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At the first lecture it was clear that many of you
understood little of what I said.
Two problems are :
(i) English
(ii) New words, new concepts, that make you stumble ...
But other problems could be :
(i) If you are a first year student then you are not
sure yet of what is expected of you as a student.
(ii) You have prior expectations of what you will learn
in this General Education course, and these
expectations are different to what you are getting.
Such an attitude will prevent you from learning.
There is a solution !
1. Stop being a high school student and start being a
university student.
2. Think with flexibility.
3. Accept that you may not understand a concept now,
but that you may later.
4. If you come across a word or concept that you don’t
understand, then do not stumble at that word or
concept, but step over it and continue to listen. You
can come back to it later. If you get stuck at one
place then you cannot learn anything.
5. READ AS MUCH AS POSSIBLE. It is your duty to
start reading and to continue to read the rest of
your life.
6. We cannot teach you to learn, you must learn by
yourself.
7. The best students are those driven by CURIOSITY.
8. The best students do not expect an answer to all of
their questions; and yet expect many answers to one
question.
9. Expect to be educated very widely at ICU, but also
expect the opportunity to learn some things at great
depth. Above all, broaden your horizons by learning
about many things. A broad knowledge of many
things, and a specialised knowledge of some will help
you in later life. But the most important is learning
how to learn.
10. Expect to increase your vocabulary by 100%, that is
to double your vocabulary in your four years as a
student.
11. All the same, you can also expect to get a great deal
of enjoyment from the university environment, and
make many friends. However, never forget your
duty to learn and do well.
Now a thought test !
I want to see how much you are aware of your
surroundings, especially the biosphere, because
what I will introduce next relates to the
biosphere.
Gas (Symbol)
Percentage
Nitrogen (N)
78.03
Oxygen (O)
20.99
Argon (Ar)
.94
Carbon dioxide (CO2).
.035 -.04
Hydrogen (H)
.01
Neon (Ne)
.012
Helium (He)
.0005
Krypton (Kr)
.0001
Ozone (O3)
.00006
Xenon (Xe)
.000009
3. TODAY: Biogeochemical cycling. The four important
elements in the biosphere related to life. Why N is an
important macronutrient.
4. Next Wednesday: The phenomenon of the
Rhizobium/legume symbiosis
5. Next Friday: How plant cells work, the biology of the
root hair
6. The following Monday: Experiments with root hairs
7. The following Tuesday: Agrobacterium and science
Biogeochemical Cycling: movement of elements within or
between ecosystems caused by organisms, by geological,
hydrological, and atmospheric forces, and by chemical reactions
Elements within a cycle can move as:
Solids, Liquids, or Gases
Elements involved in cycling can also have different
chemical forms (e.g., Carbon as CO2 (carbon dioxide)
or CO (carbon monoxide)
The chemical form of an element can vary among physical
states or within a physical state
(e.g., Nitrogen as NH4+, NH3, N2)
There are two basic terms used in cycling:
Pools: the amount of an element within a physical
location or component of a cycle (sinks)
e.g.: tree, ocean, atmosphere, soil
Fluxes: the rate of movement of an element
between pools
e.g.: evaporation, burning, dissolution of a
rock, river carrying materials from land to the
ocean
Residence Time
The length of time that an atom or molecule of a
particular element spends in a particular location or
component of a cycle
Mean Residence Time
Recycle Time
Altering cycling rates can alter mean residence times and
have the potential to lead to either depletion or pollution
Basics of nutrient cycling
• Short-term: fixed stock
available for
organisms; question of
turnover time
• Long-term: exchange
between short-term
pools and
minerals/fossils
• Anthropogenic
perturbations of
nutrient cycling, local
and global
Local and global cycling
• Local vs. global feedbacks in nutrient dynamics
(recycling vs. one-way flow) depend on physics:
gaseous phases mix, solid/liquid phases stay
– one-way: energy, water
– partly recycled: carbon, nitrogen
– mostly recycled: phosphorus
• Humans have long affected local landscapes, but
only recently affected global biogeochemical
cycles
Dynamics of recycled nutrients
• cycling: available, temporarily
unavailable, incorporated
• amount of biomass in plants =
biomass:nutrient ratio (approx. 2:1
for carbon, varies more for other
nutrients)
• fast cycling can be good for
organisms (lots of available
nutrient), but can also lead to longterm nutrient losses
organisms
uptake
available
(mineralized)
death
decomposition/
mineralization
unavailable
(organic)
The water cycle
• water goes through biological systems on essentially a
one-way trip
• cycle is fairly quick (except for aquifers, deep ocean
circulation)
Let’s consider four biogeochemical cycles
of elements required by organisms for life
Carbon
Phosphorus
Sulphur
Nitrogen
The Carbon Cycle
Carbon Forms: CO2, CO, CH4, H2CO3, organic matter, CaCO3
Major pools of the carbon
cycle in billions of tons of
carbon.
