008 engineering c4 rice - paul quick

Engineering C4 rice

C3

C4

XI Conferencia Internacional de Arroz para America Latina y el Caribe21-24 Septiembre 2010

Cali, Colombia

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Green Revolution SlowsWorld Rice Yield (1961-2010)World Rice Yield (1961 2010)

Data Source: FAO

Average yield (t ha-1) Average yearly increase overprevious 10 years (kg ha-1)

5 0 200

4.0

5.0

160

200

3.0 120

1 0

2.0

40

80

0.0

1.0

1955 1965 1975 1985 1995 2005 2015

0

40

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1955 1965 1975 1985 1995 2005 2015

Year

The relationship between rice production and l ti f A i i (1961 2004)population for Asian rice consumers (1961-2004)

Data Source: UN and FAO

Production (Mt)

4.56 B700

800

900

2050

500

600

700

300

400

100

200

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Population (Billion)

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A Second Green Revolution?A Second Green Revolution?

1

Final dry weights of crops in C3 and C4 groups correlated with the length of growing season (Monteith, 1978)

Standing dry weight at harvest (t/ha)

kaleC344 DAGMaize C4 Grain Yield = 13.9 t ha-1 kalesugar beetpotatoesricecassavaoil palm

bulrush milletmaize

C3

C4

40

60

80

C4

maizesorghumsugarcanenapier grass

IRRI Expt-riceIRRI Expt-maize

0

20

40

C3

42 DATRice C3 Grain Yield = 8.3 t ha-1

0 100 200 300

Length of growing season (days)

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C4 i ld

C4 RICEC4 rice could:

•increase rice yield by 50%

•double water-use efficiency

•improve nitrogen use efficiency •improve nitrogen-use efficiency

C4 photosynthesis is one of the few evolutionary mechanisms that could deliver th i bi ti f b fitthese superior combination of benefits.

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C4 would confer benefits on all of the global rice ecosystemsS u c : Ric Alm n c (M cl n t l 2002)Source: Rice Almanac (Maclean et al, 2002)

IRRIGATED RAINFED

Area = 79 M haProduction = 75%


UPLAND DEEP WATER



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C4 Supercharges Photosynthesis Using A TwoCompartment CO2 Concentrating Mechanism

C3 Photosynthesis

Compartment CO2 Concentrating Mechanism

C4 Photosynthesis

3 Phosphoglyceratec c c

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C4 photosynthesis involves alterations to biochemistry, cell biology and leaf anatomy

CO2

PEP

OAA

Mesophyll Cell

2HCO3

-1

= C4PEP

MalatePyruvate

3

5

RuBisCOCO2

4

Bundle Sheath Cell

Many of the genes that control these processes are unknown

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Evolutionary ChangeEvolutionary Change

Genetic alterations

C3 AnatomyChange

BiochemChange

FineTuning+++ = C4

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Despite its complexity C has Despite its complexity, C4 has evolved independently ~60 times

It can’t be that difficult?!

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The Timeline for C4 Rice

3 years

It will likely take a minimum of 15 years of coordinated research carried out in the laboratories of the C4 Rice Consortium to deliver C4 rice to plant breeders in the developing world.

Genediscovery

andTransform

rice to

3 years

3 years5 years

4 yearsand

molecular toolbox

development

rice to express Kranz

anatomy and the C4

Optimize C4 function

in transgenic

rice

Breed C4transgenic

s into local

Characterize regulatory controls

metabolic enzymes

rice varieties

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To determine the feasibility of replicating the two-cellC4 photosynthetic pathway in rice:4 p y p y

Challenge 1

LeafAnatomyAnatomy

Challenge 2Challenge 2

CellBiochemistry

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Molecular Engineering Team

Ajay KohliJulian Hibberd Jane Langdale Peter WesthoffInez Slamet-Loedin

Transgenics; over expression RNAi reduction characterisation of Transgenics; over expression, RNAi reduction, characterisation of transgenics

Identification of regulatory switches, micro dissection of leaf BSC MC primordia transcriptome and genome sequencingBSC, MC, primordia, transcriptome and genome sequencing

Identification of promoters and their regulation to give accurate cell specific and developmenal expression

Molecular Engineering - building up C4 biochemistry

Reduce Glycine decarboxylase in BSCR

elat

ive

unda

nce

of

ansc

ripts

Increase PEPC ve

nce

pts

decarboxylase in BSCRab tr

a

c ease Cin MC

Rel

ativ

abun

dan

of

tran

scri p

Increase PPDK in MCR

elat

ive

abun

danc

eof

tr

ansc

ripts

BSC =Bundle Sheath CellMS = Mesophyll Cell

Identify promoter elements to allow cell specific expression in rice

Gene Promoter

BSC

Specific regions of thenon-coding DNA sequenceof C4 genes direct Cell Specific expression

