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Five Grand Challenges
A) Feed the increasing world population
B) Meet projected energy demands
C) Control greenhouse gas emissions
D) Preserve natural ecosystems and biodiversity
E) Maintain global security
In 2008, the stock to use ratio of rice was at its lowest point in 30 years.
For wheat, the stock to use ratio is at the lowest level in 50 years.
For all grains, the stock to use ratio is at its lowest level in 45 years (FAO 2008).
National Geographic, June 2009, Global Food Crisis.
Bangladesh: A woman sweeps a harvested rice field for left-over grain to feed her family.
The End of Plenty
Green Revolution SlowsGreen Revolution SlowsRice Yield in Asia
1.0
2.0
3.0
4.0
5.0
1955 1965 1975 1985 1995 2005
Year
Average rice yield (t ha‐1)
Sl ide courtesy of John Sheehy, International Rice Research Institute
Enhancing Food and Fuel Supplies by Improving Photosynthesis
• Higher photosynthetic capacity enhances yield.
• Higher photosynthesis per unit water enhances water use efficiency (WUE).
• Higher photosynthesis per unit absorbed light enhances radiation use efficiency (RUE).
• Higher Photosynthesis per unit nitrogen enhances nitrogen use efficiency (NUE).
Photosynthesis and the Five Grand Challenges
A) Feed the increasing world population
B) Meet projected energy demands
C) Control greenhouse gas emissions
D) Preserve natural ecosystems and biodiversity
E) Maintain global security
The Advantage ofC4 Photosynthesis
Biochemical advantage• Suppression of photorespiration• Near CO2 saturation of Rubisco
Physiological advantages above 25°C• Higher Radiation Use Efficiency (RUE) • Higher Water Use Efficiency (WUE)• Higher Nitrogen Use Efficiency (NUE)• Higher yield in warm climates
THE DUAL CATALYTIC NATURE OF RUBISCO
RUBISCO
CO2O2
CO2
Photorespiration C3 Photosynthesis
RuBP RuBP
PGA
2 PGAATP + NADPH
sugar
ATPNADPH
ATP + NADPH
PGA + PG
ATP
PCRcycle
PCOcycle
The Relative Rate of Photorespiration as a Function of CO2 and Temperature
Ehleringer, Sage, Pearcy and Flanagan (1991) Trends Ecol. Evol. 6:95
Current range
Value on hot soils
during the Pleistocene
C4 PhotosynthesisA CO2 Concentrating Mechanism
C3 Photosynthesis
CO2
atmosphere
mesophyll cell
CO2
carbohydrates
RUBISCOPCR cycle
C4 Photosynthesis
CO2
atmosphere
bundle-sheath cell
mesophyll cell
CO2
CO2 HCO3-CA PEPC
RUBISCOPCR cycle
C4cycle
PEP
carbohydrates
RUBP RUBP
pyruvate malate
malate
CO2=250 ppm CO2=2000 ppm
Slide courtesy of Martha Ludwig
Kranz AnatomyC3 leaf cross sectionBrachypodium
Setaria C4 leaf cross section
Mesophyll cells
Mesophyll cells
Bundle sheath cells
Bundle sheath cells
RICEy = 2.9xr2 = 0.98
MAIZEy = 4.4xr2 = 0.98
0
500
1000
1500
2000
2500
3000
3500
0 200 400 600 800
Accumulated intercepted PAR (MJ m-2 )
Above-ground dry weight (g m-2)
Radiation Use Efficiency (RUE)2006 Dry Season Experiment
Slide courtesy of John Sheehy, International Rice Research Institute
Phra
gmite
s
Perr
enia
l rye
gras
s
Tall
fesc
ue
Bam
boo
Phal
aris
Annu
al r
yegr
ass
Aru
ndo
Switc
hgra
ss
Mis
cant
hus
Gam
ba g
rass
Sorg
hum
Eria
nthu
s
Pani