The oceans contain the
largest pool of carbon.
Recycling rate of H2O, O2 and CO2 among the atmosphere,
hydrosphere, biosphere and lithosphere
Carbon cycle
• Central “nutrient”:
– closely bound to
energy
– bound to N
– makes up structure of
most organisms: 50%
of dry biomass
• Major carbon storage, or sinks:
– slow-decomposing compounds in soil
– bicarbonate in the ocean
– fossil fuels
– wood
Carbon (2)
• Gaseous phase: well-mixed. Atmospheric
concentration  350 ppm (pre-industrial 
250 ppm)
• Aqueous phase: dissolves in ocean water
(bicarbonate buffer).
• Solid phase: residence times of carbon in
soil, and in plants, from weeks to centuries
The Phosphorus Cycle
The phosphorus cycle is much slower than that of C or N
Phosphorus
• Extremely local recycling (no
gaseous phase)
• Long-term weathering/erosion
cycle
• Most important/limiting in
aquatic ecosystems, tropical
terrestrial habitats
The Sulphur Cycle
Dimethylsulfide (DMS)
is released by
phytoplankton, is then
oxidized to sulfur
dioxide and ultimately
sulfate in the
atmosphere.
Sulfate can cause
clouds to form by having
water droplets condense
on it.
The Nitrogen Cycle
• Nitrogen: used for
proteins,
RUBISCO
• only nitrates (and
some ammonium)
available to
plants: N
mineralization by
decomposers
• long-term
loss/gain of N:
deposition,
leaching,
volatilization
Nitrogen
Nitrogen (2)
• Nitrogen dynamics depend on plant
chemistry (C:N ratio, pH, decomposability),
carbon dynamics, microbial community
• Short-cuts for plants: N-fixing organisms,
mycorrhizae, direct uptake of organic N (?)
• Human nitrogen loading: fertilizer runoff
(overflows from previously almost-closed
cycles)
What’s so important about Nitrogen cycling?
essential nutrient (fertilizers, growing legumes as crops) –
changes in native species composition of ecosystem
atmospheric pollutant (burning fuels)
groundwater pollutant
Nitrogen Cycle
N2
NONSYMBIOTIC
N2 FIXATION
SYMBIOTIC
N2 FIXATION
DECOMPOSITION
DENITRIFICATION
NO3-
PLANT UPTAKE
NITRIFICATION
AMMONIFICATION
Organic N
IMMOBILIZATION
NH4+
ASSIMILATORY or
DISSIMILATORY
NO3- REDUCTION
Forms of Organic N
Amino
sugar N
Nucleic
acid N
Acid
Insoluble N
Protein &
peptide N
Hydrolyzable
Unknown N
Labile N
Major Inorganic N Compounds
Compound
Ammonium
Hydroxylamine
Dinitrogen
Nitrous oxide
Nitric oxide
Nitrite
Nitrate
Formula Oxidation
state
Form in soil
NH4+
-3
Fixed in clay lattice, dissolved,
as gaseous ammonia (NH3)
NH2OH
N2
N2O
NO
NO2NO3-
-1
0
+1
+2
+3
+5
Not detected
Gas
Gas, dissolved
Gas, dissolved
Dissolved
Dissolved
Nitrogen Fixation
The nodules on the roots
of this bean plant contain
bacteria called
Rhizobium that help
convert nitrogen in the
soil to a form the plant
can utilize.
Dinitrogen Fixation
The alder, whose fat shadow nourisheth–
Each plant set neere him long flourisheth.
