Assimilation%20and%20fixation%20of%20nitrogen

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Assimilation and fixation of nitrogen
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Plants are

• Capable of making all necessary organic compounds
  from inorganic compounds and elements in the
  environment (autotrophic)
• Required to compete with other organisms for
  these nutrients
• Required to employ complex energetic pathways to
  convert macronutrients to useable forms

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Nitrogen in the environment

• Many biochemical compounds present in plant cells
  contain nitrogen
• Nucleoside phosphates
• Amino acids
• These form the building blocks of nucleic acids
  and protein respectively
• Only carbon, hydrogen, and oxygen are nor
  abundant in plants than nitrogen

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Nitrogen in the environment

• Present in many forms
• 78 of atmosphere is N2
• Most of this is NOT available to living organisms
• Getting N2 for the atmosphere requires breaking
  the triple bond between N2 gas to produce
• Ammonia (NH3)
• Nitrate (NO3-)
• So, N2 has to be fixed from the atmosphere so
  plants can use it

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Nitrogen in the environment

• This occurs naturally by-Lightning
• 8 splits H2O the free O and H attack N2
  forms HNO3 (nitric acid) which fall to ground
  with rain
• Photochemical reactions
• 2 photochemical reactions between NO gas and O3
  to give HNO3
• Nitrogen fixation
• 90 biological bacteria fix N2 to ammonium
  (NH4)

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Nitrogen in the environment
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Nitrogen in the environment

• Once fixed in ammonium or nitrate -
• N2 enters biochemical cycle
• Passes through several organic or inorganic forms
  before it returns to molecular nitrogen
• The ammonium (NH4) and nitrate (NO3-) ions
  generated via fixation are the object of fierce
  competition between plants and microorganisms
• Plants have developed ways to get these from the
  soil as fast as possible

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Root uptake soon depletes nutrients near the roots

• Formation of a nutrient depletion zone in the
  region of the soil near the plant root
• Forms when rate of nutrient uptake exceeds rate
  of replacement in soil by diffusion in the water
  column
• Root associations with Mycorrhizal fungi help the
  plant overcome this problem

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Mycorrhizal associations

• Not unusual
• 83 of dicots, 79 of monocots and all
  gymnosperms
• Ectotrophic Mycorrhizal fungi
• Form a thick sheath around root. Some mycelium
  penetrates the cortex cells of the root
• Root cortex cells are not penetrated, surrounded
  by a zone of hyphae called Hartig net
• The capacity of the root system to absorb
  nutrients improved by this association the
  fungal hyphae are finer than root hairs and can
  reach beyond nutrient-depleted zones in the soil
  near the root

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Mycorrhizal associations

• Vesicular arbuscular mycorrhizal fungi
• Hyphae grow in dense arrangement , both within
  the root itself and extending out from the root
  into the soil
• After entering root, either by root hair or
  through epidermis hyphae move through regions
  between cells and penetrate individual cortex
  cells.
• Within cells form oval structures vesicles
  and branched structures arbuscules (site of
  nutrient transfer)
• P, Cu, Zn absorption improved by hyphae
  reaching beyond the nutrient-depleted zones in
  the soil near the root

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Nutrients move from fungi to root cells

• Ectotrophic Mycorrhizal
• Occurs by simple diffusion from the hyphae in the
  hartig net to the root cells
• Vesicular arbuscular mycorrhizal fungi
• Occurs by simple diffusion from the arbuscules to
  the root cells
• Also, as arbuscules are degenerating as new ones
  are forming, the nutrients may be released
  directly into the host cell

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Stored ammonium can be toxic

• Plants can store high levels of nitrate or
  translocate it via the phloem without any effect.
• However, high levels of ammonium are toxic
• Dissipates transmembrane proton gradients
  required for both photosynthetic and respiratory
  electron transport
• AND movement of metabolites to vacuoles.