cum
max
imum
Sacc
haru
m
Elep
hant
gra
ss
Pea
k dr
y m
atte
r yie
ld, T
Ha-1
0
20
40
60
80 C3 CropsC4 crops
Maximum Dry Matter Yields Reported for Biofuel Crops
from El Bassam (1997) Energy Plant Species
Water Use Efficiencies (WUE)(Sage 2001, Encylopedia of Ecology)
• C3 Plants: 1.5‐2.5 g dry matter Kg H2O
• C4 Plants:3‐5 g dry matter Kg H2O
West Australia Wheat crop, October 2010
C4 plants on a salt flatMojave Desert Region,
Southern Nevada
C4 Photosynthesis allows for production in otherwise hostile landscapes
Crop Photosynthetic PathwaysC3 Crops C4 Crops
• Wheat
• Rice
• Barley, Oats, Rye• Legumes (beans, peas)
• Chile Peppers• Sunflower
• Squashes• Melons
• Potatoes
• Sweet potato, yams
• Maize
• Sorghum
• Panicum millets• Amaranth
• Sugar cane
Echinochloa C4
The Productivity Advantage of C4 Photosynthesis
42 DAT
42 DAT
44 DAGMaize C4
Rice C3
DAG = Days after germinationDAT = Days after transplanting
Grain Yield = 13.9 t ha‐1
Grain Yield = 8.3 t ha‐1
© JESSlide courtesy of John Sheehy, International Rice Research Institute
Maize Record Yield (C4), 23 t ha‐1
Rice record yield, 13.7 t ha‐1
Normal maximum attainable yieldfor rice, about 10 t ha‐2 (1990’s)
Average yields for developednations, 6‐8 t ha‐2 (after 1950)
Yields in lesser developed nations1 to 5 t ha‐1 (before 1950)
Addition of pesticides, fertilizers, breeding
Improved varieties
Optimal climate, no pests, optimal nutrition
Breaking the Yield Barrier in C3 Plants
Introducing C4 Photosynthesis
Yield data from IRRI rice almanac (1997) and Evans (1993) Crop Evolution Adaptation and Yield
2020 Yield Needs
2050 Yield Needs
Candidates Crops for C4 Engineering
Rice Leading grain cropSuffers high photorespiration Used in high population areas
Wheat Leading grain cropGrown in dry regions
Soybean Leading Legume cropSuffers high photorespirationNitrogen fixing
Water Use Efficiencies (WUE)(Sage 2001, Encylopedia of Ecology)
• C3 Plants: 1.5‐2.5 g dry matter Kg H2O
• C4 Plants:3‐5 g dry matter Kg H2O
West Australia Wheat crop, October 2010
The relationship between rice production and population for Asian rice consumers (1961-2004)
Production (Mt)
4.56 B2050
100
200
300
400
500
600
700
800
900
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Population (Billion)
© JESSlide courtesy of John Sheehy, International Rice Research Institute
The IRRI C4 Rice Consortium
Thomas Brutnell
Gerry Edwards
James Burnell
Bob Furbank
Udo Gowik
Julian Hibberd
Jane LangdaleRichard Leegood
Erik Murchie
Timothy Nelson
Rowan Sage
Susanne von Caemmerer
Peter WesthoffRichard
Bruskiewich
Hei Leung
Paul Quick
Jacque Dionora
Anaida Ferrer
John Sheehy
Inez Slamet‐Loedin
Chris Myers
Xinguang ZhuGyn An
C4 Bioengineering GoalsKnowledge of genetic controls: poor, moderate, better
• Introduce Kranz anatomy
• Introduce the C4 metabolic cycle in a tissue specific manner
• Silence expression of Rubisco and other C3 enzymes in the mesophyll tissue
• Introduce regulatory elements to coordinate mesophyll and bundle sheath metabolism
• Introduce high capacity transport networks between the mesophyll and bundle sheath cells.
