BCH/PLS/PPA 609 -- Plant
Biochemistry

Lecture Twenty-three
Nitrogen Metabolism -- nitrate reduction, ammonia assimilation

Goal: A good working knowledge and understanding of the reduction of oxidized forms of nitrogen commonly encountered by plants to ammonia and the assimilation of the reduced ammonia into major nitrogenous compounds of plant tissues.

Outline:

  1. Nitrate uptake
  2. Nitrate reduction to ammonia -- nitrate reductase & nitrite reductase
  3. Ammonia assimilation into glutamate & glutamine

 


Background Readings for the discussion on nitrate reduction and ammonia assimilation :

a)   REQUIRED:

1 -  Chapter 16, sections 16.6 - 16.10 of the Biochemistry & Molecular Biology of Plants class text.

2 - Han, Y.-L., Song, H.-X., Liao, Q., Yu, Y., Jian, S.-F., Lepo, J.E., Liu, Q., Rong, X.-M., Tian, C., Zeng, J., et al. (2016). Nitrogen use efficiency is mediated by vacuolar nitrate sequestration capacity in roots of Brassica napus. Plant Physiology 170:1684-1698.

 

b)      SUGGESTED:

1 -   Lee, J.-H., B.S. Evans, G. Li, N.L. Kelleher, and W.A. van der Donk. 2009. In Vitro Characterization of a Heterologously Expressed Nonribosomal Peptide Synthetase Involved in Phosphinothricin Tripeptide Biosynthesis. Biochemistry 48:5054-5056.. 

2 -    Crawford, Nigel M. 1995. Nitrate: Nutrient and signal for plant growth. The Plant Cell 7: 859-868.

3 -    Scheible, W.-R., R. Morcuende, T. Czechowski, C. Fritz, D. Osuna, N. Palacios-Rojas, D. Schindelasch, O. Thimm, M.K. Udvardi, and M. Stitt. 2004. Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response to Nitrogen. Plant Physiol. 136: 2483-2499.

4 - Forde, B.G. 2002. LOCAL AND LONG-RANGE SIGNALING PATHWAYS REGULATING PLANT RESPONSES TO NITRATE. Annu. Rev. Plant Biol. 53: 203-24.

5 - Lea, U.S., M.-T. Leydecker, I. Quillere, C. Meyer, and C. Lillo. 2006. Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence. Plant Physiol. 140: 1085-1094.

6 - Okamoto, M., A. Kumar, W. Li, Y. Wang, M.Y. Siddiqi, N.M. Crawford, and A.D.M. Glass. 2006. High-Affinity Nitrate Transport in Roots of Arabidopsis Depends on Expression of the NAR2-Like Gene AtNRT3.1. Plant Physiol. 140:1036-1046.

7- Remans, T., P. Nacry, M. Pervent, T. Girin, P. Tillard, M. Lepetit, and A. Gojon. 2006. A Central Role for the Nitrate Transporter NRT2.1 in the Integrated Morphological and Physiological Responses of the Root System to Nitrogen Limitation in Arabidopsis. Plant Physiol. 140:909-921


Remember from the previous class discussion, nitrogen (N2) must first be fixed usually into a reduced form such as ammonia. Ammonia is usually rapidly oxidized into nitrate by nitrifying bacteria in soils so nitrate is the usual form of nitrogen available to most plants.

 

 

 

 

  • Plant have distinct transport systems with different affinities for nitrate (Vidmar et al., 2000; Ortiz-Lopez et al., 2000).
    1. Constitutive high-affinity transport system (CHATS)
      • Responsible for nitrate uptake at low concentrations (below ~1 mM).
      • Saturation kinetics, with Km values below 300 µM.

    2. Inducible high-affinity transport system (IHATS)
      • Induced by NO3- taken up by CHATS.
      • Increases overall NO3- uptake transiently.

    3. Low affinity transport system (LATS)
      • Uptake of nitrate at high concentration (> 0.2 mM).
      • Like the high affinity systems, the low affinity systems are electrogenic and involve proton cotransport.
      • Show linear rather than saturation kinetics.
      • Not saturated even at 50 mM NO3-.

  • Studies on nitrate uptake
  • Measuring NO3- uptake has been hampered by the lack of a suitable radioactive tracer.
    1. 13N has a half-life of only 10 min.
    2. 15N is not radioactive.

    3. [not covered in 2018]

    4. When NO3- is supplied to cells it is metabolized and effluxed as equilibrium is established with preexisting pools.

  • Chlorate (ClO3-)
  • Utilizing ion-specific electrodes it has been suggested that there is a cotransport of NO3-/H+ across the plasmalemma of maize root cells and that the cytoplasmic level of nitrate is kept relatively constant by storage of nitrate in the vacuole.
  • In NO3--starved roots, uptake is dominated by a constitutively expressed, low activity, high affinity system for NO3- uptake in barley and maize roots.
  • When N-starved plants are treated with NO3-, the root develops a high rate of NO3- uptake with a greater affinity for NO3-:
  • The induction of mRNA of the IHATS transporter is longer and greater at 1 mM (a) than 10 mM (b) NO3-.
  • Induced by NO3- and somewhat less so by NO2- but inhibited by NH4+ (all at 10 mM).
  •  
  • Not only is the nitrate uptake system induced, but also nitrate induces nitrate and nitrite reductase activities by altering gene expression mainly by enhancing transcription of the respective genes.  Nitrate, light and sugars are inducers; glutamate and glutamine are repressors.

  • Why might this be?
  •  

     

  • Nitrate is a signal for developmental changes in the physiology of the plant.
  • The primary responses include:
    1. Induction of genes for nitrate and nitrite reduction.
    2. Nitrate uptake and translocation systems.
    3. DNA regulatory proteins required for expression of the secondary response gene system.

  • The secondary response include more complex phenomena such as
    1. Proliferation of the root system.
    2. Enhancement of respiration.
    3. Other changes in the physiology of the plant.

  • A constitutive 'NO3- sensor' protein would detect the presence of environmental NO3-, then NO3- induction regulatory protein(s) would be activated which would act to initiate transcription of the primary response genes by RNA polymerase, resulting in NR, NiR, NO3- transporters, NO3- translocaters, ammonia assimilation enzymes.
  • What might be expected to inhibit this?
  • The fate of NO3- taken up by a root epidermal cell.
  •  
  • Once transported into an epidermal cell, NO3- has one of four fates:
    1. It may undergo efflux to the apoplast and soil environment.
    2. It may enter the vacuole and by stored.
    3. It may be reduced to ammonium by the combined action of NR and NiR.
    4. It may be translocated via the symplast to the xylem.

  • Nitrate Reduction
  • a-Ketoglutarate
    Phosphoenolpyruvate
    Glutamine
    Malate
    Glutamine
    (2) Glutamate
    Aspartate
    Oxaloacetate
    Glucose
    Glutamine
    Malate
    NH+4
    Asparagine
    Oxaloacetate
    Sucrose