Download The maize leaf transcriptome

Integrating diverse datasets to understand
photosynthetic development in C3 and C4 plants
Tom Brutnell
Boyce Thompson Institute
Why iPlant and not iBrain?
• 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
Urgent need to develop the NextGen of food,
feed and bioenergy crops
Global food security
•
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 alltime highs
•
Major agronomic disaster (e.g.
drought in China) could have
serious consequences on food
supply
Photosynthesis: C3 to C4
C5
BS
2xC3
M
From website of Donald Ort http://www.life.uiuc.edu/pru/labs/ort.html
Models for C4 photosynthetic development
C3 state
C4 state
A) repression of gene expression
through novel trans-factor
B) Enhanced expression in BS or M
through novel cis-element
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
Using monocot leaf gradient to find networks and regulatory points for C4
Tests
Modify regulatory points
Perturb environment
Modeling
Expression/regulation
Metabolism
Comparisons
developmental stages
BS vs. M cells (LCM)
C3 and C4
Developmental inventories
transcripts
proteins
metabolites
physiology
anatomy
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)
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)
Where should we sample?
Maize inbred B73, 9 dap
L3
L2
Ligule of L2
L3
L1
Standardization of the base-to-tip leaf gradients
• extent of growth (time)
• anatomical features
• gene markers
• sink-source transition
Anatomical gradient: Maize L3 Base
Maize L3: -1 to 0 cm past L2 ligule
Maize L3: 4 to 5 cm past L2 ligule
M
BS
Maize L3: 1 cm from tip
BS
M
Locating sink-source transition
Sink-source transition is site of global changes in physiology, expression, anatomy
Label from L3 tip
Label 5 min
Chase 1 hour
from L1
Max chl accumulation
Plastid diffn complete
Sink-Source transition complete
L2 ligule
Zone of import
limited to below
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
Gene expression markers along gradient
3
2
Calibrated gradient with 20 expression markers (RT-PCR)
PPdK
• PS genes increase from S-S transition
1
0
20
16
12
8
4
0
Sheath
CycD2
• Cell cycle genes only near base
Developmental Inventories—Common samples for all inventories
Leaf
Section 1
Section 4
Section 9
Section 14
Source-sink
Histology
LCM
RNA isolated
RNA isolation and
amplification
Metabolite extraction
Total RNA
Primary
metabolites
Bundle
sheath cell
Mesophyll
cell
mRNA
aRNA
Secondary
metabolites
Small RNA library
Metabolites profiling
RNA-seq library
Solexa libraries sequencing
qPCR
validation
Solexa expression analysis
Proteomics
Systems biology
NextGen Sequencing
16
NextGen Sequencing
17
Developmental Inventories—dynamics of individual
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
Splice variants by stage
novel transcript
MapMan Pathway Viewer- see Bjoern
Base
Tip
Adapted MapMan view: does not show multiple stages, cell types, C4
Mesophyll Cell
Bundle Sheath Cell
PEP
PEP
MEP3
RPE
Xyl-5P
RPI
TKL
OAA
OAA
MEP1?
TPI
TPI
NADP+
NADP-MDH
Mal
DAP
Maltose
DIT1
ASP
PPDK: Pyruvate Orthophospho-Dikinase
PRP: PPDK – regulatory protein
PEPCase: PhosphoenolPyruvate Carboxylase
CA: Carbonic Anhydrase
NADP-MDH: NADP – malate dehydrogenase
TPT: Phosphate/triose-phosphate translocator
SBP
NADPH
Sucrose
Starch
Synthesis
OAA
Glu
NADP+
AKG
Mal
FBA
MEX1
AKG
ASP-TS
Glu
TKL
FBP
DAP
NADPH
OAA
ASP-TS
GAPB
Pyr
ATP
PRK
PGK
GAPA
AMP
Mitochondrion
HCO3-
PGK
CO2
NADP-ME
CA
RCA
Rubisco
PGA
PGA
PEP
PEPCase
CO2
PPT
?
PPDK
PRP
Chloroplast
AMP
Chloroplast
ATP
ASP
DIT2
ATP
ADP
AATP1
Pyr
MEP4
PEPCK
ATP
ADP
Pyr
Mal
ASP
NADP-ME: NADP – Malic enzyme
PPT: Phosphate/PhosphoenolPyruvate translocator
DIT: 2-oxoglutarate/malate translocator
MEP: Envelope protein
PEPCK: PhosphoenolPyruvate carboxykinase
Rubisco: Ribulose-1,5-bisphosphate carboxylase/oxygenase
RCA: Rubisco activase
PGK: Phosphoglycerate kinase
PRK: Phosphoribulokinase
TPI: Triosephosphate isomerase
RPI: ribose-5-phosphate isomerase
GAPA: Glyceraldehyde-3-phosphate dehydrogenas
TKL: Transketolase
GAPB: Glyceraldehyde-3-phosphate dehydrogena
SBP: Sedoheptulose-1,7-bisphosphatase
ASP-TS: Aspartate Aminotransferase
FBA: Fructose-bisphosphate aldolase
DAP: dihydroxyacetone phosphate
Lin 02/2009
δ
α
β
AT
P
α
PsbC
δ
α
β
AT
P
PSII
H2 +O2
α
PsaF
PC
PSI
Lumen
stroma
cytosol
H+
NDH complex
H+
Cyt b6f complex
PC
PsaA
PsaB
PsaN
PsaF
PSI
Lhca 1-4
ISPa
3H+
C, E, H, J, K
IV
M, I, L, O, D, G
PQH2
Cyt
f
NdhA NdhB
ISPb
ATP synthase
PsaN
FD
Cyt b6
a
PsaB
N M
H
NdhL
NdhJ
NdhD
NdhF
C12
H+
Cyt b6f complex
O
γ
thylakoid
membrane
ISPa
PsaA
FNR2
bb
ε
PsbR
ISPb
Lhca 1-4
ADP +
Pi
PsbQ
IV
C, E, H, J, K
H2O
PsbO
M, I, L, O, D, G
3H+
PsbP
Cyt
f
PsbA PsbD
Cyt
a
PsbB
b6
C12
FD
L, S, N, H, M, X
γ
ATP synthase
Bundle Sheath
PQH2
bb
ε
thylakoid
membrane
FNR1
hv
J, K, W, I, E, F
Lhcb 1-6
Mesophyll
ADP +
Pi
NADP+
NADPH
Lumen
stroma
cytosol
Lin 02/2009
PageMan view of developmental gradient, BS/M specificity
gradient
BS/M
Down in M
Up in BS
C4 pathway viewer with plasmodesmata, transporters, etc
TFs clustered by developmental dynamics
base -1
4
tip
A
B
C
D
E
F
G
H
I
TF types in dynamic classes
A
B
C
D
E
F
G
H
I
XXXX
Input
gene ID
0h
6h
12h
18h
24h
Gene expression
base
-1
+4
tip
“a slider view”
0h
6h
12h
18h
24h
Gene expression
base
-1
+4
tip
“a slider view”
0h
6h
12h
18h
24h
Gene expression
base
-1
+4
tip
“a slider view”
0h
6h
12h
18h
24h
Gene expression
base
-1
+4
tip
“a slider view”
0h
6h
12h
18h
24h
Molecular inventories correspond well to leaf gradient features
Plastid
differentiation
Cell division
Cell wall
Polymers
Plasmodesmata
Cell patterning
Respiration
Transcr. factors
PS light & dark reactions (C4)
PS Pigments
PS Activities
PS metabolites
Plastid number
Transcription factors
L3
base
tip
Sink-Source transition
L2 ligule