10.2 Nitrate reduction and amino acid biosynthesis in plants

Plants are nitrogen-hungry machines, constantly transforming nitrate into usable forms. This process, called nitrate reduction, happens in two steps: nitrate to nitrite in the cytosol, then nitrite to ammonium in plastids. It's a crucial part of nitrogen metabolism.

Once plants have ammonium, they can make all 20 amino acids needed for proteins. This happens mainly in plastids and is tightly linked to carbon metabolism. Plants regulate this process at multiple levels, responding to light, stress, and nutrient availability.

Nitrate Reduction in Plants

Two-Step Process and Cellular Localization

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• Nitrate reduction converts nitrate (NO3-) to nitrite (NO2-) and then to ammonium (NH4+) through a two-step process
• Process occurs in different cellular compartments
  • Nitrate reduction takes place in the cytosol
  • Nitrite reduction happens in plastids
• Essential for nitrogen assimilation by converting inorganic nitrogen into a usable form for amino acid synthesis
• Requires energy in the form of reducing agents (NADH or NADPH)

Regulation and Influencing Factors

• Tightly regulated process influenced by various factors
  • Light availability affects enzyme activity and gene expression
  • Carbon metabolism interacts with nitrogen assimilation (C/N balance)
  • Nitrogen availability in the environment modulates the process
• Environmental stresses impact nitrate reduction
  • Drought conditions can alter nitrogen uptake and assimilation
  • Temperature extremes affect enzyme activity and overall metabolism

Enzymes of Nitrate Reduction

Key Enzymes and Their Functions

• Nitrate reductase (NR) catalyzes nitrate to nitrite reduction in the cytosol
• Nitrite reductase (NiR) converts nitrite to ammonium in plastids
• Two forms of nitrite reductase exist
  • Ferredoxin-dependent NiR predominates in photosynthetic tissues (leaves)
  • NAD(P)H-dependent NiR found in non-photosynthetic tissues and roots
• Glutamine synthetase (GS) and glutamate synthase (GOGAT) assimilate ammonium into amino acids
  • GS catalyzes the formation of glutamine from glutamate and ammonium
  • GOGAT transfers the amide group from glutamine to 2-oxoglutarate, forming two molecules of glutamate

Enzyme Regulation and Cellular Localization

• Nitrate reductase activity regulated by phosphorylation and dephosphorylation
  • Phosphorylation inactivates NR in darkness or under stress conditions
  • Dephosphorylation activates NR in light or favorable conditions
• Nitrite reductase localized in plastids to prevent accumulation of toxic nitrite in the cytosol
• GS exists in cytosolic (GS1) and plastidic (GS2) isoforms
  • GS1 predominant in roots and non-photosynthetic tissues
  • GS2 major isoform in leaves, involved in photorespiratory ammonium reassimilation

Amino Acid Biosynthesis in Plants

Essential and Non-Essential Amino Acids

• Plants synthesize all 20 proteinogenic amino acids, unlike animals
• Amino acid biosynthesis primarily occurs in plastids, linked to carbon metabolism
• Classification of amino acids based on biosynthetic pathways
  • Aromatic amino acids (phenylalanine, tyrosine, tryptophan) synthesized via shikimate pathway
  • Aspartate-derived amino acids (lysine, threonine, methionine, isoleucine) share common steps
  • Glutamate-derived amino acids include proline, arginine, and glutamine
  • Branched-chain amino acids (valine, leucine, isoleucine) synthesized from pyruvate
  • Serine, glycine, and cysteine biosynthesis involves multiple cellular compartments

Key Biosynthetic Pathways

• Shikimate pathway crucial for aromatic amino acid synthesis
  • Involves seven enzymatic steps from phosphoenolpyruvate and erythrose 4-phosphate
  • Produces chorismate, a precursor for phenylalanine, tyrosine, and tryptophan
• Aspartate-derived amino acid synthesis
  • Aspartate kinase catalyzes the first step, forming aspartyl phosphate
  • Branching pathways lead to lysine, threonine, and methionine
• Glutamate serves as a central precursor
  • Proline synthesized via glutamate-5-semialdehyde and pyrroline-5-carboxylate
  • Arginine formed through the ornithine cycle
• Branched-chain amino acid synthesis
  • Begins with pyruvate and involves several shared enzymatic steps
  • Dihydroxyacid dehydratase and branched-chain aminotransferase are key enzymes

Regulation of Amino Acid Biosynthesis

Multilevel Regulation Mechanisms

• Transcriptional regulation controls enzyme gene expression
  • Light-responsive elements in promoter regions of key genes (NR, NiR)
  • Nitrogen-responsive elements modulate expression based on N availability
• Post-transcriptional regulation fine-tunes enzyme activity
  • mRNA stability and translation efficiency affected by environmental cues
  • Protein phosphorylation/dephosphorylation (NR regulation)
• Allosteric regulation provides rapid response to metabolic changes
  • Feedback inhibition common in amino acid biosynthetic pathways
  • Example: Threonine inhibits aspartate kinase in its own biosynthetic pathway

Environmental and Hormonal Influences

• Light plays crucial role in regulating nitrate assimilation and amino acid biosynthesis
  • Activates nitrate reductase and increases expression of biosynthetic genes
  • Influences carbon metabolism, affecting precursor availability
• C/N balance sensing mechanism integrates carbon and nitrogen metabolism
  • High C/N ratio promotes amino acid biosynthesis
  • Low C/N ratio downregulates biosynthetic pathways
• Environmental stresses alter amino acid biosynthesis and accumulation
  • Drought stress often leads to proline accumulation (osmotic adjustment)
  • Salt stress can increase glycine betaine synthesis in some species
• Hormonal signals modulate amino acid metabolism
  • Cytokinins generally promote nitrogen assimilation and amino acid biosynthesis
  • Abscisic acid influences amino acid transport and metabolism under stress
• Target of rapamycin (TOR) kinase pathway integrates nutrient and energy signals
  • Regulates amino acid metabolism and protein synthesis
  • Responds to changes in cellular energy status and nutrient availability