HsiangChun LinPDF – C4 Rice Center

International Rice Research Instituteh.lin@irri.org

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.

The International Rice Research Institute (IRRI)

Los Baňos

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.

First Green Revolution in rice

50 years of IR8

Extreme climates

SA

LT

FL

OO

DH

EA

TD

RO

UG

HT

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

C3 and C4 Photosynthesis

C3 Calvin-Benson CycleRice

NADP-ME C4 CycleSorghum

M

BS BS

M

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

The C4 Rice group at IRRI

We work with the C4 Consortium from 12 institutions in 8 countries.

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.

Molecular engineering: the installation of C4 biochemistry into rice

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

Gene Discovery: C4 leaf anatomyIRRI Genetic Screens

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

Vein density screen

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

Rice activation tagged lines

35Senhancers

GeneT-DNA

35Senhancers

GeneT-DNA

Upstream

Downstream

Field Screen

33,076 Mutant Lines screened for increased vein spacing (VS). Trait identified in 48 mutant lines.

TRIM lines reducing vein density

CVS

WT

Chaterjee, Coe et al. (in prep)

50 µm

Mesophyll Cell lobing

Rice C4 Rice

Chaterjee, Coe et al. (in prep)

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

Fine tuning

Low CO2 screen of Setaria for loss of C4

function

Low CO2 chambers

PlantScreen: Fv/Fm

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

MIT Technology Review10 Breakthrough Technologies

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