–William Browne (1613), Brittania’s Pastorals, Book I, Song 2
Treatment
No N added
Non-inoculated
Inoculated with legume soil
Inoculated with sterile soil
112 mg NO3-–N per pot added
Non-inoculated
Inoculated with legume soil
Hellriegel and Wilfarth (1888)
Yield (g)
Oats
Peas
0.6
0.7
—
0.8
16.4
0.9
12.0
11.6
12.9
15.3
Types of Biological Nitrogen Fixation
Free-living (asymbiotic)
• Cyanobacteria
• Azotobacter
Associative
• Rhizosphere–Azospirillum
• Lichens–cyanobacteria
• Leaf nodules
Symbiotic
• Legume-rhizobia
• Actinorhizal-Frankia
Free-living N2 Fixation
Energy
• 20-120 g C used to fix 1 g N
Combined Nitrogen
• nif genes tightly regulated
• Inhibited at low NH4+ and NO3- (1 μg g-1 soil, 300 μM)
Oxygen
•
•
•
•
•
•
Avoidance (anaerobes)
Microaerophilly
Respiratory protection
Specialized cells (heterocysts, vesicles)
Spatial/temporal separation
Conformational protection
Associative N2 Fixation
•
•
•
•
•
•
Phyllosphere or rhizosphere (tropical grasses)
Azosprillum, Acetobacter
1 to 10% of rhizosphere population
Some establish within root
Same energy and oxygen limitations as free-living
Acetobacter diazotrophicus lives in internal tissue of sugar
cane, grows in 30% sucrose, can reach populations of 106
to 107 cells g-1 tissue, and fix 100 to 150 kg N ha-1 y-1
Estimated Average Rates of Biological N2 Fixation
Organism or system
Free-living microorganisms
Cyanobacteria
Azotobacter
Clostridium pasteurianum
N2 fixed (kg ha-1 y-1)
25
0.3
0.1-0.5
Grass-Bacteria associative symbioses
Azospirillum
5-25
Cyanobacterial associations
Gunnera
Azolla
Lichens
10-20
300
40-80
Leguminous plant symbioses with rhizobia
Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus)
Pasture legumes (Trifolium, Medicago, Lupinus)
50-100
100-600
Actinorhizal plant symbioses with Frankia
Alnus
Hippophaë
Ceanothus
Coriaria
Casuarina
40-300
1-150
1-50
50-150
50
• Nitrogen Fixation
•
•
•
•
•
Almost all N is in the atmosphere
90-190 Tg N fixed by terrestrial systems
40-200 Tg N fixed by aquatic systems
3-10 Tg N fixed by lightning
32-53 Tg N fixed by crops
Some biogeochemical cycling key points:
• cycling occurs at local to global scales
• biogeochemical cycles have 2 basic parts: pools and
fluxes
• elements are recycled among the biosphere, atmosphere,
lithosphere and hydrosphere
• cycles of each element differ (chemistry, rates, pools, fluxes,
interactions)
• cycling is important because it can affect many other
aspects of the environment and the quality of our lives
Nitrogen Metabolism
Plant Nutrition
• Plant metabolism is based on
sunlight and inorganic
elements present in water,
air, and soil.
• C, H, and O and energy are
used to generate organic
molecules via
photosynthesis.
• Other chemical elements,
such as mineral nutrients,
are also absorbed from soil.
Plant Nutrients
• Plants absorb many elements, some of which they
do not need.
• An element is considered an essential nutrient if it
meets three criteria:
• It is necessary for complete, normal plant
development through a full life cycle.
• It itself is necessary; no substitute can be
effective.
• It must be acting within the plant, not outside it.
• Many roles in plant metabolism.
Types of Essential Nutrients
• Nine essential nutrients, called macronutrients, are
needed in very large amounts
• Eight other essential nutrients, called
micronutrients, are needed only in small amounts.
Essential Nutrients to Most Plants
Macronutrient
Carbon (C )
% Dry
Weight Component/Function
Organic compounds
45.0
Oxygen (O)
Hydrogen (H)
Nitrogen (N)
Organic compounds
45.0
Organic compounds
6.0
1.0-4.0 Amino acids; nucleic acids, chlorophyll
Potassium (K)
1.0
Amino acids; regulates stomata
opening/closing
Calcium (Ca)
0.5
Enzyme cofactor; influences cell
permeability
Phosphorus (P)
0.2
ATP; proteins; nucleic acids;
phosphoplipids
Magnesium (Mg) 0.2
Sulfur (S)
0.1
Chlorophyll; enzyme activator
CoA; amino acids
Essential Nutrients to Most Plants
Micronutrient
Iron (Fe)
Chlorine (Cl)
Component/Function
Copper (Cu)
Manganese (Mn)
Zinc (Zn)
Plastocyanin; enzyme activator
Cytochromes; chlorophyll synthesis
Osmosis; water-splitting in photosynthesis
Enzyme activator; component of chlorophyll
Enzyme activator
Molybdenum (Mo) Nitrogen fixation
Boron (B)
Nickel (Ni)
Cofactor in chlorophyll synthesis
Cofactor for enzyme functioning in nitrogen
metabolism
Nitrogen: An Essential
Macronutrient
• N is not present in rock, but is abundant in the atmosphere as a
gas, N2.
• The process of converting N2 to chemically active forms of N is
nitrogen metabolism.
