous lamina tissue from illuminated plants well supplied with nitrate is normally used as a source material because of its high activity. However, monchlorophyllous organs, such as corn scutella or roots, have been used. Soybean leaves, corn scutella, and cultured rice seedlings are the best known sources of NAD(P)H nitrate reductase. For extraction of the enzyme, the material is homogenized for 30 to 90 seconds in a medium of 1 mM EDTA, 1 to 25 mM cysteine, and 25 mM potassium phosphate, adjusted to a final pH of 8.8 with KOH. Six milliliters of grinding medium are added for each gram of fresh weight of tissue. The homogenate is pressed through four layers of cheesecloth or a single layer of Miracloth and the filtrate centrifuged for 15 minutes at 30,000 g. The supernatant fluid is then decanted through glass wool and used for assays. The homogenates and extracts are kept cold (2° to 3°) throughout. The optimum concentration of cysteine must be verified for each type of tissue and homogenization technique. Glutathione and dithiothreitol are usually slightly more effective (10%) than cysteine.

[24] Nitrate reductase from higher plants

Publisher Summary
This chapter focuses on ammonia assimilation and discusses the mechanisms by which the ammonia produced is incorporated into organic compounds. Nitrate and dinitrogen gas, two of the major sources of inorganic nitrogen available to plants, are reduced to ammonia before the nitrogen is incorporated into organic matter. Ammonia is released and reassimilated in large amounts at different stages of the plant's metabolism. Nitrate is the major source of inorganic N available to the leaves of most land plants; ammonia is not present in the xylem stream leaving the roots. Thus, the ammonia assimilated is normally generated in situ from the reduction of nitrate through nitrite. Ammonia is not normally transported into a leaf but is rather a product of nitrate reduction; however, it can also be generated within the leaf by two other ways: (1) by the breakdown of asparagine through transaminase or asparaginase and (2) in photorespiration.

4 – Ammonia Assimilation

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-beta-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

https://hal.archives-ouvertes.fr/hal-00356462/document

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport.

The assimilation of nitrate, nitrite and ammonia in barley, wheat, corn and bean leaves was studied using 15N-labelled molecules and either leaf chamber experiments with the uptake of the nitrogen species in the transpiration stream, or vacuum-infiltration experiments. The assimilation of 15NO3− into amino nitrogen was strictly dependent on light and ceased abruptly when the light was extinguished. If the leaves were exposed to air, CO2-free air or N2 there was no effect on the rate of NO3− assimilation over 0.5 h. After 1.25 h of CO2-free air, NO3− assimilation into amino acids was sharply reduced. Resupply of air at this time stimulated NO3− assimilation and restored it to the rate observed in leaves exposed to air only. There was no recovery by tissue pretreated for 1.25 h in N2 and subsequently resupplied with air. Incorporation of 15NO2− was also markedly dependent on light with little reduction occurring in the dark. Incorporation of 15NH4+ into amino acids was stimulated 5 fold by light but considerable incorporation occurred in the dark. The presence of 100 mM NO3− had no effect on the rate of incorporation of 15NO2− or 15NH4+. Nitrite at 1 mM had no effect on 15NO3− incorporation but at 10 mM inhibited it completely after 0.5 h. Ammonia at 1 mM had no effect on 15NO3− or 15NO2− incorporation and while 10 mM inhibited incorporation for 0.5 h this inhibition did not persist.

Nitrate, Nitrite and Ammonia Assimilation by Leaves : Effect of Light, Carbon Dioxide and Oxygen

The assimilation of H(14)CO3 (-), (15)NO3 (-), (15)NO2 (-) and (15)NH4 (+) by barley (Hordeum vulgare L.) leaf segments in the presence of a number of metabolic inhibitors was studied in experiments where the substrates and inhibitors were vacuum infiltrated into the tissue. 3-(3',4'-Dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl-cyanide-m-chlorophenylhydrazone (CCCP), and iodoacetate (IOA) inhibited (14)CO2 fixation and (15)NO3 (-) and (15)NO2 (-) assimilation in the light. (15)NH4 (+) assimilation in the light was only inhibited 70% by 10(-4)M DCMU. (15)NH4 (+) assimilation was stimulated by 10(-5)M CCCP but was inhibited by concentrations of CCCP above 5×10(-5)M. In double-label experiments (15)NO3 (-) assimilation was less sensitive than (14)CO2 fixation to both DCMU and CCCP. CCCP but not DCMU stimulated NO2 (-) accumulation in dark and IOA was inhibitory. The rate of NO2 (-) accumulation in the light in the presence of DCMU or atrazine was similar to that in the dark and in all cases NO2 (-) accumulation was inhibited about 90% by oxygenation of the medium. The results indicate that the assimilation of all nitrogen species is closely linked to photosynthetic electron transport, that rate of assimilation of nitrogen species is independent of rate of assimilation of CO2, and that the dark in-vivo nitrate reduction is a useful analogue of the mechanism operating in the light only if electron flow to oxygen is impaired.

D. T. Canvin

Papers

Nitrate, Nitrite and Ammonia Assimilation by Leaves : Effect of Light, Carbon Dioxide and Oxygen

Nitrate, nitrite and ammonia assimilation by leaves: Effects of inhibitors.