More Related Content Similar to 008 engineering c4 rice - paul quick (14) More from FLAR - Fondo Latinoamericano para Arroz de Riego (20) 008 engineering c4 rice - paul quick1. Engineering C4 rice
C3
C4
XI Conferencia Internacional de Arroz para America Latina y el Caribe
21-24 Septiembre 2010
Cali, Colombia
© WPQ
2. Green Revolution Slows
World Rice Yield (1961-2010)
(1961 2010)
Data Source: FAO
Average yield (t ha-1) Average yearly increase over
previous 10 years (kg ha-1)
5.0
5 0 200
4.0 160
3.0 120
2.0 80
1.0
1 0 40
0.0 0
1955 1965 1975 1985 1995 2005 2015
Year
© WPQ
3. The relationship between rice production and
population f A i rice consumers (1961 2004)
l ti for Asian i (1961-2004)
Data Source: UN and FAO
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)
© WPQ
4. A Second Green Revolution?
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)
C3 kale
Maize C4 Grain Yield = 13.9 t ha-1 44 DAG
1 80 sugar beet
potatoes
rice
cassava
60 C4 oil palm
C4 bulrush millet
40 maize
sorghum
sugarcane
C3 napier grass
20
IRRI Expt-rice
IRRI Expt-maize
0
0 100 200 300
Length of growing season (days)
Rice C3 Grain Yield = 8.3 t ha-1 42 DAT
© WPQ
5. C4 RICE
C4 rice could:
i ld
•increase rice yield by 50%
•double water-use efficiency
•improve nitrogen use efficiency
nitrogen-use
C4 photosynthesis is one of the few
evolutionary mechanisms that could deliver
these superior combination of b
th i bi ti f benefits.
fit
© WPQ
6. C4 would confer benefits on all of the global rice ecosystems
Source: Ric Alm n c (Maclean t l
S u c : Rice Almanac (M cl n et al, 2002)
IRRIGATED RAINFED
Area = 79 M ha Area = 36 M ha
Production = 75% Production = 18%
UPLAND DEEP WATER
Area = 19 M ha Area = 12 M ha
Production = 4% Production = 3%
© WPQ
7. C4 Supercharges Photosynthesis Using A Two
Compartment CO2 Concentrating Mechanism
C3 Photosynthesis C4 Photosynthesis
3 Phosphoglycerate
c c c
© WPQ
8. C4 photosynthesis involves alterations to biochemistry, cell
biology and leaf anatomy
CO2
Mesophyll Cell
HCO3-
1 OAA
2
PEP
3
5
Pyruvate
Malate
= C4
4
CO2
RuBisCO
Bundle Sheath Cell
Many of the genes that control
these processes are unknown
© WPQ
9. Evolutionary Change
Genetic alterations
C3 + Anatomy
Change + Biochem
Change + Fine
Tuning = C4
© WPQ
10. Despite its complexity C4 has
complexity,
evolved independently ~60
times
It can’t be that difficult?!
© WPQ
12. The Timeline for C4 Rice
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.
3 years
3 years
Gene 5 years
discovery Transform 4 years
and rice to
Optimize
molecular express Breed C4
C4 function
toolbox Kranz transgenic
in
development anatomy s
transgenic
and the C4 into local
rice
Characterize metabolic varieties
regulatory enzymes
controls
© WPQ
13. To determine the feasibility of replicating the two-cell
C4 photosynthetic pathway in rice:
p y p y
Challenge 1
Leaf
Anatomy
Challenge 2
Cell
Biochemistry
© WPQ
14. Molecular Engineering Team
Julian Hibberd Ajay Kohli Jane Langdale Inez Slamet-Loedin Peter Westhoff
Transgenics; over expression, RNAi reduction, characterisation of