• Nitrogen metabolism consists of 3 stages:
• Nitrogen Fixation (N2 -> NO3-)
• Nitrogen Reduction (NO3- -> NO2- -> NH3 -> NH4+)
• Nitrogen Assimilation (transfer of NH2 groups)
• Runoff, leaching, denitrification, and harvested crops reduce soil
nitrogen.
Nitrogen Cycling Processes
Nitrogen Fixation – bacteria convert nitrogen gas (N2)
to ammonia (NH3).
Decomposition – dead nitrogen fixers release Ncontaining compounds.
Ammonification – bacteria and fungi decompose dead
plants and animals and release excess NH3 and
ammonium ions (NH4+).
Nitrification – type of chemosynthesis where NH3 or
NH4+ is converted to nitrite (NO2-); other bacteria
convert NO2- to nitrate (NO3-).
Denitrification – bacteria convert NO2- and NO3- to N2.
Means of Nitrogen Fixation
1) Human manufacturing of synthetic fertilizers
2) Lightning
3) Nitrogen-fixing bacteria and cyanobacteria
Nitrogen Fixing Bacteria and
Cyanobacteria
• Some are free-living in soil (E.g., Nostoc,
Azotobacter); others live symbiotically with plants
(E.g., Frankia, Rhizobium).
• These organisms have nitrogenase, an enzyme
that uses N2 as a substrate.
• N2 + 8e- + 8H+ + 16ATP -> 2NH3 + H2 + 16ADP + 16Pi
•
•
•
•
NH3 is immediately converted to NH4+.
Bacterial enzymes sensitive to O2.
Leghemoglobin binds to O2 and protects enzymes.
Symbiotic fixation rate depends on plant stage.
Natural Sources of Organic N
Source
%N
Dried blood
12
Peruvian guano
12
Dried fish meal
10
Peanut meal
7
Cottonseed meal
7
Sludge from sewer treatment plant
6
Poultry manure
5
Bone meal
4
Cattle manure
2
Symbiotic Nitrogen
Fixation
• Nitrogen-fixing bacteria fix N
(E.g., Rhizobium)
• Plants fix sugars (E.g.,legumes).
• Plants form swellings that house
N-fixing bacteria, called root
nodules.
• Mutualistic association.
• Excess NH3 is released into soil.
• Crop rotation maintains soil
fertility.
Development of a Root Nodule
• Bacteria enter the
root through an
infection thread.
• Bacteria are then
released into cell
and assume form
called bacteroids,
contained within
vesicles.
Symbiotic Nitrogen
Fixation
The Rhizobium-legume
association
Bacterial associations with certain
plant families, primarily legume
species, make the largest single
contribution to biological nitrogen
fixation in the biosphere
When this association is not present or functional, we
apply nitrogen-containing fertilizers to replace reduced
nitrogen removed from the soil during repeated cycles
of crop production.
This practice consumes fossil fuels, both in fertilizer
production and application.
Biological nitrogen fixation is the reduction of
atmospheric nitrogen gas (N2) to ammonium ions (NH4+)
by the oxygen-sensitive enzyme, nitrogenase. Reducing
power is provided by NAPH/ferredoxin, via an Fe/Mo
centre.
Plant genomes lack any genes encoding this enzyme,
which occurs only in prokaryotes (bacteria).
Even within the bacteria, only certain free-living
bacteria (Klebsiella, Azospirillum, Azotobacter),
blue-green bacteria (Anabaena) and a few symbiotic
Rhizobial species are known nitrogen-fixers.
Another nitrogen-fixing association exists between
an Actinomycete (Frankia spp.) and alder (Alnus spp.)
The enzyme nitrogenase catalyses the conversion of
atmospheric, gaseous dinitrogen (N2) and dihydrogen (H2)
to ammonia (NH3), as shown in the chemical equation below:
N2 + 3 H2  2 NH3
The above reaction seems simple enough and the
atmosphere is 78% N2, so why is this enzyme so important?
The incredibly strong (triple) bond in N2 makes this
reaction very difficult to carry out efficiently.
In fact, nitrogenase consumes ~16 moles of ATP for
every molecule of N2 it reduces to NH3, which makes it
one of the most energy-expensive processes known
in Nature.
S
Fe
Mo
homocitrate
Fe - S - Mo electron transfer cofactor
in nitrogenase
Biological NH3 creation (nitrogen fixation) accounts for
an estimated 170 x 109 kg of ammonia every year.
Human industrial production amounts to some 80 x 109 kg
of ammonia yearly.
The industrial process (Haber-Bosh process) uses an Fe
catalyst to dissociate molecules of N2 to atomic nitrogen
on the catalyst surface, followed by reaction with H2
to form ammonia. This reaction typically runs at ~450º C
and 500 atmospheres pressure.