CommercialC4 Rice
Genediscovery
andmolecular toolbox
development
Characterize regulatory controls
Transform rice to
express Kranz anatomy and
the C4
metabolic enzymes
Optimize C4
function in trangenic
rice
Breed C4
from trangenics into local varieties
Phase1
Phase2
Phase4
2010 2015 2020 20252030
Phase3
Year
The Roadmap To C4 Rice
Molecular Toolbox DevelopmentC4 engineering requires the modification of dozens to
hundreds of genes in target C3 crops
• Gene stacking – sequentially introducing genes of choice into transferable unit of DNA
• Artificial chromosomes – a “C4” chromosome
• Transformation induced selection sweeps
Gene Discovery
Establish a known pool of genesthat confers C4 traits
Screennaturaldiversity
C3 to C4lineages
Ricerelatives
Ricevarieties
Screenmutagenized
lines
Sorghum EMS lines
Screenmodel
organisms
Forward and reverse genetics
with Arabidopsis, Setaria,
Brachypodium, Sorghum
Screentranscriptomes
C3 to C4lineages
Sorghum and maize mesophyll, bundle
sheath, and husks
Rice activationtagged lines
C3 AnatomyChange
BiochemChange
FineTuning+++ = C4
IRRI is screening thousands of sorghum EMS and gamma
irradiated mutants for ‘revertants’ in Kranz anatomy and C4
physiology
REVERSION
Slide courtesy of John Sheehy, International Rice Research Institute
C4(Maize)
C3(IR72)
Screens of Activation Tagged Lines May Identify Genes Controlling Bundle Sheath Size and Vein Density
Images courtesy of John Sheehy, International Rice Research Institute
Laser Dissection Allows for Tissue Specific Transcriptome Analysis
Transcriptomeassay Transcriptome
assay
Photos of Zea mays courtesy of John Sheehy, IRRI
When placed in rice, the promoters of some maize genes generate accumulation of GUS reporters in mesophyll cells only
Matsuoka et al. 1993&1994
PEPC
PPDK
One Option is to Exploit Existing C4 Promoters, Transcription Factors and Structural Genes
Slide courtesy of Julian Hibberd, Cambridge University
A Second Option is to Engineer Rice Genes to Resemble Genes from C4 Plants
PEP carboxylase
from separate C4 grass
lineages show similar shifts in 21 amino acids.
Christin et al. (2008) Current Biology 17, 1241-1247
Alanine to serine at site 780alters PEP affinity
Alanine to glutamateat site 579:
function unknown
PEP carboxylase
from separate C4 grass
lineages show similar shifts in 21 amino acids.
Christin et al. (2008) Current Biology 17, 1241-1247
Alanine to serine at site 780alters PEP affinity
Alanine to glutamateat site 579:
function unknown
Comparisons of the gene sequence for PEP carboxylase from 18 distinct C4 grass lineages showed which amino acids are altered in creating the C4 form of the enzyme.
Gene Discovery
Establish a known pool of genesthat confers C4 traits
Screennaturaldiversity
C3 to C4lineages
Ricerelatives
Ricevarieties
Screenmutagenized
lines
Sorghum EMS lines
Screenmodel
organisms
Forward and reverse genetics
with Arabidopsis, Setaria,
Brachypodium, Sorghum
Screentranscriptomes
C3 to C4lineages
Sorghum and maize mesophyll, bundle
sheath, and husks
Rice activationtagged lines
Objectives of the C3 to C4 Natural Lineage Studies
• Compare the Pattern of C4 Evolution‐ Identify the sequence of key trait changes
• Identify genetic control over C4 Evolution– Compare transcriptomes for shared alterations
Flowering Plant Families with C4 Species and the Estimated Number of Evolutionary Origins of C4 Photosynthesis as of 2007
Adapted from Muhaidat, Sage and Dengler, American Journal of Botany 94:362
Dicots 38Acanthaceae 1Aizoaceae 3Amaranthaceae 5Asteraceae 5Boraginaceae 2Capparidaceae 1Caryophyllaceae 1Chenopodiaceae 10Euphorbiaceae 1Molluginaceae 1Nyctaginaceae 2Polygonaceae 1Portulacaceae 2Scrophulariaceae 1Zygophyllaceae 2
Monocots 24
Poaceae 18Cyperaceae 5Hydrocharitaceae 1
Total origins ~60
Families in blue also contain C3‐C4 intermediate species
Gomphrenoids
Alternanthera
Amaranthus
Tidestromia
Aerva
The Occurrence of C4 Photosynthesis in theAmaranthaceae sensu strictoSage, Sage, Pearcy and Borsch (2007)
American Journal of Botany 94:1992‐2003.
