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

What is C4 Photosynthesis?

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

C4 grasses

Warm seasonC3 grasses

Relative Growth Rate, % day

‐1

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

West Australia Spinifex grassland

The Engineering of C4Photosynthesis into C3 crops

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

Neurachne (Poaceae)

Neurachne lanigeraC3

Neurachne minorC3-C4

Neurachne munroiC4

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. 

$340 millioneach

F‐22 Raptor

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