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Stored ammonium can be toxic

• At high pH in stroma, matrix or cytoplasm
• Ammonium reacts with OH- to produce NH3.
• NH3 is membrane permeable and diffuses freely
  across a membrane down a concentration gradient
• At low pH in intermembrane space, lumen, or
  vacuole
• NH3 reacts with H to form ammonium

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Remember Nitrogen the most important mineral
nutrient in the soil

• Nitrogen is frequently limiting in in terrestrial
  systems terrestrial systems
• Microbial activity is continually converting N to
  lower energy forms
• Conversion to organic form requires raising N to
  higher energy levels

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Nitrate Assimilation
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Deficiency Symptoms - N

• General chlorosis.
• Chlorosis progresses from light green to yellow.
• Entire plant becomes yellow under prolonged
  stress.
• Growth is immediately restricted and plants soon
  become spindly and drop older leaves.

http//plantsci.sdstate.edu/woodardh/soilfert/Nutr
ient_Deficiency_Pages/soy_def/SOY-N1.JPG
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Nitrogen assimilation

• NO3 NO2 NH4 amino acids
• nitrate nitrite ammonium
• Requires large input of energy
• Forms toxic intermediates
• Mediated by specialized enzymes that are closely
  regulated are closely regulated
• Doesnt have to start at the beginning

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Nitrogen assimilation

• Plants assimilate most of the nitrate absorbed by
  their roots into organic nitrogen compounds.
• The first step of this process is the reduction
  of nitrate to nitrite in the cytosol by the
  enzyme nitrate reductase.

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Nitrogen assimilation

• NAD(P)H induces NADH or NADPH
• The most common form of nitrate reductase uses
  only NADH as an electron donor
• The nitrate reductases of higher plants are
  composed of two identical sub-units, each
  containing three prosthetic groups
• FADflavin adenine dinucleotide
• Heme
• Molybdenumorganic molecule called pterion

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Nitrate Assimilation

• Nitrate reductase is the main molybdenum
  containing protein in vegetative tissues
• Nitrate levels, light intensity, and
  concentration of carbohydrates all influence the
  activity of nitrate reductases at the
  transcription and translation levels
• These factors stimulate a protein, phosphatase,
  that dephosphorylates several serine residues on
  the nitrate reductase protein thereby activating
  the enzyme
• This dephosphorylation/phosphorylation cycle
  provides more rapid control over this enzyme than
  degredation/synthesis of new enzyme would achieve
  
• ( minutes versus hours)

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Nitrite Reductase Converts Nitrite to Ammonium

• Nitrite (NO2-)is highly reactive
• Plant cells immediately transport the nitrite
  generated by nitrite reduction from the cytosol
  into chloroplasts in leaves and plastids in roots
• In these organelles, nitrite reductase reduces
  nitrite to ammonium

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Nitrite Reductase Converts Nitrite to Ammonium

• Chloroplast and root plastids contain different
  forms of the enzyme, but both forms consist of a
  single polypeptide containing an iron sulfur
  cluster and a specialized heme group
• The heme does redox reactions and electron flow,
  just like the reaction sites of chlorophyll

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Nitrite Reductase Converts Nitrite to Ammonium

• Nitrite is highly reactive
• Plant cells immediately transport the nitrite
  generated by nitrite reduction from the cytosol
  into chloroplasts in leaves and plastids in roots
• In these organelles, nitrite reductase reduces
  nitrite to ammonium
• Chloroplast and root plastids contain different
  forms of the enzyme, but both forms consist of a
  single polypeptide containing an iron sulfur
  cluster and a specialized heme group
• The heme does redox reactions and electron flow,
  just like the reaction sites of chlorophyll

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Plants assimilate nitrate in both roots and shoots

• In many plants, when the roots receive small
  amounts of nitrate, this nitrate is reduced
  primarily in the roots
• As nitrate supply increases, a greater proportion
  of the absorbed nitrate is translocated to the
  shoot and assimilated there
• Generally, species native to temperate rely more
  heavily on nitrate assimilation by the roots than
  do species of tropical or subtropical origins

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Ammonium Assimilation

• Plants cells avoid ammonium toxicity by rapidly
  converting the ammonium generated from nitrate
  assimilation or photorespiration into amino acids
• This requires the action of two enzymes
• Glutamine synthetase combines ammonium with
  glutamate to form glutamine
• Glutamate synthase stimulated by elevated
  levels of glutamine synthetase
• Transfers the amino group of glutamine to an
  intermediate yielding two molecules of glutamate

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Transamination Reaction Transfer Nitrogen