expression reduction
transgenics
Identification of regulatory switches, micro dissection of leaf
BSC, MC primordia
BSC MC, primordia, transcriptome and genome sequencing
Identification of promoters and their regulation to
give accurate cell specific and developmenal
expression
15. Molecular Engineering - building up C4 biochemistry
abundance
anscripts
Relative
Reduce Glycine
of
decarboxylase in BSC
R
tra
nce
pts
Increase PEPC
c ease C
ve
transcrip
abundan
Relativ
of
in MC
abundance
transcripts
Relative
Increase PPDK
of
in MC
BSC =Bundle Sheath Cell
MS = Mesophyll Cell
16. Identify promoter elements to allow cell specific expression in rice
Gene Promoter
BSC
Specific regions of the
non-coding DNA sequence MS
of C4 genes direct
Cell-Specific
Cell Specific expression
BSC
© WPQ
17. Molecular Physiology Team
Bob Furbank Jim Burnell Gerry Edwards Richard Leegood Rowan Sage Tammy Sage Susanne
von Caemmerer
high throughput screen development, detailed mechanistic
physiology of C3 and C4
gene specific antibody production and biochemical
characterization of enzymology of transgenics
detailed microscopy of C3 and C4 anatomy and
characterisation i th mutant and t
h t i ti in the t t d transgenic li
i lines
18. Bioinformatics and Systems Biology Team
Xinguang Zhu Richard Bruskiewich Chris Myers Tom Brutnell Tim Nelson
data
d t analysis of sequencing projects t
l i f i j t transcriptome/genome
i t /
modelling the C3 and C4 pathways
analysis of maize t
l i f i transciptome along l f d
i t l leaf developmental
l t l
gradients in BSC and MC
bioinformatics of vascular development in model
systems and search f rice analogues
d h for l
develop the C4 web platform for the consortium
19. Transcriptome and genome of closely related C3 and C4 species
- increasing phylogenetic coverage
The 1000 plant transcriptomes project - Gane Wong and Beijing Genome Institute
The 100 plant genome project
Molluginaceae, Amaranthaceae, Aizoaceae,
Chenopodiaceae, Nyctaginaceae, Portulacaceae
26 transcriptomes
20 genomes
Euphorbiaceae, Chamocyceae
Cleomaceae
Boraginaceae, Zygophyllaceae
Scrophulariaceae
Asteraceae
© WPQ
20. Standardized Maize leaf developmental gradient for 9 day old Leaf 3
Leaf 1
Sink Transition Source
Base ‐1 cm +4 cm Tip
Nelson and Brutnell
N l dB ll
Cornell USA
© WPQ
21. Identify development related
Adaxial/abaxial polarity
HB-PHB, ZmRLD1, REV ARF-ARF3, ARF4
transcription factors
YABBY-ZmYAB2, ZmYAB14, ZmYAB15
GARP G2- KAN1, ZmMWP1 Myb- ZmRS2
G1 G2 G3
Stomatal development/movement
bHLH-FAMA, MUTE, ICE1 Myb-MYB60, MYB61
GeBP 10
Leaf morphogenesis/development
GRF 11 Cell fate: GeBP YABBY-DL1, DL2 MADS-AGL
Cell expansion/growth: GRF family Trihelix-GTL
Alfin-like 13
Cell differentiation: TCP Myb-LOF1 SBP-ZmLG1, SPL
Early = anatomy Trihelix 22
G1
Vascular development: ARF-MP HB-HB15, ZmRS1,
KANT7
MADS 11 1
(base) Metabolic process
C2C2-YABBY 7 1 Wax/lignin/carbon: AP2/EREBP-SHN1, WRI1NAC NST1
Wa /lignin/carbon AP2/EREBP SHN1 WRI1NAC-NST1
TCP 19 1 2
Signaling
Hormone: GRAS-SLR1,GAI1, SCL3 ARF ARR
Aux/IAA 17 3 Sugar: bZip-ABF2