MS

Cell-Specific expression

BSC

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Molecular Physiology Team

Bob Furbank Jim Burnell Susannevon Caemmerer

Gerry Edwards Richard Leegood Tammy SageRowan Sage

high throughput screen development, detailed mechanistic physiology of C3 and C4

gene specific antibody production and biochemical gene specific antibody production and biochemical characterization of enzymology of transgenics

detailed microscopy of C3 and C4 anatomy and h t i ti i th t t d t i licharacterisation in the mutant and transgenic lines

Bioinformatics and Systems Biology Team

Richard Bruskiewich

d t l i f i j t t i t /

Xinguang Zhu Tom Brutnell Tim NelsonChris Myers

data analysis of sequencing projects transcriptome/genome

modelling the C3 and C4 pathways

l i f i t i t l l f d l t l analysis of maize transciptome along leaf developmental gradients in BSC and MC

bioinformatics of vascular development in model d h f lsystems and search for rice analogues

develop the C4 web platform for the consortium

Transcriptome and genome of closely related C3 and C4 species - increasing phylogenetic coverage

The 1000 plant transcriptomes project - Gane Wong and Beijing Genome InstituteThe 100 plant genome project

Molluginaceae, Amaranthaceae, Aizoaceae,

Chenopodiaceae, Nyctaginaceae, Portulacaceae

26 transcriptomes20 genomes

Euphorbiaceae, Chamocyceae

Cleomaceae

20 genomes

Asteraceae

Boraginaceae, ZygophyllaceaeScrophulariaceae

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Standardized Maize leaf developmental gradient for 9 day old Leaf 3

Leaf 1

Base ‐1 cm +4 cm TipSourceSink Transition

N l d B ll

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Nelson and BrutnellCornell USA

GeBP 10

G1 G2 G3

Adaxial/abaxial polarityHB-PHB, ZmRLD1, REV ARF-ARF3, ARF4YABBY-ZmYAB2, ZmYAB14, ZmYAB15

GARP G2- KAN1, ZmMWP1 Myb- ZmRS2 Stomatal development/movement

bHLH-FAMA, MUTE, ICE1 Myb-MYB60, MYB61Leaf morphogenesis/development

Identify development related transcription factors

MADS

Trihelix

Alfin-like

GRF

11

22

13

11

1

Leaf morphogenesis/developmentCell fate: GeBP YABBY-DL1, DL2 MADS-AGLCell expansion/growth: GRF family Trihelix-GTLCell differentiation: TCP Myb-LOF1 SBP-ZmLG1, SPLVascular development: ARF-MP HB-HB15, ZmRS1,

KANT7Metabolic process

Wa /lignin/carbon AP2/EREBP SHN1 WRI1NAC NST1

G1(base)

Early = anatomy

SBP

zf-HD

Aux/IAA

TCP

C2C2-YABBY

10

11

17

19

7

2

1

1

1

1

3

2

Wax/lignin/carbon: AP2/EREBP-SHN1, WRI1NAC-NST1

SignalingHormone: GRAS-SLR1,GAI1, SCL3 ARF ARR Sugar: bZip-ABF2

Chromatin regulationAlfin-like

Middle = cell function

bZIP

ARF

GRAS

C2C2-GATA

SBP

42

21

23

22

10

3

3

3

2

2

13

5

5

4Secondary cell wall

NAC-SND1, SND2HB-KNAT

MYB-MYB52, MYB54, MYB63, MYB85Lipid (VLCFA): MYB-MYB30

Light signaling: bHLH-PIL6, PIF3 GRAS-PAT1G2

(transition)

Middle = cell function

C2H2 -zinc

HSF

HB

bHLH

42

11

41

61

2

9

10

23

4

12

18 Leaf morphogenesis/developmentARF-ARF19AS2-ZmRA2

PhotosyntheticApparatusGARP G2-ZmG2, ZmGLK

Light signaling

( )

Circadian photoperiodDOF-CDF3 MYB-LHY

Late = photosynthesis

NAC

MYB

C2C2-DOF

WRKY

AP2/EREBP

18

56

13

28

25

8

15

2

7

5

17

28

6

9

9g t s g a g

C2C2 CO-STO, COL3 DOF-OBP3 bHLH-PIL5, PIL6 bZIP-HY5, CPRF2

DOF CDF3 MYB LHY

DevelopmentNAC-NAC1, VND7 HB-BEL1 MYB-ZmMybst1 GARP G2-APL

Photoprotection: C2H2 Zinc-ZAT10

Photosynthetic Apparatus

G3(tip)