These extreme reaction conditions consume a huge
amount of energy each year, considering the scale at which
NH3 is produced industrially.
The Dream…..
If
a way could be found to mimic nitrogenase catalysis
(a reaction conducted at 0.78 atmospheres N2 pressure
and ambient temperatures), huge amounts of energy
(and money) could be saved in industrial ammonia production.
If a way could be found to
transfer the capacity to form N-fixing symbioses
from a typical legume host
to an important non-host crop species such as corn or wheat,
far less fertilizer
would be needed to be produced and applied
in order to sustain crop yields
Because of its current and potential economic importance,
the interaction between Rhizobia and leguminous plants
has been intensively studied.
Our understanding of the process by which these two
symbionts establish a functional association is still not
complete, but it has provided a paradigm for many
aspects of cell-to-cell communication between microbes
and plants (e.g. during pathogen attack), and even
between cells within plants (e.g. developmental signals;
fertilization by pollen).
Symbiotic Rhizobia are classified in two groups:
Fast-growing Rhizobium spp. whose nodulation functions
(nif, fix) are encoded on their symbiotic
megaplasmids (pSym)
Slow-growing Bradyrhizobium spp. whose N-fixation and
nodulation functions are encoded on their chromosome.
There are also two types of nodule that can be formed:
determinate
and
indeterminate
This outcome is controlled by the plant host
Determinate nodules
Formed on tropical legumes by
Rhizobium and Bradyrhizobium
Meristematic activity not persistent - present only
during early stage of nodule formation;
after that, cells simply expand rather than divide, to
form globose nodules.
Nodules arise just below epidermis; largely internal
vascular system
Uninfected cells dispersed throughout nodule;
equipped to assimilate NH4+ as ureides
(allantoin and allantoic acid)
allantoin
allantoic acid
Indeterminate nodules
Formed on temperate legumes
(pea, clover, alfalfa);
typically by Rhizobium spp.
Cylindrical nodules with a persistent meristem;
nodule growth creates zones of different developmental
stages
Nodule arises near endodermis, and nodule vasculature
clearly connected with root vascular system
Uninfected cells of indeterminate nodules
assimilate NH4+ as amides (asparagine, glutamine)
Typical Associations
(cross-inoculation groups)
R.l. biovar viciae
colonizes pea (Pisum spp.) and vetch
(temperate; indeterminate nodules)
R.l. biovar trifolii
colonizes clover (Trifolium spp.)
(temperate; indeterminate nodules)
Rhizobium leguminosarum biovar phaseoli
colonizes bean (Phaseolus spp.)
(tropical; determinate nodules)
Rhizobium meliloti
colonizes alfalfa (Medicago sativa)
temperate; indeterminate nodules
Rhizobium fredii
colonizes soybean (Glycine max)
tropical; determinate nodules
Bradyrhizobium japonicum
colonizes soybean
tropical; determinate nodules
Rhizobium NGR 234
colonizes Parasponia and tropicals;
very broad host range
Nodule development process
1. Bacteria encounter root;
they are chemotactically attracted toward specific
plant chemicals (flavonoids) exuding from root tissue,
especially in response to nitrogen limitation
naringenin
(a flavanone)
daidzein
(an isoflavone)
2. Bacteria attracted to the root attach themselves to
the root hair surface and secrete specific oligosaccharide
signal molecules (nod factors).
nod factor
Examples of different
nod factors
3. In response to oligosaccharide signals, the root hair
becomes deformed and curls at the tip; bacteria become
enclosed in small pocket.
Cortical cell division is induced within the root.
4. Bacteria then invade the root hair cell and move along
an internal, plant-derived “infection thread”,
multiplying, and secreting polysaccharides that fill
the channel.
Rhizobium cells
expressing GFP
(green fluorescent
protein) invade a host
root hair
infection thread
5. Infection thread penetrates through several layers
of cortical cells and then ramifies within the cortex.
Cells in advance of the thread divide and organize
themselves into a nodule primordium.
6. The branched infection thread enters the nodule
primordium zone and penetrates individual primordium
cells.
7. Bacteria are released from the infection thread
into the cytoplasm of the host cells, but remain
surrounded by the peribacteroid membrane. Failure to
form the PBM results in the activation of host defenses
and/or the formation of ineffective nodules.
8. Infected root cells swell and cease dividing. Bacteria
within the swollen cells change form to become
endosymbiotic bacteroids, which begin to fix nitrogen.
