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 End of Plenty 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).

Bangladesh: A woman sweeps a harvested rice National Geographic, June 2009, Global field for left-over grain to feed her family. Food Crisis. Green Revolution Slows Rice Yield in Asia

Average rice yield (t ha‐1) 5.0

4.0

3.0

2.0

1.0 1955 1965 1975 1985 1995 2005 Year Slide 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 of C4 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

Photorespiration C3 Photosynthesis

O2 CO2 RuBP RuBP ATP ATP NADPH

PGA PCO PCR cycle RUBISCO cycle

PGA sugar

CO2 + PG 2 PGA ATP + NADPH ATP + NADPH The Relative Rate of Photorespiration as a

Function of CO2 and Temperature

Value on hot soils during the Pleistocene

Current range

Ehleringer, Sage, Pearcy and Flanagan (1991) Trends Ecol. Evol. 6:95 C4 Photosynthesis A CO2 Concentrating Mechanism

C3 Photosynthesis C4 Photosynthesis CO =250 ppm CO =2000 ppm 2 carbohydrates 2 carbohydrates RUBP RUBP CO PCR cycle CO2 PCR cycle 2 RUBISCO RUBISCO malate

mesophyll cell bundle-sheath cell pyruvate C4 malate cycle atmosphere PEP PEPC CO2 CA - CO2 HCO3

mesophyll cell

atmosphere

CO Slide courtesy of Martha Ludwig 2 Kranz Anatomy

Brachypodium C3 leaf cross section

Mesophyll cells

Bundle sheath cells

Setaria C4 leaf cross section

Bundle sheath cells

Mesophyll cells C4 grasses 1 ‐ day

Rate, Warm season

C3 grasses Growth

Relative Radiation Use Efficiency (RUE) 2006 Dry Season Experiment

Above-ground dry weight (g m-2) 3500 MAIZE y = 4.4x 3000 r2 = 0.98

2500 RICE y = 2.9x r2 = 0.98 2000

1500

1000

500

0 0 200 400 600 800 Accumulated intercepted PAR (MJ m -2 )

Slide courtesy of John Sheehy, International Rice Research Institute Maximum

Peak dry matter yield, T Ha-1 from 20 40 60 80 0

Dry

Phragmites Bassam

Perrenial ryegrass Matter C C Tall fescue 4 3 crops Crops Bamboo

(1997) Phalaris Crops

Yields Annual ryegrass

Arundo Energy Switchgrass

Miscanthus Reported

Gamba grass Plant Sorghum

Erianthus Species Panicum maximum

Saccharum for Elephant grass

Biofuel

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 Photosynthesis allows for production in otherwise hostile landscapes

C4 plants on a salt flat Mojave Desert Region, Southern Nevada West Australia Spinifex grassland The Engineering of C4 Photosynthesis into C3 crops Crop Photosynthetic Pathways

C3 Crops C4 Crops • Wheat • Maize • Rice • Sorghum • Barley, Oats, Rye • Panicum millets • Legumes (beans, peas) • Amaranth • Chile Peppers • Sugar cane • Sunflower • Squashes • Melons • Potatoes • Sweet potato, yams The Productivity Advantage of C4 Photosynthesis

‐1 Maize C4 Grain Yield = 13.9 t ha 44 DAG

Echinochloa C4 42 DAT

‐1 Rice C3 Grain Yield = 8.3 t ha 42 DAT

DAG = Days after germination Slide courtesy of John Sheehy, International Rice Research Institute DAT = Days after transplanting © JES Breaking the Yield ‐1 Maize Record Yield (C4), 23 t ha Barrier in C3 Plants

Introducing C4 Photosynthesis 2050 Yield Needs

Rice record yield, 13.7 t ha‐1 Optimal climate, no pests, optimal nutrition 2020 Yield Needs Normal maximum attainable yield for rice, about 10 t ha‐2 (1990’s) Improved varieties Average yields for developed nations, 6‐8 t ha‐2 (after 1950) Addition of pesticides, fertilizers, breeding

Yields in lesser developed nations 1 to 5 t ha‐1 (before 1950)

Yield data from IRRI rice almanac (1997) and Evans (1993) Crop Evolution Adaptation and Yield Candidates Crops for C4 Engineering

Rice Leading grain crop Suffers high photorespiration Used in high population areas

Wheat Leading grain crop Grown in dry regions

Soybean Leading Legume crop Suffers high photorespiration Nitrogen 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) 900

800 4.56 B 700 2050 600

500

400

300

200

100 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Population (Billion)

Slide courtesy of John Sheehy, International Rice Research Institute © JES The IRRI C4 Rice Consortium

