Integrating diverse datasets to understand photosynthetic development in C3 and C4 plants

Tom Brutnell Boyce Thompson Institute

•World population is expected to reach 9 billion by 2050 (currently approx. 7 billion) = more mouths to feed, cars to fuel and bodies to clothe

• We are near or past peak oil production = food production is more expensive (fertilizer and tractors)

• Global warming is reshaping land use (bioenergy) and contributing to a more unpredictable environment

Why iPlant and not iBrain?

Urgent need to develop the NextGen of food, feed and bioenergy crops

• In 46 developing countries food prices are higher than 12 months ago, despite global recession

• In sub-Saharian Africa price of maize, millet and sorghum are at all-time highs

• Major agronomic disaster (e.g. drought in China) could have serious consequences on food supply

Global food security

From website of Donald Ort http://www.life.uiuc.edu/pru/labs/ort.html

C5

2xC3

BS

M

Photosynthesis: C3 to C4

Models for C4 photosynthetic development

A) repression of gene expression through novel trans-factor

B) Enhanced expression in BS or M through novel cis-element

C3 state C4 state

Models for C4 photosynthetic development

C3 state C4 state

C) Destabilization of protein due to novel cellular environment (e.g. redox, missing complex)

D) Expression directly or indirectly linked to metabolite Y

Physiology & cell biology—Bob Turgeon (Cornell)Photosynthesis—Richard Peterson (CAES)Transcriptomics—Tom Brutnell (Boyce Thompson Inst)Proteomics—Klaas van Wijk (Cornell) Laser microdissection—Tim Nelson (Yale)

Developmental inventoriestranscripts

proteinsmetabolitesphysiologyanatomy

Developmental inventoriestranscripts

proteinsmetabolitesphysiologyanatomy

Comparisons developmental stagesBS vs. M cells (LCM)

C3 and C4

Comparisons developmental stagesBS vs. M cells (LCM)

C3 and C4

ModelingExpression/regulation

Metabolism

ModelingExpression/regulation

Metabolism

TestsModify regulatory points

Perturb environment

TestsModify regulatory points

Perturb environment

Metabolites—Alisdair Fernie (MPI-Potsdam Golm)Statistics—Peng Liu (Iowa St U)Informatics—Qi Sun (Cornell), Pankaj Jaiswal (Oregon St )Modeling, systems—Chris Meyers (Cornell)

Using monocot leaf gradient to find networks and regulatory points for C4

Standardization of the base-to-tip leaf gradients• extent of growth (time)• anatomical features• gene markers• sink-source transition

Ligule of L2

Maize inbred B73, 9 dap

L3

L1

L2

L3

Where should we sample?

Anatomical gradient: Maize L3 Base

Maize L3: -1 to 0 cm past L2 ligule

Maize L3: 4 to 5 cm past L2 ligule

BSM

Maize L3: 1 cm from tip

BS M

Label 5 minChase 1 hour

Label from L3 tip from L1

Zone of import limited to below

L2 ligule

Max chl accumulation

Plastid diffn complete

Sink-Source transition complete

L2 ligule

• L2 ligule is an accurate physical marker for sink-source transition in L3• Ligule site confirmed accurate for both maize and rice L3

Locating sink-source transition

Sink-source transition is site of global changes in physiology, expression, anatomy

Sheath

0

1

2

3

PPdK

0

4

8

12

16

20

CycD2

Calibrated gradient with 20 expression markers (RT-PCR)

• Cell cycle genes only near base

• PS genes increase from S-S transition

Gene expression markers along gradient

Section 1Section 1

RNA isolatedRNA isolated

Solexa libraries sequencingSolexa libraries sequencing

Total RNA Total RNA mRNA

mRNA

Small RNA librarySmall RNA library RNA-seq library RNA-seq library

Solexa expression analysisSolexa expression analysis

Metabolite extractionMetabolite extraction

Metabolites profilingMetabolites profiling

Systems biologySystems biology

Primary metabolites

Primary metabolites

Secondary metabolitesSecondary

metabolites

Mesophyll cell

Mesophyll cell

Bundle sheath cell

Bundle sheath cell

RNA isolation and amplification

RNA isolation and amplification

aRNA aRNA

Section 4Section 4 Section 9Section 9 Section 14Section 14LCMLCM

qPCR validation

ProteomicsProteomics

LeafLeaf

Developmental Inventories—Common samples for all inventories

Source-sinkHistology

Source-sinkHistology

16

NextGen Sequencing

17

NextGen Sequencing

Splice variants by stage

novel transcript

RNA-seq profiles• Resolves closely related

members of a gene family

• Identifies alternative splicing patterns

• Allele-specific transcription

• Less dependent on gene annotation

• More dynamic fold change

Adapted MapMan view: does not show multiple stages, cell types, C4

BaseBase

TipTip

MapMan Pathway Viewer- see Bjoern

Mesophyll Cell Bundle Sheath Cell

ATP

AMP

Pyr

PEP

HCO3-

OAA

NADPH

NADP+

Mal Mal

Pyr

CO2

Mal

Chlo

ropl

ast

PyrPEP

OAA

Chlo

ropl

ast

Lin 02/2009

PPDKPPDK

DIT1DIT1

DIT

2D

IT2

MEP

3M

EP3

MEP4MEP4

PPT?PPT?