The nodule provides an oxygen-controlled environment
(leghemoglobin = pink nodule interior) structured to
facilitate transport of reduced nitrogen metabolites
from the bacteroids to the plant vascular system, and of
photosynthate from the host plant to the bacteroids.
Sinorhizobium meliloti
Bacteroids
transporters
bacteroid
peribacteroid
membrane
Types of bacterial functions involved in
nodulation and nitrogen fixation
nod (nodulation) and nol (nod locus) genes
mutations in these genes block nodule formation or
alter host range
most have been identified by transposon mutagenesis,
DNA sequencing and protein analysis, in R. meliloti,
R. leguminosarum bv viciae and trifolii
fall into four classes:
nodD
nodA, B and C (common nod genes)
hsn (host-specific nod genes)
other nod genes
Gene clusters on R. meliloti pSym plasmid
(nol)
(nod)
(nif)
(fix)
F G H I N D1 A B C I J Q P G E F H D 3 E K D H A B C
NML REF D ABCIJT CBA HDK E N
Gene clusters on R. leguminosarum bv trifolii pSym plasmid
- - - D2 D1 Y A B C S U I J - - -
Gene cluster on Bradyrhizobium japonicum chromosome
Nod D (the sensor)
the nod D gene product recognizes molecules
(phenylpropanoid-derived flavonoids)
produced by plant roots and becomes
activated as a result
of that binding
activated nodD protein positively
controls the expression of the
other genes in the nod gene
“regulon” (signal transduction)
naringenin
(a flavanone)
different nodD alleles recognize various flavonoid
structures with different affinities, and respond with
differential patterns of nod gene activation
Common nod genes - nod ABC
mutations in nodA,B or C completely abolish the ability
of the bacteria to nodulate the host plant; they are found
as part of the nod gene “regulon” in all Rhizobia
( common)
products of these genes are required for bacterial
induction of root cell hair deformation and root
cortical cell division
The nod ABC gene products are enzymes responsible for
synthesis of diffusible nod factors, whcih are sulfated
and acylated beta-1,4-oligosaccharides of glucosamine
(other gene products, e.g. NodH, may also be needed
for special modifications)
SO3=
[ 1, 2 or 3 ]
[C16 or C18 fatty acid]
nod factors are active on host plants at very low
concentrations (10-8 to 10-11 M) but have no effect
on non-host species
Host-specific nod genes
mutations in these genes elicit abnormal root reactions
on their usual hosts, and sometimes elicit root hair
deformation reactions on plants that are not usually hosts
Example:
loss of nodH function in R. meliloti results in
synthesis of a nod factor that is no longer effective on
alfalfa but has gained activity on vetch
The nodH nod factor is now more hydrophobic than the
normal factor - no sulfate group on the oligosaccharide.
The role of the nodH gene product is therefore to
add a specific sulfate group, and thereby
change host specificity
Other nod genes
May be involved in the attachment of the bacteria to
the plant surface, or in export of signal molecules, or
proteins needed for a successful symbiotic relationship
exo (exopolysaccharide) genes
Encode proteins needed for exopolysaccharide synthesis
and secretion
In Rhizobium-legume interactions that lead to
indeterminate nodules, exo mutants cannot invade the
plant properly. However, they do provoke the typical
plant cell division pattern and root deformation, and
can even lead to nodule formation, although these are
often empty (no bacteroids).
In interactions that usually produce determinate nodules,
exo mutations tend to have no effect on the process.
Exopolysaccharides may provide substrate for signal
production, osmotic matrix needed during invasion,
and/or a recognition or masking function during invasion
example of
Rhizobial
exopolysaccharide
nif (nitrogen fixation) genes
Gene products are required for symbiotic nitrogen
fixation, and for nitrogen fixation in free-living N-fixing
species
Example: subunits of nitrogenase
fix (fixation) genes
Gene products required to successfully establish a
functional N-fixing nodule.
No fix homologues have been identified in free-living
N-fixing bacteria.
Example: regulatory proteins that monitor and control
oxygen levels within the bacteroids
FixL senses the oxygen level; at low oxygen tensions, it
acts as a kinase on FixJ, which regulates expression of
two more transcriptional regulators:
NifA, the upstream activator of nif and some fix genes;
FixK, the regulator of fixN (another oxgen sensor?)
This key transducing protein, FixL, is a novel hemoprotein
kinase with a complex structure. It has an N-terminal
membrane-anchoring domain, followed by the heme binding
section, and a C-terminal kinase catalytic domain.
Result?
Low oxygen tension activates nif gene transcription and
permits the oxygen-sensitive nitrogenase to function.
Metabolic genes and transporters
Dicarboxylic acid (malate) transport and metabolism
Genes for other functions yet to be identified….