• Once assimilated into glutamine and glutamate,
  nitrogen is incorporated into other amino acids
  via transamination reactions
• The enzymes involved in these reactions are known
  as aminotransferases
• Best known aspartate aminotransferase
• The amino group of glutamate is transferred to
  the carboxyl atom of aspartate
• Aspartate is the amino acid which shuttles
  reducing agents from the mitochondrion and
  chloroplast into the cytosol and in the transport
  of carbon from mesophyll to bundle sheath of C4
  carbon fixation
• All this requires vitamin B6 to act as a cofactor

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Biological nitrogen Fixation

• This accounts for most of the fixation of
  atmospheric N2 into ammonium
• Represents the key entry point of molecular
  nitrogen into the biogeochemical cycle of
  nitrogen
• Free living and symbiotic bacteria are
  responsible for converting atmospheric nitrogen
  into ammonium
• Most of these are free living in the soil, a few
  form symbiotic associations with higher plants
• The prokaryote directly provides the host plant
  with nitrogen in exchange for other nutrients and
  carbohydrates
• The most common association is between members of
  the plant family leguminosae and bacteria of the
  genera Azorhizobium

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Nitrogen Fixation Requires Anaerobic Conditions

• As oxygen irreversibly inactivates the
  nitrogenase enzymes involved in nitrogen
  fixation, nitrogen must be fixed under anaerobic
  conditions
• Therefore each of the nitrogen-fixing organisms
  either functions under natural anaerobic
  conditions or can create an internal anaerobic
  environment in the presence of oxygen

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Nitrogen Fixation Requires Anaerobic Conditions

• In cyanobacteria, anaerobic conditions are
  created in specialized cells called heterocysts
• These are thick-walled cells which lack
  photosystem IIthe oxygen producing photosystem
  of chloroplasts
• Cyanobacteria can fix nitrogen under anarobic
  conditions such as those that occur in flooded
  fields
• In Asian countries, nitrogen fixing cyanobacteria
  of both the heterocyst and non-heterocyst types
  are the major means of maintaining an adequate
  nitrogen supply in rice fields
• They fix nitrogen when the fields are flooded,
  and die as the fields dry, releasing the fixed
  nitrogen into the soil

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Symbiotic Nitrogen Fixation Occurs in Specialized
Structures

• Symbiotic nitrogen-fixing prokaryotes dwell
  within nodules
• Special organs of the plant host that enclose the
  nitrogen-fixing bacteria
• Grasses can also develop symbiotic relationships
  with nitrogen-fixing organisms, but these
  associations do not lead to the formation of root
  nodules
• Nitrogen-fixing bacteria seem to colonize plant
  tissues or anchor to the root surface, mainly
  around the elongation zone and the root hairs
• Known as actinorhizal plants

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Symbiotic Nitrogen Fixation Occurs in Specialized
Structures

• Both legumes and actinorhizal plants regulated
  gas permeability in their root nodules
• Maintaining a level of oxygen within the nodule
  that can support cellular respiration for the
  bacteria, but still sufficiently low to avoid
  inactivation of the nitrogenase

Nodules Contain an oxygen binding heme
proteinleghemoglobin Leghemoglobin produces a
pink color Helps transport oxygen to the
respiring symbiotic bacteria cells in a manner
analogous to hemoglobin transporting oxygen to
respiring tissues in animals
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Establishing Symbiosis Requires a Change of
Signals

• Legumes seedlings germinate without any
  association to rhizobia
• Under nitrogen limiting conditions, the plant and
  the bacteria seek each other out by an elaborate
  exchange of signals
• Plant genes specific to nodules are called
  nodulin (nod) genes
• Rhizobial genes that participate in nodule
  formation are called nodulation (nod) genes
• The nod genes are classified as common nod genes
  or host specific nod genes

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Establishing Symbiosis Requires a Change of
Signals

• Common nod genes
• nodA, nodB, and nodC found in all rhizobial
  strains
• Host specific non genes
• nodP, nodQ, nodH, nodE, and nodF differ among
  rhizobial species and determine the host range
• The first stage of the association is the
  migration of the bacteria through the soil
  towards the host plant

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Nod Factors produces by bacteria act as signals
for symbiosis

• nodD is constitutively expressedhas a role in
  the activation of all other nod genes by
  signaling the formation of nod factors
• Lipochitin oligosacharides with a chitin-b-1,4
  linked N-acetyl-D-glucosamine
• nodA, nodB, and nodC encode for the formation of
  this structure

