Engineering C4 rice
C3
C4
XI Conferencia Internacional de Arroz para America Latina y el Caribe21-24 Septiembre 2010
Cali, Colombia
© WPQ
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
© WPQ
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)
© WPQ
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)
© WPQ
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.
© WPQ
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%
Area = 36 M haProduction = 18%
UPLAND DEEP WATER
Area = 19 M haProduction = 4%
Area = 12 M haProduction = 3%
© WPQ
C4 Supercharges Photosynthesis Using A TwoCompartment CO2 Concentrating Mechanism
C3 Photosynthesis
Compartment CO2 Concentrating Mechanism
C4 Photosynthesis
3 Phosphoglyceratec c c
© WPQ
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
© WPQ
Evolutionary ChangeEvolutionary Change
Genetic alterations
C3 AnatomyChange
BiochemChange
FineTuning+++ = C4
© WPQ
Despite its complexity C has Despite its complexity, C4 has evolved independently ~60 times
It can’t be that difficult?!
© WPQ
© WPQ
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
© WPQ
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
© WPQ
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
© WPQ
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
© WPQ
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
© WPQ
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
© WPQ0% 50% 100%
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
© WPQ
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
© WPQ
Simple High Throughput ScreensScreens
© WPQ
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)
© WPQ
Low CO2 Screening Chamber
© WPQ
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
© WPQ
Simple and Detailed anatomical characterization of (A) rice and (B) sorghum A B(B) sorghum. A B
© WPQ
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
© WPQ
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
© WPQ
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
© WPQ
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
© WPQ
Crinkly
Some vein density changes are associated with h t
Grass-likeAsymmetric PaleDwarf
phenotypes
© WPQ
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.
© WPQ
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
© WPQ
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 ( )
© WPQ