Rowan Sage Richard Leegood Jane Langdale Udo Gowik Gyn An Xinguang Zhu John Sheehy

Thomas Brutnell Erik Murchie Paul Quick

Chris Myers Richard Anaida Ferrer Peter Westhoff Bruskiewich

Inez Gerry Edwards Timothy Nelson Jacque Dionora Julian Hibberd Slamet‐Loedin Hei Leung

Bob Furbank

Susanne von Caemmerer James Burnell C4 Bioengineering Goals Knowledge 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. The Roadmap To C4 Rice

Phase Phase Phase Phase 1 2 3 4

Gene Transform discovery rice to and Optimize C4 express Kranz function in Breed C4 molecular from trangenics Commercial anatomy and trangenic toolbox Rice the C into local C4 development 4 rice metabolic varieties enzymes Characterize regulatory controls

Year 2030 2010 2015 2020 2025 Molecular Toolbox Development C4 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

Screen Screen Screen Screen natural mutagenized transcriptomes model diversity lines organisms

Rice activation C3 to C4 Rice tagged lines lineages varieties Sorghum Rice Sorghum EMS and maize Forward and relatives lines mesophyll, reverse genetics bundle with sheath, and Arabidopsis, husks C3 to C4 Setaria, lineages Brachypodium, Sorghum

Establish a known pool of genes that confers C4 traits IRRI is screening thousands of sorghum EMS and gamma irradiated mutants for ‘revertants’

in Kranz anatomy and C4 physiology

Anatomy Biochem Fine C3 +=Change + Change + Tuning C4

REVERSION

Slide courtesy of John Sheehy, International Rice Research Institute Screens of Activation Tagged Lines May Identify Genes Controlling Bundle Sheath Size and Vein Density

C3 (IR72)

C4 (Maize)

Images courtesy of John Sheehy, International Rice Research Institute Laser Dissection Allows for Tissue Specific Transcriptome Analysis

Transcriptome Transcriptome assay assay

Photos of Zea mays courtesy of John Sheehy, IRRI One Option is to Exploit Existing C4 Promoters, Transcription Factors and Structural Genes

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

Slide courtesy of Julian Hibberd, Cambridge University A Second Option is to Engineer Rice Genes

to Resemble Genes from C4 Plants

Alanine to glutamate Alanine to serine at site 780 at site 579: alters PEP affinity 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 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.

Alanine to glutamate Alanine to serine at site 780 at site 579: alters PEP affinity 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 Gene Discovery

Screen Screen Screen Screen natural mutagenized transcriptomes model diversity lines organisms

Establish a known pool of genes that confers C4 traits 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

Monocots 24 Dicots 38 Acanthaceae 1 Poaceae 18 Aizoaceae 3 Cyperaceae 5 Amaranthaceae 5 Hydrocharitaceae 1 Asteraceae 5 Boraginaceae 2 Capparidaceae 1 Caryophyllaceae 1 Chenopodiaceae 10 Total origins ~60 Euphorbiaceae 1 Molluginaceae 1 Nyctaginaceae 2 Families in blue also contain C ‐ 3 Polygonaceae 1 C intermediate species 4 Portulacaceae 2 Scrophulariaceae 1 Zygophyllaceae 2 Tidestromia Bold lines are C4 lineages Alternanthera Gomphrenoids Aerva Amaranthus

The Occurrence of C4 Photosynthesis in the Amaranthaceae sensu stricto Sage, Sage, Pearcy and Borsch (2007) American Journal of Botany 94:1992‐2003. Genes analyzed Heliotropium section Orthostachys phylogeny based on ITS (I), rbcL (R), and matK, (M) sequences. Photosynthetic pathway Old world by isotopes and gas exchange C4 clades Frohlich, Vogan, Chase, Sage et al. in progress

C3‐C4 New world C4 C3‐C4 clades

New world C4 clades

Possible C3‐C4 C3 Other clades of C3‐C4 Heliotropium New world are largely C 3 C3 clades H.H. europaeum europaeum–C–C33 H. calcicola –C3 H. procumbens –C3‐C4 Evolutionary Progression of Leaf Anatomy in Heliotropium section Orthostachys