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Nodule formation involves several phytohormones

• During root nodule formation, two process occur
  simultaneously
• Infection and Nodule Organogenesis
• (A) Rhizobia attach to the root hairs and release
  nod factors that produce a pronounced curling of
  the root hair cell
• (B) Rhizobia get caught and curl, degrade the
  root hair cell wall allowing the bacterial cells
  direct access to the outer surface of the plant
  plasma membrane

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Nodule formation involves several phytohormones

• (C) Then the infection thread forms
• Formed from Golgi depositing material at the tip
  at the site of infection. Local degradation of
  root hair cell wall also occurs
• (D) Infection thread reaches the end of the cell,
  and thread plasma membrane fuses with plasma
  membrane of root hair cell
• Then bacterial cells are released into the fused
  plasma membranes

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Nodule formation involves several phytohormones

• (E) Rhizobia are released into the apoplast and
  enter the middle lamella,
• This leads to the formation of a new infection
  thread, which forms an open channel with the
  first
• (F) Infection thread expands and branches until
  it reaches target cells
• Vesicles composed of plant membrane enclose
  bacterial cells and they are released into the
  cytoplasm

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Nodule formation involves several phytohormones

• At first bacteria continue to grow with vesicles
  expanding by fusing with smaller vesicles
• Following an as yet determined chemical signal
  from the plant, bacteria stop dividing and
  differentiate
• Forms nitrogen-fixing organelles called
  bacteroids
• The nodule itself develops a vascular system
• To exchange fixed nitrogen for nutrients from the
  plant
• And a layer of cells to exclude O2 from the rood
  nodule interior

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The nitrogenase enzyme complex fixes N2

• Biological nitrogen fixation produces ammonium
  (NH3) from molecular nitrogen.
• N2 8e- 8H 16 ATP 2NH3 H2 16 ADP
  16 Pi
• Note that the reduction of N2 to 2NH3 is a
  six-electron transfer, and is coupled to the
  reduction of two protons to evolve H2
• This reaction is catalyzed by nitrogenase enzyme
  complex

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The nitrogenase enzyme complex fixes N2

• Can be separated into two components
• The Fe protein
• The MoFe protein
• Neither of which has catalytic activity by itself

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The nitrogenase enzyme complex fixes N2

• Ferredoxin reduces the Fe protein
• Binding and hydrolysis of ATP to the Fe protein
  is thought to cause a conformational change of
  the Fe protein that facilitates the REDOX
  reactions
• The Fe protein reduces the MoFe protein, and the
  MoFe protein reduces the N2

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The MoFe protein can reduce many substances

• The MoFe protein can reduce many substrates
• Although under natural conditions the MoFe only
  reacts with N2 and H.

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Summary

• Nutrient assimilation is the process by which
  nutrients acquired by plants are incorporated
  into the carbon constituents necessary for growth
  and development.
• For Nitrogen
• Assimilation is but one in a series of steps that
  constitute the nitrogen cycle.
• The principal sources of nitrogen available to
  plants are nitrate (NO3-) and ammonia (NH4).
• Nitrate absorbed by roots is assimilated in
  either shoots or roots
• depending on nitrate availability and plant
  species

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Summary

• In nitrate assimilation, nitrate (NO3-) is
  reduced to nitrite (NO2-) in the cytosol via the
  enzyme nitrate reductase.
• Then nitrite is reduced to ammonium (NH4) in
  roots by nitrite reductase.
• Ammonium (NH4) from either root absorption or
  generated through nitrate assimilation or
  photorespiration is converted glutamine or
  glutamate through the sequential actions of
  glutamine synthase and glutamate synthase.
• Once assimilated into either glutamine or
  glutamate, nitrogen mat be transferred to many
  other organic compounds
• Via transaminatation reactions

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Summary

• Many plants form a symbiotic relationship with
  nitrogen fixing bacteria that contain an enzyme
  complex, nitrogenase, that can reduce atmospheric
  nitrogen to ammonia.
• Legumes and actinorhizal plants form associations
  with rhizobia.
• These associations result from a finely tuned
  interaction between the bacteria and the host
  plant
• Involves the recognition of specific signals
  between the symbiotic bacteria and the host plant

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Any Questions?