Bold lines areC4 lineages
Other clades ofHeliotropiumare largely C3
Old world C4 clades
Genes analyzed
New world C4 clades
New world C3‐C4 clades
C3‐C4
New world C3 clades
C3
Heliotropium section Orthostachys phylogeny based on ITS (I), rbcL (R),
and matK, (M)sequences.
Photosynthetic pathway by isotopes and gas
exchangeFrohlich, Vogan, Chase, Sage
et al. in progress
C3
C4
C3‐C4
Possible C3‐C4
H. europaeum– C3 H. calcicola – C3H. procumbens – C3‐C4
100 µm
H. europaeum– C3
H. karwinskyi – C3 H. convolvulaceum C3‐C4H. tenellum – C3
H. gregii – C3‐C4 H. texanum C4 H. Polyphyllum ‐ C4
Evolutionary Progression of
Leaf Anatomy in HeliotropiumsectionOrthostachys
Vein
den
sity
(mm
.mm
- 2)
0
2
4
6
8
10
12
14
16
18
H. europaeumH. calcicola
H. tenellumH. procumbens
H. karwinskyi
H. convolvulaceumH. greggii
H. texanumH. polyphyllum
M:B
S ar
ea ra
tio
0
2
4
6
8
10
12
14
16
Vein DensityMesophyll:Bundle Sheath size
C3 C3‐C4 C4C3 C3‐C4 C4
Leaf Anatomical Properties in Heliotropium
Phylogenetic progression
NAD-
ME
activ
ity (µ
mol
mg
chl-1
h-1
)
0
200
400
600
800
1000
1200
PEPC
act
ivity
(µm
ol m
g ch
l-1 h
-1)
0
200
400
600
800
1000
1200
H. tenellumH. procumbens
H convolvulaceumH. greggiiH. texanum
H. polyphyllum
NADP
-ME
activ
ity (µ
mol
mg
chl-1
h-1
)
0
200
400
600
800
1000
1200(C) NADP‐ME
12.6 15.2 16.4 75.4
805.8
525.6
a b bc
e
d
11.817.3 28.1 32.7 21.0
135.8
a b c d be
(B) NAD‐ME
25.978.4 112.6
222.7
910.81015.8
a b cd
ee(A) PEPC
H. tenellumH. procumbens
H convolvulaceumH. greggiiH. texanum
H. polyphyllum
PEPC
K a
ctiv
ity (µ
mol
mg
chl-1
h-1
)
0
200
400
600
800
1000
1200(D) PEP‐CK
a b c d ea
15.8 19.8 27.0 58.7 93.914.5
C4 Enzyme Activities in Heliotropium
Phase #
P1
1A
1B
1C
2A
2B
2C
Anatomical preconditioning (e.g. close veins)
Enlargement of Bundle Sheath Cells
PHOTORESPIRATORY CO2 PUMP
Glycine decarboxylase to BSC
Enhancement of PEPCase activity
Establishment of a C4 cycle
C4 Photosynthesis
Optimization
Heliotropium greggii
H. convolvulaceum
H. karwinskyi
Heliotropium tenellum
H. calcicola
Organelle localization to inner BSC wall
H. procumbens
Heliotropium texanumH. polyphyllum
C3
C3‐C4
C4
Flaveria ramosissima
Flaveria palmeri
Flaveria trinerviaFlaveria bidentis
A Phylogenetically Robust Model of C4 Evolution
Genomics and C4 Evolution
Compare transcriptomes and genomes of C3 to C4 evolutionary lineages
C3 C3‐C4 C4Alternathera sessilis tenella caracasana
Atriplex prostrata ‐‐ rosea
Flaveria robusta ramossissima bidentis
Heliotropium calcicola convolvulaceum texanum
Mollugo pentaphylla nudicaulis cerviana
Neurachne lanigera minor munroi