 DNA microarray analysis of gene expression
patterns
 Proteomic analysis of bacteroids and
peribacteroid membrane preparations
Host plant role in nodulation
1. Production and release of nod gene inducers
- flavonoids
2. Activation of plant genes specifically required for
successful nodule formation - nodulins
3. Suppression of genes normally involved in repelling
microbial invaders - host defense genes
Nodulins
Bacteroid
development
Nitrogen
fixation
Nodule
senescence
Root hair
invasion
Bacterial
attachment
late
nodulins
early nodulins
Pre-infection
Infection
and nodule
formation
Nodulins?
Nodule
function and
maintenance
Nodule
senescence
Early nodulins
At least 20 nodule-specific or nodule-enhanced genes
are expressed in plant roots during nodule formation;
most of these appear after the initiation of the visible
nodule.
Five different nodulins are expressed only in cells
containing growing infection threads.
These may encode proteins that are part of the
plasmalemma surrounding the infection thread, or
enzymes needed to make or modify other molecules
Twelve nodulins are expressed in root hairs and in
cortical cells that contain growing infection threads.
They are also expressed in host cells a few layers ahead
of the growing infection thread.
Late nodulins
The best studied and most abundant late nodulin is
the protein component of leghemoglobin.
The heme component of leghemoglobin appears to be
synthesized by the bacteroids.
Treatment of Lotus japonicus roots with nod factor
from Mesorhizobium loti (NF), or infection with wt M. loti,
(+) or an ineffective nodC strain of M. loti (-)
Other late nodulins are enzymes or subunits of enzymes
that function in nitrogen metabolism (glutamine
synthetase; uricase) or carbon metabolism (sucrose
synthase). Others are associated with the peribacteroid
membrane, and probably are involved in transport
functions.
These late nodulin gene products are usually
not unique to nodule function, but are found in other
parts of the plant as well. This is consistent with the
hypothesis that nodule formation evolved as a
specialized form of root differentiation.
There must be many other host gene functions
that are needed for successful nodule formation.
Example: what is the receptor for the nod factor?
These are being sought through genomic and proteomic
analyses, and through generation of plant mutants
that fail to nodulate properly
The full genome sequencing of Medicago truncatula
and Lotus japonicus , both currently underway, will
greatly speed up this discovery process.
A plant receptor-like kinase required for
both bacterial and fungal symbiosis
S. Stracke et al
Nature 417:959 (2002)
Screened mutagenized populations of the legume
Lotus japonicus for mutants that showed an inability
to be colonized by VAM
Mutants found to also be affected in their ability to
be colonized by nitrogen-fixing bacteria
(“symbiotic mutants”)
WT
mutant
Mutant
LRRs
PM
cytosol
Protein kinase
catalytic domain
Inducers of nodulation in Rhizobium leguminosarum bv viciae
luteolin
eriodictyol
Inhibitor of nodulation
genistein
Figure 19.70
Genetics of Nitrogenase
Gene
nifH
nifDK
nifA
nifB
nifEN
nifS
fixABCX
fixK
fixLJ
fixNOQP
fixGHIS
Properties and function
Dinitrogenase reductase
Dinitrogenase
Regulatory, activator of most nif and fix genes
FeMo cofactor biosynthesis
FeMo cofactor biosynthesis
Unknown
Electron transfer
Regulatory
Regulatory, two-component sensor/effector
Electron transfer
Transmembrane complex
Nitrogenase
FeMo Cofactor
Fd(ox)
N2 + 8H+
Fd(red)
8e-
2NH3 + H2
nMgATP
nMgADP + nPi
Dinitrogenase
reductase
4C2H2 + 8H+
4C2H2
Dinitrogenase
N2 + 8H+ + 8e- + 16 MgATP  2NH3 + H2 + 16MgADP
Taxonomy of Rhizobia
Genus
Species
Host plant
Rhizobium
leguminosarum bv. trifolii
“
bv. viciae
“
bv. phaseoli
tropici
etli
Trifolium (clovers)
Pisum (peas), Vicia (field
beans), Lens (lentils),
Lathyrus
Phaseolus (bean)
Phaseolus (bean), Leucaena
Phaseolus (bean)
Sinorhizobium
meliloti
fredii
saheli
teranga
Melilotus (sweetclover),
Medicago (alfalfa), Trigonella
Glycine (soybean)
Sesbania
Sesbania, Acacia
Bradyrhizobium
japonicum
elkanii
liaoningense
Glycine (soybean)
Glycine (soybean)
Glycine (soybean)
Azorhizobium
caulinodans
Sesbania (stem nodule)
‘Meso rhizobium’
loti
huakuii
ciceri
tianshanense
mediterraneum
Lotus (trefoil)
Astragalus (milkvetch)
Cicer (chickpea)
[Rhizobium]
galegae
Galega (goat’s rue), Leucaena
Photorhizobium
spp.
