Download Nitrogen and humanity 2

The Players
+
Soybean
=
Bradyrhizobium
japonicum
N2 fixation in nodules
Soybeans twice protein per acre as any other major vegetable or
grain crop, 5 to 10 times than land used for dairy, and 15 times land
for meat production.
Nitrogen cycle
•Nitrogen cycles between inert N2 and biologically
available NH3
•Nitrogen main limiter of biological production
•50-60% of biospheres nitrogen comes bacterial
N2 fixation in legume nodules
Legumes
•
•
•
•
Legume Family = Fabaceae (or Leguminosae)
3rd largest family of plants 19,400 species
Three subfamilies
Papilionoideae 1:Mimosoideae
2:Caesalpinioideae
3:Faboideae (Papilionoideae)
• Not all nodulate, rare in Caesalpinioideae
• Parasponia only non-legume host
Pisum
sativum
• 7 chromosomes diploid 5 Gbp (human 3.2 Gbp)
• Mendel examined 7 traits on 4 chromosomes
• Bateson championed Mendel’s work and coined Genetics
Plant models and
crops
• Both M. truncatula and L. japonicus have small
diploid genomes, easy to transform
• M. truncatula genome size 375 Mb
• Glycine max (soybean) now sequenced 1.1.Gb
polyploid genome
European civilisation and
legumes
• Umberto Eco considers the bean saved Western
civilisation (New York Times)
• From 1000-1500AD Europe's population tripled
because of bean cultivation (Animal manure not
sufficient)
Oil of the 19th century
• Chincha Islands Guano
• War of the Pacific 1879-1883
Greatest Scientific
development
in last 200 years?
“It is the chemist who must come to the rescue of the threatened
communities“
Sir William Crookes 1898 to BAAS
N2(g) + 3H2(g) _ 2NH3(g), ΔH = −92.4 kJmol-1
CH4 + H2O → CO + 3H2
•Haber discovered 1909 Nobel prize 1918
•Bosch scaled up and won Nobel in 1931
Nitrogen and population
• Addition of nitrogen
and phosphorous:
Haber Bosch process
1909
• Dwarf varieties of
wheat and rice (short
stem, large grain):
Green revolution
1960’s
Nitrogen
cascade
•Massive application of Haber-Bosch Nitrogen for agriculture (120 Mt year)
•Reactive Nitrogen in biosphere now 2X pre-industrial level
•Nitrate leaches to ground water
•Denitrifiers convert excess soil ammonia to N2O (nitrous oxide)
•N2O 300X more potent greenhouse gas than CO2
Rockstrom et al. Nature 2009: 473
Global grain
yields
Nutrient limitation
A=maize
B=wheat
C=rice
Mueller et al. Nature 490, 254 (2012)
We have a
problem
Nitrogen cycle
•Nitrogen cycles between inert N2 and biologically
available NH3
•50-60% of biospheres nitrogen comes bacterial
N2 fixation in legume nodules
•Nitrogen main limiter of biological production
Getting inside a nodule
Gage and Margolin Current Opinions in Microbiology
2000, 3: 613-617
Nodulation signalling pathway
(shared with mycorrihzal fungi)
Nod Factor
Receptors
LRR-kinase
Cation channel
Nucleoporins
Nod Gene
induction
Ca++
spiking
TFs
CCaMK
TFs
Myc Gene
induction
Myc Factor
The exchange that limits life
rose
Suc
CO2
Oxaloacetate
Malate
ATP
Dct?
DctA
Aap
Pyr AldA
Bra
Ala
AcetylCoA
cycle
?
Sst1
Mol
SO42
-
?
?
2
4
MoO
-
N2
e
uplicat
d
e
r
o
d
En
osome
d chrom
Fe2+
Acetyl-CoA
Fen1
α-ketoglutarate
Homocitrate
GOGAT
NH3
H
Mg
+
2
Asn
Glu
+
NH4
GS
+
Gln
AS
Bac
A
MgtE
?
Dmt1
?
H+
Hexoses
?
TCA
Aap
Bra
H+
ADP+
Pi
PH
Amino acid
B Nitogenasepool
GS/
e-& ATP
complex
OAA
Ile
Leu
Val
H+
H+
PEP
ER
golgi
nucleus
NCR
SPC
Symbiotic plant cell
Udvardi and Poole, Downie Annu Rev Plant Biology (2013)
ENGINEERING SYNTHETIC SYMBIOSES
• Engineer tailored nitrogen-fixing plant microbe
associations using established endophytic plant
microbes
• Use reconstruction to identify essential and ratelimiting factors
The challenges
1. Nitrogen fixation needs lots of energy
-Plant must provide carbon source for nitrogen fixers
1. Nitrogenase is inactivated by oxygen
- Engineer O2 protection for nitrogenase
1. Deliver lots of ammonium to the plant
- Induce bacteria to secret ammonia to plant
The Vision
Endophyte / Exophyte
LCOs
Plant
LCOs
Rhizopine
Rhizopine
ATP
N2
NH4+
O2
Aminoacids
NH4+
The Synthetic modules
Nitrogenase (Azotobacter)
σ1
Rhizopine
Sensor
(mocR)
σ2
Oxygen
Sensor
(fixLJ)
nifH
nifD
nifK …..
Respiratory protection
e.g., cytochrome c oxidases
σ3
Rhizopine Catabolism
Constitutive
σ4
Nod Genes (LCO production)
Model for studying
associative interactions
Setaria viridis: transformable, rapid life
cycle, sequenced genome, emerging
genetic platforms
Pseudomonas florescens: exophyte
Rhizobium sp. IRBG74: endophyte
Translation into maize
Improving nitrogen availability for
developed and developing world
farmers
Beans in Africa
Problem with beans
• Beans in Africa get nodulated by rhizobia that fix little nitrogen
• How can we identify the good nitrogen fixing bacteria?
Glowing bacteria
light up nodules
• nifH::luxCDABE fusion in pea/bean nodulating bacteria
• Bacteria glow when they fix N2, i.e. report how effective
3. Dry peas (Pisum spp.)
892
Garden pea
Pisum sativum var. sativum
Protein pea
Pisum sativum var. arvense
4. Chickpea
Cicer arietinum 478
5. Dry cowpea, blackeye pea, blackeye bean Vigna
unguiculata
ssp. dekindtiana 350
6. Pigeon pea, cajan pea, congo bean
Cajanus cajan
103
7. Lentil Lens culinaris
199
8. Bambara groundnut, earth pea Vigna subterranea
9. Vetch, common vetch Vicia sativa
99
10. Lupins
Lupinus spp.
45
11. Minor pulses:
Lablab, hyacinth bean
Lablab purpureus
Jack bean, sword bean
Canvalia ensiformis, gladiata
Winged bean
Psophocarpus teragonolobus
Velvet bean, cowitch
Mucuna pruiens var. utilis
Yam bean
Pachyrrizus erosus
Non-pulses (oil-crops):
SoybeanGlycine max
6209
Peanut Arachis hypogaea
1874