zf-HD 11 1 1 Chromatin regulation
Alfin-like
Middle = cell function SBP 10 2
Secondary cell wall
C2C2-GATA 22 2 4
NAC-SND1, SND2
GRAS 23 3 5
HB-KNAT
MYB-MYB52, MYB54, MYB63, MYB85
ARF 21 3 5
G2 Lipid (VLCFA): MYB-MYB30
bZIP 42 3 13
(
(transition)
) Light signaling: bHLH-PIL6, PIF3 GRAS-PAT1
bHLH 61 10 18 Leaf morphogenesis/development
ARF-ARF19
AS2-ZmRA2
Late = photosynthesis
HB 41 9 12
Photosynthetic
HSF 11 2 4
Apparatus
Circadian
GARP G2-ZmG2, ZmGLK
C2H2 -zinc 42 23
photoperiod
Light s g a g
g t signaling DOF CDF3 MYB LHY
DOF-CDF3 MYB-LHY
AP2/EREBP 25 5 9
C2C2 CO-STO, COL3 DOF-OBP3
WRKY 28 7 9 G3 bHLH-PIL5, PIL6 bZIP-HY5, CPRF2
C2C2-DOF 13 2 6
(tip) Development Photoprotection: C2H2 Zinc-ZAT10
NAC-NAC1, VND7 HB-BEL1 MYB-ZmMybst1 GARP G2-APL
MYB 56 15 28
NAC 18 8 17 Photosynthetic Apparatus Photosynthetic Apparatus
GARP G2-ZmG2 TCP-PTF1 GARP G2-GLK
ARR 4 1 5
Light signaling Light signaling
GARP-G2 10 2 13 DOF- OBP3, DAG1
Development
BS M AUX/IAA-PAP2 bZIP-HY5
bHLH-PIL5 C2C2 CO-STO
4 5 17
C2C2-CO
TCP –TCP5 NAC family GRAS-PAT1
0% 50% 100% © WPQ
22. Genetic Screening Team
Paul Quick Hei Leung Gynheung An 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 lines
gg
screening activation tagged rice lines
23. Phase 1: Gene Discovery
y
Screen Screen
transcriptomes genomes
Screen mutagenized
g
and transgenic
lines
C3 to C4 lineages
Rice activation-
activation- Sorghum and maize
S h d i C3 and C4 related species
tagged lines mesophyll, bundle Model species Arabidopsis,
sheath, leaf development Setaria, Brachypodium,
Sorghum, Rice
Sorghum
mutant lines
Establish a pool of genetic
diversity that confers
C4 traits
Gene candidates tested
transgenically in rice
© WPQ
24. Rice activation tagged lines
Ri ti ti t d li
ACTIVATION
C3 + Anatomy
Change + Biochem
Change + Fine
Tuning = C4
REVERSION
Sorghum mutant lines
© WPQ
26. Compensation point
CO2 response curve Microscopy images
Leaf Gross Photosynthesis (mmol CO2 m-2 s-1)
15
Rice
Maize
10 Sorghum
5
0
-10 0 10 20 30 40 50 60 70 80 90 100 110
CO2 (ppm)
-5
-10
Rice Maize Sorghum
© WPQ
28. Vein spacing?
Identify C4 genes that regulate vein spacing
C4 plants
-have narrower vein spacing with 7 or more veins per mm
C3 plants
- have wider vein spacing, there are about 5 veins per mm
Currently, these genes are largely unknown
Mutate C4 genes – Sorghum or Activate C3 genes - Rice
Vein BS M M BS Vein
Strategy
Vein BS M M M M M M M M BS Vein
Mutate C3 plants - Rice
© WPQ
29. Simple and Detailed anatomical
characterization of (A) rice and
(B) sorghum
sorghum.
A B
© WPQ
30. Activation tagged lines of rice
Leaf Sampling in Taiwan –
flag leaf samples collected from each of the 12 replicates of the
5,050 mutant lines
Su-May Yu (Academia Sinica, Taiwan)
© WPQ
31. Interesting Rice mutants
TRIM # Vein Spacing
Tainung67 (WT) 104656 7 + 0.0
vein spacing = 5.5/mm 1 2 3 4 5 6 7 8 9 10 11
108615 7+01
0.1
110321 7 + 0.1
105588 8 + 0.4
106332 6.5 + 0.2
106602 6.5 + 0.4
TRIM Mutant 108615
vein spacing = 7/
i i 7/mm 1 2 3 4 5 6 7 8 91011 12 1314 110124 6.0 0.3
60+03
Frequency of mutation:
about 1 in 1000
© WPQ
32. Some interesting mutants are starting to emerge!
Tainung67
7 mesophyll cells
p y
between veins
b i
vein vein
TRIM Mutant 108615
5 mesophyll cells
between veins
vein
vein
© WPQ
33. Secondary screen - Morphological characterization of high
vein density mutants at 7th leaf stage
y g
30 cm
WT-T67 M104656 M110321 M110124 M105588
1 2 3 4 5 1 2 3 4 5 6 78 1 2 3 4 5 6 78 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8
Vein density mutants exhibit shorter plant h i ht
V i d it t t hibit h t l t height
compared with the wildtype
34. C4 - Sorghum: Generation of Mutant Resources
EMS mutagenised M2 population created 2009
Gamma Irradiated M2 population created 2010
36. end
MACRO SCREEN Discard
No
Vein density of
start 1,000 M2 seed lines Score for pale and albino 5th leaf Vein density/mm
≤7
Yes
Possible
candidates
Phase 1
one leaf
Phase 2
all leaves
Vein density
candidates
Detailed leaf
anatomy
37. Leaf vein density of wild type and interesting
y yp g
EMS mutants of sorghum (BTx623)
1 3 5 7 9 11 13 15 17
2 4 6 8 10 12 14 16 18
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 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
38. Sorghum mutant with increased Sorghum mutant
mesophyll cells between veins
5
1 4
2 3
vein vein
vein vein
1 2
Sorghum wildtype
g m yp
39. T4R2-028 plant no. 4 T3R1-028 plant no. 18
4-5 mesophyll cells
p y 4 mesophyll cells
p y
1 2 1 2
3 4 3 4
1 2 3 4 5
4
1 2 3
© WPQ
40. Crinkly
Some vein density changes are associated with
phenotypes
h t
Asymmetric Dwarf Grass-like Pale
© WPQ
41. Conclusions
We have assembled a global network of scientists of
diverse disciplines to tackle a complex interdisciplinary
problem with significant implications for agriculture
Transcriptome and genome screens are progressing and
T d d
have already revealed many candidates for us to test.
Much more sequencing is in progress.
Some hi h th
S high throughput screens are in place for leaf anatomy
h t i l f l f t
and photosynthetic compensation point.
We have already identified in rice and sorghum putative
mutant candidates with phenotypic variants i l f
t t did t ith h t i i t in leaf
anatomy.
© WPQ
42. The IRRI C4 Team
Acknowledgements
k l d
John Sheehy
The C4 Rice Consortium
BMGF for funding
We welcome collaborations and our plan is to expand
the consortium as far as possible:
• to enhance our current efforts
• to bring in new ideas
• to introduce additional sources of funding
© WPQ
43. Thank you for listening
http://www.amazon.com/books-used-books-textbooks/
C4 Rice web site: http://beta.irri.org/projects15/c4rice
44. Phenotype 1
45 CO2 response curve
40
35
30
nthesis
25
Rooney
20 ml14-40
Photosyn
15 IR72
10 Ml 14-40 r2
5
0
0 50 100 150 200 250 300 350
-5
-10 Ci (ppm)
CO2 Compensation point: 11.36 Vein Density
No. of hits with vein density ≤14 = 4
Status: Healthy
45. Phenotype 2 45
CO2 response curve
40
35
30
nthesis
25 Rooney
20 Ml 26-10
Photosyn
15 ml26-10
IR72
10
5
0
0 50 100 150 200 250 300 350
-5
-10
Ci (ppm)
CO2 Compensation point: 11.62 Vein Density
No. of hits with vein density ≤14 = 4
Status: Healthy
46. Radiation Use Efficiency is improved by 50%
Source: Sheehy et al 2007
al,
Above-ground dry weight (g m-2)
3500
3000 MAIZE
y = 4.4x
2500 r2 = 0.98
2000
1500
RICE
1000 y = 2.9x
r2 = 0.98
500
0
0 200 400 600 800
Accumulated intercepted PAR (MJ m-2)
2
© WPQ
47. C
(mmol CO2 mol-1 H2O)
0.0
1.5
3.0
4.5
6.0
7.5
F. cro
onquisti
C3
F. pringlei
p
C3
Water use efficiency
F. robusta
r
C4-like
F. angu
ustifolia
Annual C4
F. chlor
raefolia
Type I C3-C4
Perennial C4
Type II C3-C4
F. sono
orensis
F. an
nomala
C3-like
F. flo
oridana
F. ramos
sissima
F. brownii
F. palmeri
p
C4-like
F. va
aginata
greater than C3 plants
F. ko
ochiana
F. bidentis
b
F. tr
rinervia
C4
F. austr
ralasica
Water Use Efficiency is 1.5 to 3 times
© WPQ
48. Nitrogen Use Efficiency is enhanced by 260%
Source: Evans and von Caemmerer 2000
Caemmerer,
Rate of CO2 assimilation (µmol m-2 s-1)
60
C4
50 Maize
Sorghum
40
30
20 C3
Wheat
10 Rice
0
0 20 40 60 80 100 120 140 160 180 200
(mmol m-2)
Leaf nitrogen content (
g
© WPQ