Transcript Nitrogen availability

Microbial symbioses
Root-nodule symbiosis
Nitrogen availability
• Nitrogen is required in large amounts as an essential
component of proteins, nucleic acids and other cellular
constituents.
• Abundant supply of nitrogen in the earth's atmosphere
-nearly 79% in the form of N2 gas
• N2 is unavailable for use by most organisms
-triple bond makes N2 molecule almost inert
• N2 must be fixed to ammonium (NH4+) or nitrate (NO3-)
ions to make it available for growth and metabolism
Sources of Available Nitrogen
- weathering of rocks releases negligible amounts of
ammonium (NH4+) or nitrate (NO3-) ions
- small amount of ammonia/nitrate is produced by lightning
- ammonia/nitrate is also produced industrially
Terrestrial ecosystems, 80% to 90% of nitrogen available
to plants originates from biological nitrogen fixation
80% of the total comes from symbiotic associations
Three Major Types of Nitrogen Fixing symbioses
1) Rhizobia – Gram negative bacteria that form nodules mainly
on roots.
legume host plants: peanut, soybean, lentil, bean, pea, clover
and alfalfa.
non-legume host: Parasponia
2) Actinomycetes - Gram positive bacteria (genus Frankia)
diverse hosts: many trees and woody shrubs
Important role in nitrogen economies of forests and other
natural Ecosystems.
3) Cyanobacteria – Gram negative photosynthetic bacteria
diverse hosts: cycads, ferns, liverworts and hornworts
Anabaena associates with the water fern Azolla and is used as
a co-crop in rice paddies allowing sustainable rice cultivation
Morphological variations in the rhizobium–legume symbiosis.
(a) Cupriavidus taiwanensis – Mimosa pudica and occasionally on the stems of
legumes
(b) Azorhizobium caulinodans – Sesbania rostrata
(c) Bradyrhizobium sp. ORS322 – Aeschynomene afraspera
(d) Bradyrhizobium sp. ORS278 – Aeschynomene sensitiva
Nodules display various shapes:
-round (e) Sinorhizobium fredii – soybean
-coralloid (f) Methylobacterium nodulans – Crotalaria perrottetii
- elongated (g) Sinorhizobium meliloti – Medicago sativa
Cortical tissue infection proceeds via transcellular infection threads (h) S.
meliloti (tagged with red and green fluorescent proteins)– M. sativa
or
crack-entry at emergence of lateral roots (i) Bradyrhizobium sp.
ORS285 (green tagged) – Aeschynomene indica
Invasion of plant roots
Barrel medic
Rhizobium
Flavonoids (2-phenyl-1,4-benzopyrone derivatives)
Flavonoids bind bacterial NodD proteins, which are members of the LysR family of
transcriptional regulators, and activate these proteins to induce the transcription of
rhizobial genes, namely the nod genes. Flavonoids from non-host plants inhibit nod
gene transcription.
Structure of Nod factors
Nod factors (or lipochitooligosaccharides) have:
-a backbone of β 1–4-linked N-acetylglucosamine residues
(black) with N-linked acyl groups (green) and other hostspecific decorations ( red ).
-Each species produces multiple Nod factors; e.g., the
number of glucosamine residues can be four or five, the
acyl chains of the Nod factors from S. meliloti and R.
leguminosarum bv. viciae can be C18:1 instead of C16:2
and C18:4 as shown, and not all Nod factors necessarily
carry all the host-specific decorations
-The decoration groups can be fucosyl, sulphuryl, acetyl,
methyl, carbamoyl and arabinosyl residues
Nod factors responses:
- Increase in the intracellular levels of calcium in root hairs,
followed by strong calcium oscillations (spiking) and alterations
to root hair cytoskeleton
- Curling of the root hairs, which traps rhizobial bacteria within
what is known as a tight colonized curled root hair (CCRH)
- Simultaneously, Nod factors stimulate root cortex cells to
reinitiate mitosis and these cells will form the nodule
primordium, and give rise to the cells that will receive the
invading bacteria
Root hair curling and cortical cell divisions
Nod factor signal transduction system
Infection thread development
Bacterial mutants unable to produce cyclic b-glucans cannot attach to the
root hairs
Bacteria have to produce a simbiotically active exopolysaccharide
Reorganization of cellular polarity causes the inversion of the tip growth
and the formation of the infection thread
Only bacteria at the tip of the infection thread are actively dividing
S. meliloti produces the exopolysaccharides succinoglycan (EPS I) and
galactoglucan (EPS II)
Succinoglycan is more efficient than galactoglucan in mediating infection
thread formation in Medicago sativa and the only one in M. truncatula
Infection thread failure can be caused by plant or bacterial defects
A- wild-type S. meliloti
B- S. meliloti exoY
mutant
C- M. truncatula lin
mutant
D- S. meliloti nodF nodL
mutant
E- M. trucatula partially
depleted of MtNFP
F- S. meliloti nodF nodL
mutant on M. truncatula
partially depleted for
MtLYK3
Reactive oxygen species (ROS) are generated in the
infection thread but their importance on the progression of
the symbiosis is unknown
Endocytosis of bacteria and bacteroid differentiation
Nitrogen fixation
 Overall stoichiometry of dinitrogen reduction:
N2 + 8H+ + 8e- + 16ATP
2NH4+ + 16ADP + 16Pi + H2
 N2 is reduced to NH4+ by the enzymatic complex
nitrogenase:
- dinitrogenase (a2b2 tetramer, 8Fe-7S,
MoFe7S9.homocitrate)
- dinitrogenase reductase (homodimer, 4Fe-4S)
 It has a slow activity with 3 molecules of N2 per second
Structure of the nitrogenase complex
Genes involved in nitrogenase biosynthesis
and nitrogen fixation regulation
Turnover cycle of the nitrogenase
 8 electrons: 6 to reduce N2
and 2 for H2
 The electron source is
ferredoxin or flavodoxin
 One electron is transferred in
each cycle of oxidation/reduction
 2 ATP are used per electron
transferred
 In the presence of oxygen, the
dinitrogenase reductase has a halflife of 30 s, and dinitrogenase has
10 min
Distribution of oxygen in symbiotic nitrogen-fixing nodules
Nif gene
expression
O2
N2 + 8e- + 10H+
nitrogenase
16 ATP
2NH4+ + H2
16 Pi + 16 ADP
 Nitrogenase is regulated by oxygen and ammonia
availability and also by the cell energetic status
Regulatory cascade
controlling nif genes
transcription
FixL/FixJ- two
component regulatory
system
NifA- is the master
regulator of nitrogen
fixation
Metabolic reactions within the symbiosome
Nodulation strategies in rhizobia
Rhizobia induce the formation of nodules on legumes using either a NF-dependent (a)
or a NF-independent (b) process.
(b) The need for one initial plant signal remains to be demonstrated. The bacteria enter
in the plant via cracks in the epidermis which result from the emergence of lateral
roots. Accumulation in these infection zones of cytokinin-like compounds synthesized
by the bacteria might directly bypass the early NF signaling pathway and trigger nodule
organogenesis.