Nitrogen Fixation

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Nitrogen Fixation

• A symbiotic association between nodule-forming
  bacteria (Rhizobiacae) and legumes
• Three genera of bacteria
• Rhizobium, Bradyrhizobium, Azorhizobium
• 12000 species of legumes
• includes all important protein crops
• soybean, lentil, pea, lupin, chickpea etc

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Nitrogen Fixation

• Other plant/microbe interactions fix nitrogen as
  well
• Waterfern Azola Anabaena sp
• Important for rice production
• Woody plants and actinomycetes Frankia
• Includes some alders

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Rhizobia/Legume Interaction

• The interaction is species specific
• e.g. R. phaseolibean B. japonicumsoybean
• Initiation of process is by plants and dictates
  specificity
• Secretion of flavonoids attracts bacteria
• luteolin alfalfa R. meliloti
• naringenin genestein soybean

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Attachment

• The presence of these flavonoids causes genes to
  be expressed in the Rhizobium sp
• These genes are largely extrachromosomal and are
  called nod genes (except in Bradyrhizobium)
• One of the products of the nod genes is a large
  lipopolysaccharide molecule
• This molecule interacts with lectins on the root
  hairs

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Nodulation Factor
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Infection

• In response to the nod factor the plant genes are
  expressed
• At the point of attachment, root hair begins to
  deform
• Cell wall begin to disintegrate at base of root
  hair
• Root hair deformation cause curling

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Infection

• Cell division is induced in the root cortical
  cells in response to nod factor
• Cell wall-like synthesis occurs and surrounds the
  attached Rhizobium
• Synthesis continues back toward the base of the
  root hair where it merges with existing cell wall

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Infection

• This synthesis is referred to as the infection
  thread
• Bacteria are engulfed within the cortical cell
• Cell membrane invagination at point of entry into
  the cell
• The bacteria are now encased in the membrane
  (peribacteroid membrane) inside the cell

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Nodule Formation

• Once inside the coritcal cell, the bacteria
  multiple and dedifferentiate
• Infected root cells swell and stop dividing
• After dedifferentiation, the Rhizobium are
  referred to as a bacteroid
• After formation, the bacteroid begin to express
  nif and fix genes and the plant expresses late
  nodulin genes

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Nodule Formation

• The bacteroid are in the nodule
• Nodule can be either spherical or indeterminate
• The indeterminate are found on perennial crops

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Metabolite Exchange

• Many of the late nodulin genes encode enzymes
  involved in the exchange of metabolites between
  bacteriod and plant
• Specific channel proteins are synthesized to
  traverse the perisomal membrane
• Plants provide carbon to the bacteroid
• Most common from is malate

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Metabolite Exchange

• The bacteroid fixes N2 and exports it into the
  nodule cells as ammonium
• Is assimilated by normal pathway in many plants
• In some plants the nitrogen is converted to
  purines
• It is then converted to ureides for transprt and
  storage

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Nitrogen Fixation

• After bacteroid has stopped dividing, nif and fix
  genes are expressed
• These genes encode proteins associated with the
  formation of the nitrogenase enzyme complex
• The complex is comprised of 2 multiple subunit
  enzymes, each with multiple bound cofactors

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Nitrogenase

• The enzymes in the complex are nitrogenase
  reductase dinitrogenase
• The dinitrogenase catalyzes the reduction of N2
  to NH3
• N2 4H 4NADPH 16ATP ? 2 NH3 4NADP 16
  ADP 16Pi
• Therefore 14 ATP per NH3

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Nitrogenase

• The enzyme also catalyzes several other reactions
• For every NH3 produced 1 molecule of H2 is
  synthesized
• 2H 2e- ? H2
• The enzyme also converts acetylene to ethylene
• H2 is an explosive gas

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Hydrogenases

• Bacteroids possess hydrogenases which are coupled
  to electron transport
• 2 H2 O2 ? 2 H2 O
• Whether any ATP is generated in this reaction is
  unknown

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The Oxygen Paradox

• Major problem
• nitrogenase is inhibited by oxygen
• bacteroids need oxygen for aerobic respiration
  and energy synthesis
• How are both conditions satisfied?
• Oxygen entry into bacteroid is controlled by the
  plant

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Partitioning the Components

• The dilemma is resolved via 2 mechanisms
• First - the oxygen requiring respiratory
  components are embedded in the outrer membrane of
  the bacteroid and the nitrogenase is on the
  interior of the bacteroid
• Second - oxygen is scavenged and released
  directly to the ETS

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Do Plants Bleed?

• Animals control oxygen transport and exchange
  oxygen via hemoglobin myoglobin
• In the space between the plant cell membrane and
  the bacteroid membrane, a plant protein called
  leghemoglobin accumulates
• Structurally similar to hemoglobin

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Leghemoglobin

• It is a late nodulin gene
• The heme is synthesized by the bacteroid
• Binds and concentrates oxygen in the region of
  the bacteroid ETS
• Releases oxygen into ETS at concentration
  adequate to maintain energy production
• Leghemoglobin found in non-legumes

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Plant Defense Genes

• Infection of plant tissues generally results in
  defense response
• Early nodule gene expression represses typical
  defense genes
• Allows for infection
• Permits opportunistic infection

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Nodule Senescence

• In annual (determinate nodules) plants senescence
  of above ground tissues initiates nodule
  breakdown
• Leghemoglobin isnt synthesized
• Perisomal membrane disintegrates
• Bacteroid disintegrates
• Nitrogen enters rhizosphere

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Improving Mineral Nutrition

• Our major sources of fertilizer exploit non
  renewable resource
• Maintaining high productivity will require
  continued high inputs
• Can plants or the environment be modified to
  improve mineral uptake?
• Numerous targets under consideration

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Microbial Inoculants

• Inoculants currently available for improved
  nitrogen, phosphorous, zinc, magnesium, and iron
  uptake
• Work on principles of acidification,
  solubilization, and symbiosis
• Engineering enhanced microbes

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Modifying Plants

• Engineering increased acidifying ability
• Engineering enhanced root growth
• Engineered carrier proteins
• Engineering for improved symbiosis