MEP1?MEP1?

NADP-ME: NADP – Malic enzymePPT: Phosphate/PhosphoenolPyruvate translocator

DIT: 2-oxoglutarate/malate translocatorMEP: Envelope protein

PEPCK: PhosphoenolPyruvate carboxykinaseRubisco: Ribulose-1,5-bisphosphate carboxylase/oxygenase

PPDK: Pyruvate Orthophospho-DikinasePRP: PPDK – regulatory protein

PEPCase: PhosphoenolPyruvate Carboxylase CA: Carbonic Anhydrase

NADP-MDH: NADP – malate dehydrogenaseTPT: Phosphate/triose-phosphate translocator

NAD

P-MD

H

NADPH

NADP+

CO2

NAD

P-ME

PRK

RPI

TKL

SBP

PGK

TPI

DAP

TPTTPTDAP

TPI

GAPB

PGK

PGA

PPTPPT

PGA

ASP

Glu

AKG

ASP

Mitochondrion

ASP-TS

OAA

PEPPEPCK

ATP

AMP

RCA

RCA: Rubisco activasePRK: Phosphoribulokinase

RPI: ribose-5-phosphate isomeraseTKL: Transketolase

SBP: Sedoheptulose-1,7-bisphosphataseFBA: Fructose-bisphosphate aldolase

PGK: Phosphoglycerate kinaseTPI: Triosephosphate isomerase

GAPA: Glyceraldehyde-3-phosphate dehydrogenase AGAPB: Glyceraldehyde-3-phosphate dehydrogenase B

ASP-TS: Aspartate AminotransferaseDAP: dihydroxyacetone phosphate

PEPCase

PRP

CAGAPA

AATP1ATPADP

ATPADP

Starch Synthesis

Sucrose

MEX1

ASP

OAA

Glu

AKG

ASP-TS

Rubisco

FBP

TKL

Xyl-5P RPE

FBA

Maltose

stroma

Mes

ophy

ll

Lumen

thylakoid membrane

ADP + Pi

ATP

3H+

α αβ

γ

δ

ε bb

C12 a

stromaLumenBu

ndle

She

ath

thylakoid membrane

ADP + Pi

ATP

3H+

α αβ

γ

δ

ε bb

C12 a

cytosol

cytosol

Lin 02/2009

PsbB

PsbA PsbD

PsbP PsbO

PsbRPsbQ

PsbC

J, K, W, I, E, F

L, S, N, H

, M,

X

ATP synthase PSIICyt

f

ISPa

Cyt b6 IV

ISPb

Cyt b6f complex

Lhcb 1-6

hv

H2O H2 +O2 H+

PQH2

PsaA PsaB

PsaFPsaNM

, I, L, O, D, G

C, E, H, J, K

Lhca 1-4

PC

FNR1

FD

NADPH NADP+

PSI

Cyt f

ISPa

Cyt b6 IV

ISPb

Cyt b6f complexH+

PQH2

PsaA PsaB

PsaFPsaN

M, I, L, O

, D, G

C, E, H, J, K

Lhca 1-4

PC

FNR2

FD

PSI

NdhA NdhB

N MO

NdhLNdhJ

H

NdhF

NdhD

H+

NDH complexATP synthase

1.5

Up in BS

Down in MPageMan view of developmental gradient, BS/M specificity

gradientBS/M

C4 pathway viewer with plasmodesmata, transporters, etc

base -1 4 tip

A

B

C

D

E

F

G

H

I

TFs clustered by developmental dynamics

A

B

C

D

E

F

G

H

I

TF types in dynamic classes

0h 24h12h6h 18h

XXXXInput gene

ID

0h 24h12h6h 18h

“a slider view”

base tip-1 +4

Gen

e e

xp

ress

ion

0h 24h12h6h 18h

base tip-1 +4

Gen

e e

xp

ress

ion

“a slider view”

0h 24h12h6h 18h

base tip-1 +4

Gen

e e

xp

ress

ion

“a slider view”

0h 24h12h6h 18h

base tip-1 +4

Gen

e e

xp

ress

ion

“a slider view”

Molecular inventories correspond well to leaf gradient features

L3

Cell divisionCell wallPolymers

PlasmodesmataCell patterning

RespirationTranscr. factors

Cell divisionCell wallPolymers

PlasmodesmataCell patterning

RespirationTranscr. factors

PS light & dark reactions (C4)PS Pigments PS Activities

PS metabolitesPlastid number

Transcription factors

PS light & dark reactions (C4)PS Pigments PS Activities

PS metabolitesPlastid number

Transcription factors

Sink-Source transitionL2 ligule

Plastid differentiation

Plastid differentiation

base tip