Photosynthetic Apparatus

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C2C2-CO

GARP-G2

ARR

4

10

4

5

2

1

17

13

5GARP G2-ZmG2 TCP-PTF1

Light signalingDOF- OBP3, DAG1

DevelopmentTCP –TCP5 NAC family

BS M

GARP G2-GLKLight signaling

AUX/IAA-PAP2 bZIP-HY5bHLH-PIL5 C2C2 CO-STOGRAS-PAT1

Genetic Screening Team

Paul Quick Gynheung AnHei Leung Caroline Hsing Erik Murchie Su-May Yu John Sheehy

EMS mutagenised rice

generation and screening of mutagenised sorghum

production of transgenic activation tagged rice populations for non-targeted screening and provision of targeted activation tagged linesgg

screening activation tagged rice lines

Phase 1: Gene Discovery

Screen mutagenized Screen mutagenized

Screengenomes

Screentranscriptomes

y

ggand transgenic and transgenic

lineslines C3 to C4 lineages

S h d i

Sorghum Sorghum mutant linesmutant lines

C3 and C4 related species Model species Arabidopsis,

Setaria, Brachypodium,Sorghum, Rice

Sorghum and maize mesophyll, bundle

sheath, leaf development

Rice activationRice activation--tagged linestagged lines

Establish a pool of geneticdiversity that confers

mutant linesmutant lines

diversity that confersC4 traits

Gene candidates tested

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transgenically in rice

Ri ti ti t d li

ACTIVATION

Rice activation tagged lines

C3 Anatomy Biochem Fine C4

ACTIVATION

C3 AnatomyChange

BiochemChange

FineTuning+++ = C4

REVERSION

Sorghum mutant lines

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Simple High Throughput ScreensScreens

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Compensation pointCO2 response curve Microscopy images

Compensation point

10

15

Leaf Gross Photosynthesis (mmol CO2 m-2 s-1)

RiceMaizeSorghum

0

5

-10 0 10 20 30 40 50 60 70 80 90 100 110

Rice Maize Sorghum-10

-5CO2 (ppm)

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Low CO2 Screening Chamber

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Vein spacing?

C4 plants-have narrower vein spacing with 7 or more veins per mm

Identify C4 genes that regulate vein spacing

C3 plants- have wider vein spacing, there are about 5 veins per mm

Currently, these genes are largely unknownMutate C4 genes – Sorghum or Activate C3 genes - Rice

Strategy

Vein VeinBS BSM M

Mutate C3 plants - Rice

Vein VeinBS BSM M M MM MM M

Mutate C3 plants Rice

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Simple and Detailed anatomical characterization of (A) rice and (B) sorghum A B(B) sorghum. A B

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Activation tagged lines of rice

Leaf Sampling in Taiwan –

flag leaf samples collected from each of the 12 replicates of the flag leaf samples collected from each of the 12 replicates of the 5,050 mutant lines

© WPQ

Su-May Yu (Academia Sinica, Taiwan)

Interesting Rice mutants

1 2 3 4 5 6 7 8 9 10 11Tainung67 (WT)vein spacing = 5.5/mm

TRIM #

104656

108615

Vein Spacing

7 + 0.0

7 + 0 1108615

110321

105588

7 + 0.1

7 + 0.1

8 + 0.4

TRIM Mutant 108615i i 7/

106332

106602

110124

6.5 + 0.2

6.5 + 0.4

6 0 0 31 2 3 4 5 6 7 8 91011121314vein spacing = 7/mm 110124 6.0 + 0.3

Frequency of mutation: about 1 in 1000

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Some interesting mutants are starting to emerge!

Tainung677 mesophyll cells

b ip y

between veins

vein vein

TRIM Mutant 1086155 mesophyll cells

between veins

vein vein

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Secondary screen - Morphological characterization of high vein density mutants at 7th leaf stagey g

30 cm

WT-T67 M105588M104656 M110124M110321WT-T67 M105588M104656 M110124M110321

1 2 3 4 5 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 71 2 3 4 5 6 7 8

V i d it t t hibit h t l t h i htVein density mutants exhibit shorter plant height compared with the wildtype

C4 - Sorghum: Generation of Mutant ResourcesEMS mutagenised M2 population created 2009EMS mutagenised M2 population created 2009Gamma Irradiated M2 population created 2010

Screening of M2 EMS and Gamma mutant populations

of sorghum (2010)of sorghum (2010)

