integrating diverse datasets to understand photosynthetic development in c3 and c4 plants
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Integrating diverse datasets to understand photosynthetic development in C3 and C4 plants. Tom Brutnell Boyce Thompson Institute. Why iPlant and not iBrain?. - PowerPoint PPT PresentationTRANSCRIPT
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