Aeschynomene (stem nodule)
Cicer (chickpea)
Nitrogen Fixation
• Energy intensive process :
• N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 +
16ADP + 16 Pi
• Performed only by selected bacteria and
actinomycetes
• Performed in nitrogen fixing crops
(ex: soybeans)
Nodulation in Legumes
Role of Root Exudates
General
• Amino sugars, sugars
Specific
• Flavones (luteolin), isoflavones
(genistein), flavanones, chalcones
• Inducers/repressors of nod genes
• Vary by plant species
• Responsiveness varies by rhizobia
species
nod Gene Expression
Common
nod genes
Nod factor–LCO
(lipo-chitin oligosaccharide)
Infection Process
• Attachment
• Root hair curling
• Localized cell wall
degradation
• Infection thread
• Cortical cell
differentiation
• Rhizobia released into
cytoplasm
• Bacterioid differentiation
(symbiosome formation)
• Induction of nodulins
Nodule Metabolism
Oxygen metabolism
• Variable diffusion barrier
• Leghemoglobin
Nitrogen metabolism
• NH3 diffuses to cytosol
• Assimilation by GOGAT
• Conversion to organic-N for
transport
Carbon metabolism
• Sucrose converted to
dicarboxylic acids
• Functioning TCA in
bacteroids
• C stored in nodules as starch
Nitrogen Cycle
Sources
•
•
•
•
•
•
Lightning
Inorganic fertilizers
Nitrogen Fixation
Animal Residues
Crop residues
Organic fertilizers
Forms of Nitrogen
•
•
•
•
•
•
•
Urea  CO(NH2)2
Ammonia  NH3 (gaseous)
Ammonium  NH4
Nitrate  NO3
Nitrite  NO2
Atmospheric Dinitrogen N2
Organic N
Global Nitrogen Reservoirs
Nitrogen
Reservoir
Atmosphere
Metric tons
nitrogen
3.9*1015
Actively cycled
Ocean 
soluble salts
Biomass
6.9*1011
5.2*108
Yes
Yes
Land  organic
matter
 Biota
1.1*1011
2.5*1010
Slow
Yes
No
Roles of Nitrogen
• Plants and bacteria use nitrogen in the
form of NH4+ or NO3• It serves as an electron acceptor in
anaerobic environment
• Nitrogen is often the most limiting
nutrient in soil and water.
Nitrogen is a key element for
• amino acids
• nucleic acids (purine, pyrimidine)
• cell wall components of bacteria (NAM).
Nitrogen Cycles
•
•
•
•
•
Ammonification/mineralization
Immobilization
Nitrogen Fixation
Nitrification
Denitrification
Mineralization or Ammonification
• Decomposers: earthworms, termites, slugs,
snails, bacteria, and fungi
• Uses extracellular enzymes  initiate
degradation of plant polymers
• Microorganisms uses:
• Proteases, lysozymes, nucleases to degrade
nitrogen containing molecules
• Plants die or bacterial cells lyse  release of organic
nitrogen
• Organic nitrogen is converted to inorganic nitrogen
(NH3)
• When pH<7.5, converted rapidly to NH4
• Example:
Urea
NH3 + 2 CO2
Immobilization
•
•
•
•
•
•
The opposite of mineralization
Happens when nitrogen is limiting in the environment
Nitrogen limitation is governed by C/N ratio
C/N typical for soil microbial biomass is 20
C/N < 20 Mineralization
C/N > 20 Immobilization
Microorganisms fixing
•
•
•
•
•
Azobacter
Beijerinckia
Azospirillum
Clostridium
Cyanobacteria
• Require the enzyme
nitrogenase
• Inhibited by oxygen
• Inhibited by
ammonia (end
product)
Rates of Nitrogen Fixation
N2 fixing system
Rhizobium-legume
Nitrogen Fixation (kg
N/hect/year)
200-300
Cyanobacteria- moss
30-40
Rhizosphere
associations
Free- living
2-25
1-2
Bacterial Fixation
• Occurs mostly in salt marshes
• Is absent from low pH peat of northern
bogs
• Cyanobacteria found in waterlogged
soils
Denitrification
• Removes a limiting nutrient from the
environment
• 4NO3 + C6H12O6 2N2 + 6 H20
• Inhibited by O2
• Not inhibited by ammonia
• Microbial reaction
• Nitrate is the terminal electron acceptor