Discard

end

MACRO SCREENstart 1,000 M2 seed lines Score for pale and albino

Vein density of 5th leaf Vein density/mm

≤ 7

NoMACRO SCREEN

Possible candidates

≤ 7

Yes

candidates

Phase 1 one leaf

Phase 2 all leaves

Vein density candidates

Detailed leaf anatomy

Leaf vein density of wild type and interestingy yp gEMS mutants of sorghum (BTx623)

1 3 5 7 9 11 13 15 17

1 2 3 4 5 6 7 8 9 10 11 12 13 142 4 6 8 10 12 14 16 181 2 3 4 5 6 7 8 9 10 11 12 13

a. Wild type, VD = 18 b. Mutant ID no. 161, VD = 14 c. Mutant ID No. 279, VD = 13

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Sorghum mutant with increased mesophyll cells between veins

Sorghum mutant

1 2 34 5

vein vein

vein veinvein vein

1 2

Sorghum wildtypeg m yp

T4R2-028 plant no. 44-5 mesophyll cells

T3R1-028 plant no. 184 mesophyll cellsp y p y

1 2 3 41 2 3 43 4 3

1 2 3 4 5

1 2 3 4

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Conclusions

We have assembled a global network of scientists of diverse disciplines to tackle a complex interdisciplinary problem with significant implications for agriculture

T d d Transcriptome and genome screens are progressing and have already revealed many candidates for us to test. Much more sequencing is in progress.

S hi h th h t i l f l f t Some high throughput screens are in place for leaf anatomy and photosynthetic compensation point.

We have already identified in rice and sorghum putative t t did t ith h t i i t i l f mutant candidates with phenotypic variants in leaf

anatomy.

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k l d

The IRRI C4 Team

Acknowledgements

John SheehyThe C4 Rice ConsortiumThe C4 Rice ConsortiumBMGF for funding

We welcome collaborations and our plan is to expand the consortium as far as possible:

• to enhance our current effortsto enhance our current efforts• to bring in new ideas• to introduce additional sources of funding

© WPQ

Thank you for listening

http://www.amazon.com/books-used-books-textbooks/

C4 Rice web site: http://beta.irri.org/projects15/c4rice

CO2 response curvePhenotype 1

40

45

nthe

sis

20

25

30

35

Rooney

Phot

osyn

5

10

15

20 ml14-40

IR72

Ml 14-40 r2

Ci (ppm)-10

-5

00 50 100 150 200 250 300 350

CO2 Compensation point: 11.36No. of hits with vein density ≤14 = 4Status: Healthy

Vein Density

Status: Healthy

CO2 response curvePhenotype 2

35

40

45

20

25

30

RooneyMl 26-10

nthe

sis

0

5

10

15 ml26-10IR72

Phot

osyn

-10

-5

00 50 100 150 200 250 300 350

Ci (ppm)

CO2 Compensation point: 11.62No. of hits with vein density ≤14 = 4Status: Healthy

Vein Density

Status: Healthy

Radiation Use Efficiency is improved by 50%Source: Sheehy et al 2007Source: Sheehy et al, 2007

Above-ground dry weight (g m-2)

3500

2500

3000 MAIZEy = 4.4xr2 = 0.98

1500

2000

500

1000RICEy = 2.9xr2 = 0.98

Accumulated intercepted PAR (MJ m 2)

00 200 400 600 800

Accumulated intercepted PAR (MJ m-2)

© WPQ

Water Use Efficiency is 1.5 to 3 times greater than C plantsgreater than C3 plants

Water use efficiencyC4C4-likeC3-likeC3

6.0

7.5C3Type I C3-C4Type II C3-C4C4-like Perennial C2O

)

4.5

Perennial C4Annual C4

CO

2m

ol-1

H2

1.5

3.0

(mm

ol C

onqu

isti

prin

glei

robu

sta

ustif

olia

raef

olia

oren

sis

nom

ala

orid

ana

siss

ima

brow

nii

palm

eri

agin

ata

ochi

ana

bide

ntis

riner

via

rala

sica

0.0

F. c

ro F. p

F. r

F. a

ngu

F. c

hlor

F. s

ono

F. a

n

F. fl

o

F. ra

mos F.

F. p

F. v

a

F. k

o

F. b

F. tr

F. a

ustr

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Nitrogen Use Efficiency is enhanced by 260%Source: Evans and von Caemmerer 2000Source: Evans and von Caemmerer, 2000

Rate of CO2 assimilation (µmol m-2 s-1)

60

MaizeSorghum

C4

40

50

60

30

40

10

20WheatRice

C3

Leaf nitrogen content (mmol m-2)

00 20 40 60 80 100 120 140 160 180 200

g ( )

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008 engineering c4 rice - paul quick

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