100 µm

H. tenellum –C3 H. karwinskyi –C3 H. convolvulaceum C3‐C4

H. gregii –C3‐C4 H. texanum C4 H. Polyphyllum ‐ C4 H M:BS area ratio .

e 10 12 14 16 u 0 2 4 6 8 r o

a H e . u c m a Mesophyll:Bundle Phylogenetic l c

H i c . o H t l e . a n p e r l o l u c m u C3

H m H . . b Leaf k c e a o n r n w s

v i o n

l s progression v k

u y

l i

a Anatomical

H u

. m Sheath

g C3

r H e

g ‐ . g C4 H t e i . i x p a

o n

l u

y m

p size

y C4 l l u

Properties

Vein density (mm.mm-2) 10 12 14 16 18 0 2 4 6 8

in C3

Heliotropium Vein

Density C3 ‐ C4 C4 -1 -1 NADP-ME activity (µmol mg chl h ) PEPC activity (µmol mg chl-1 h-1) 1000 1200 H 1000 1200 200 400 600 800 . 200 400 600 800

e 0 0

n H e

. l

l p 25.9 (A) (C) u 12.6

r m

o a

H a

m PEPC NADP o

b n 78.4

e 15.2 v

o n C b l s b v

u 4 l a

c ‐ 112.6 e Enzyme ME 16.4

m c b H

e 222.7 g 75.4

g H

i d c . i

H a

n . 910.8

805.8 p u Activities

o m e e l y

y 1015.8

l l

u 525.6

m e d

-1 -1 -1 -1 PEPCK activity (µmol mg chl h ) NAD-ME activity (µmol mg chl h ) in

H 1000 1200 1000 1200 200 400 600 800 . 200 400 600 800

t e 0 0

n H e Heliotropium

. l

p l (D) u 15.8 (B) 11.8

r m o ab H a c

c m NAD o PEP

n b

v e 19.8 17.3

o n

l b v

u ‐ ‐ l CK a ME

e 27.0 u 28.1

m c c H

r e

g 58.7 32.7 g H d i d . i

t e

H a

. n

93.9

p u 21.0

o m b e l y

l 135.8

l 14.5 u

m a e A Phylogenetically Robust Model of C4 Evolution

Heliotropium texanum H. polyphyllum C4 Photosynthesis C4 Flaveria trinervia Flaveria bidentis

2C Optimization

Establishment of Flaveria palmeri

2B a C4 cycle

Enhancement of Flaveria ramosissima 2A Phase # PEPCase activity Heliotropium greggii

PHOTORESPIRATORY CO2 PUMP C ‐C 1C H. convolvulaceum 3 4 Glycine decarboxylase to BSC H. procumbens

1B Organelle localization to inner BSC wall H. karwinskyi Heliotropium C3 1A Enlargement of Bundle Sheath Cells tenellum

P1 Anatomical preconditioning (e.g. close veins) H. calcicola Genomics and C4 Evolution

Compare transcriptomes and genomes of C3 to C4 evolutionary lineages

C3 C3‐C4 C4 Alternathera 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 lanigera Neurachne minor Neurachne munroi C3 C3-C4 C4 The benefits of a C4 rice versus a C3 rice. Increase in rice production (50%) = 300 million tonnes (modeled by John Sheehy, IRRI) Benefit ($ in US $ per annum)

Increase in revenue (300 $/t) 90 Billion $

Water saved by using C4 rice (10$/Ml) 645 Million $

Nitrogen saving (10$/50kg urea) 13 Billion $

Total benefit 104 Billion $

If C4 rice could be engineered for $1 billion USD, the return on the investment would be over 1000 times every decade.

$340 million each 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 Engineering Yellow fill indicates an active organization, red a proposed organization, and blue an idea only CGIAR China USA? E.U.

Shanghai IRRI C4 DOE European Centre Centre for Rice Program Center for for Photosynthesis C4 Engineering Photosynthetic Engineering Expertise in Expertise in molecular Breeding, screening Computational engineering, tranformation Biology USA C4 physiology, Transport, Australia Promoter analysis NESCENT Center metabolomics CSIRO/ANU Centre CYMMT for C4 Evolution For Photosynthetic Wheat 4P Improv ement Program

Rubisco, Phenomics India Protein engineering Canada Photosynthetic theory Breeding, screening NSERC Indian Centre Centre of Excellence For Single-Celled Japan in Leaf Development C4 plants ICARTA Japanese Centre Dryland For Rice Photosynthesis Photosynthesis Program

Transporters Genetic engineering United Nations/FAO Program for Advanced Training in Agricultural Biotechnology (Graduate scholarship and PDF program for students from developing nations) 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

Mesophyll Tissue Bundle Sheath Tissue

Pco2~150 µbar Pco2~1500 µbar CHL C3 acid RUBISCO

Pyruvate CO2

ATP

PCR xylem 2 Pi PPDK DC cycle RUBISCO phloem PPi AMP

Export C4 acid PEP

sugars OAA ‐ PEPC RUBISCO CO HCO3 2 Cytosol

Abbreviations: DC, decarboxylating enzyme; PEPC, PEP carboxylase; PPDK, pyruvate, phosphate dikinase