HsiangChun Lin PDF – C 4 Rice Center International Rice Research Institute [email protected] 5 th Annual South Asia Biosafety Conference Sep 13, 2017 Engineering a C 4 rice prototype C3 C4
HsiangChun LinPDF – C4 Rice Center
International Rice Research [email protected]
5th Annual South Asia Biosafety ConferenceSep 13, 2017
Engineering a C4 rice prototype
C3
C4
Overview
(1) Introduction to rice, photosynthesis and The C4 Rice Project
(2) Describe the problems we face by engineering a C4 rice
(3) Molecular engineering –the installation of C4 biochemistry into rice
(4) Gene discovery –screen for genes controlling C4 anatomy and C4 function
An IRRI-orientated presentation reporting the work of multiple scientists.
Rice is Life
The contribution of rice to total global calorie intake
90% of rice produced
here
70% of the world’s most poor live here
Source: FAO and World Bank 2010
Rice is a staple crop for the world’s poor.
Global rice production
Yield Gap
Potential current increase in production
Reproduced from GRiSP 2010
Next Green Revolution
The potential of a C4 rice
Photosynthesis is doubled Yield increased by ~50%
WUE 1.5-3 times higher NUE 2.5 times higher RUE 50% higher
Sheehy et al. 2009
Challenges faced with making a C4 rice
Plasmodesmata
Bundle Sheath Size
Organelle localization, number and size
Wall thickness
Vein spacing
C4 cycle
Photorespiration
Rubisco
Starch synthesis
Metabolite transport
C3 cycle
Structure Function
Mechanism
C4 Photosynthesis
Genetic Modification
Engineering a two-celled C4
photosynthetic pathway in rice
C3
RiceAnatomy Change
C4
Rice+ =Biochemical
ChangeFine Tuning++
C4 photosynthesis involves alterations to biochemistry, cell biology and leafanatomy.
Installation of C4 biochemistry into rice
cytosolcytosol
chloroplast
CO2
CO2
CA
OAA malate
PEP pyruvatePPDK
3-PGA
triose-P
3-PGA
CO2
NA
DP
-ME
RuBP
PPT
bundle sheathmesophyll
triose-PGdcH
Photorespiration &Calvin Cycle
Rubisco
OAA
PEP
HCO3-
malate
pyruvate
©©©©
©©©
©©©©
©
©©©
chloroplast
TPT TPT
MEP
DiT2DiT1OMT
RubiscoMEP
PEP
C
NADP-MDH
Cell specific expression of C4 maize photosynthetic proteins in rice
CA MDH ME
PEPCPPDK
OMT DiT1
MEP PPT
RBCS GDCH
X
X
C4 Biochemistry
Down regulation of C3 genes
C4 Transporters
Crossing strategy
Most advanced cross line: 10 genes• Expressed eight primary C4 enzymes and transporters • Knockdown two C3 pathway enzymes
Our most advance cross lines: 10-fold line
Wild-type 10-fold line
Maize
Multi-channel using RFP, GFP, and DAPI filters
BSC
MCBSC
MC
PEPC OE line
ZmPEPC mesophyll cytosolic specific expression
MC
BSC
Wild-type
Immunolocalization of Maize protein expressions in transgenic plants
BSC
MC
BSCMC
BSC
Quality control of transgenic lines overexpressingC4 photosynthetic genes
0
50
100
150Enzyme Activity of MDH
Maize
M 1 2 1 2 3
Wild-type
MDH OE lines
Enzyme Activity assay of C4 enzymes
Metabolite profile in leaves GC-MS Analysis
Photosynthesis measurementLight response curve
Verification of C4 pathway function
Rubisco
3-PGA
CH2O
Mesophyll cell
Calvin cycle
Bundle sheath cell
oxaloacetate
CO2
malate
pyruvatePEP
HCO3-
13CO2
PEPC
12CO2
0
20
40
60
80
100
0 10 30 60 120
% 1
3C
En
rich
me
nt
Labeling time (s)
PGA, % Label
0
5
10
15
20
0 10 30 60 120
% 1
3C
En
rich
me
nt
labeling time (s)
Malate, Label
13C labeling: Metabolite flux into the C4 cycle
Calculations suggest that we have about 5% of the carbon fixed during photosynthesis is moving through malate.
13C enrichment of Malate
Wild-type
Quintuple crosses
Genetic Screens
Biochemical Change
Strategy One: REVERSION
Sorghum and Setaria mutant lines
Strategy Two: ACTIVATION
Rice activation tagged lines
+ =Anatomy Change
Fine Tuning++C3 C4
Leaf Anatomy
VMBS
VMBS
1 2 3 4 5 6 7
C3 Oryza sativa IR64 C4 Sorghum bicolor
5.5 vein mm-1 8.5 vein mm-1
1 2
1 2 3 4 5 6 7 8 9 10 11
2.0 mm
1 2 3 4 5 6 7 8 9 101112 13141516 17 18
2.0 mm
Loss of function mutations
Sorghum bicolor
1.6 millionRizal et al. 2015, The Plant Journal. 84, 257-266
Sorghum with an increased number of mesophyll cells between veins
50 µm 50 µm
Rizal et al. 2015, The Plant Journal. 84, 257-266
Gene ID
CYP90D2 - Brassinosteroid biosynthesis
Premature stop codon
M30
M8
3.3 Mb Inversion break point
Rizal et al. 2015, The Plant Journal. 84, 257-266
Field Screen
33,076 Mutant Lines screened for increased vein spacing (VS). Trait identified in 48 mutant lines.
Insertion in chromosome 9
8260k 8270k 8280k 8290k 8300k
LOC_Os09g14010
glycosyl hydrolase family 9 protein
LOC_Os09g14019
disease resistance protein (RPS2)
Chatterjee, Coe et al. (in prep)
3211 bp 14936 bp
C4
Seta
ria
C3
Ric
e
30 40 50 60 70 100 200 300 400 800 ambientCO2 Concentration (PPM)
19 day old seedlings
Г = 50 ppm
Г = 6 ppm
Compensation Point Screens
Rapid screen of mutant plants
F v/F m
0.83
0.50
6,425 M3 Lines = 615 candidate lines – 5 homozygous
Chatterjee, Acebron and Coe et al.
48 hours at 30 ppm96 hours at 30 ppm
A loss of C4 function
Г = 2.06 ± 0.216
Г = 47.60 ± 14.36
δ1
3C
o/ o
o
Higher CO2 compensation pointLower photosynthetic rate
Lower 13C isotope composition
Chatterjee, Acebron and Coe et al.
Possible Mutations
Rubisco
Lower CO2
concentration in the BS
BS cell wall composition
Positioning of
chloroplasts
Positioning of C4
decarboxylation
Outcomes & Perspectives
• Substantial progress has been made:– Engineering C4 biochemistry into rice
– Screening for C4 genes controlling leaf anatomy
– Research being undertaken in the wider consortium
• Our results are a step in the right direction but there is more work to do:– Fine-tuning biochemistry
– Engineering leaf anatomy
• There is value in what we have learnt along the way -the future of rice science.
Acknowledgements
The C4 Rice Consortium Thank you
Funding donors
IRRI C4 Rice CenterPaul QuickRob CoeAnindya BandyopadhyayEfren BagunuMae Antonette MercadoIsaiah Paolo SalazarJoanne JereniceLeanilyn CastanarJolly ChaterjeeJacque DionoraXiaojia Yin Shanta KarkiGovinda RizalJohn Sheehy
Past and present members worldwide