The benefits of a CThe benefits of a C44 rice versus a Crice versus a C33 rice.rice.Increase in rice production (50%) = 300 million tonnesIncrease in rice production (50%) = 300 million tonnes
(modeled by John Sheehy, IRRI)(modeled by John Sheehy, IRRI)
Benefit($ in US $ per annum)
Increase in revenue (300 $/t)Increase in revenue (300 $/t)
Water saved by using CWater saved by using C44 rice (10$/Ml)rice (10$/Ml)
Nitrogen saving (10$/50kg urea)Nitrogen saving (10$/50kg urea)
Total benefitTotal benefit
90 Billion $90 Billion $
645 Million $645 Million $
13 Billion $13 Billion $
104 Billion $104 Billion $
© JES
If C4 rice could be engineered for $1 billion USD, the return on the investment would be over 1000 times every decade.
Acknowledgements
• NSERC – National Science and Engineering Research Council of Canada
• John Sheehy, IRRI• Julian Hibberd, University of Cambridge
• Tom Brutnell, Cornell University• Bob Furbank, CSIRO, Canberra Australia• Tammy Sage, University of Toronto
• Graduate Students: Patrick Vogan, Riyadh Muhaidat, Athena McKown
A Global Network for Photosynthetic EngineeringYellow fill indicates an active organization, red a proposed organization, and blue an idea only
Shanghai Centre for
C4 Engineering
China
Expertise in Computational
Biology
CSIRO/ANU CentreFor Photosynthetic
Improv ement
Rubisco, PhenomicsProtein engineeringPhotosynthetic theory
Australia
Japanese CentreFor Rice
Photosynthesis
Japan
TransportersGenetic engineering
IRRI C4 Rice Program
Breeding, screeningtranformation
CYMMTWheat 4P Program
Breeding, screening
ICARTA Dryland
PhotosynthesisProgram
DOE Center for
Photosynthetic Engineering
NESCENT Center for C4 Evolution
USA?CGIAR
NSERCCentre of Excellence in Leaf Dev elopment
Canada
United Nations/FAO Program for Advanced Training in Agricultural Biotechnology(Graduate scholarship and PDF program for students from developing nations)
European Centre for Photosynthesis
Expertise in molecular
engineering, C4 physiology,
Transport, Promoter analysis
metabolomics
Indian CentreFor Single-Celled
C4 plants
India
E.U.
USA
All enzymes of the C4 pathway have counterparts in C3 plants
C4 isoforms versus C3 isoforms
1) C4 isoforms typically expressed at higher levels in C4 species than C3 isoforms of both C3 and C4 plants
2) Isoforms have different tissue- and cell-specific expression patterns
A Schematic of C4 Photosynthesis
CO2 HCO3‐
PEP
CO2
PCRcycle
sugars
xylem
phloem
Export
OAA
C4 acid
DC
C3 acid
Pyruvate
ATP
AMPPPi
2 Pi
Cytosol
Mesophyll Tissue Bundle Sheath Tissue
PPDK
RUBISCO
RUBISCO
RUBISCO
Pco2~150 µbar Pco2~1500 µbarCHL
PEPC
Abbreviations: DC, decarboxylating enzyme; PEPC, PEP carboxylase; PPDK, pyruvate, phosphate dikinase