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Aruna Kilaru
East Tennessee State University
Johnson City, TN, USA
Understanding the regulation of oil biosynthesis in oil-rich tissues
(for the purpose of enriching plant oil content to generate biofuels)
Significance of Plant Oils
Food, Feed and Fuel!
Oils Are the Most Energy-Dense Plant Products
Glucose (CHOH)n
Oleic acid CH3(CH2)nCOOH
More Reduced Carbon
Biomass crop with 20% oil content will almost double energy content available for liquid fuel
Is this possible?
Biomass = Bioenergy?
3 tons oil (~3400 L biodiesel) ~113 GJ energy
12 tons lignocellulose (~5000 L ethanol) ~117 GJ energy
15 tons dry matter/ha 20% oil content
Bio-Refinery
To Increase Energy Content of Biomass – Add Oil by Engineering
Assumptions: 22 tons Miscanthus/ha (Avg. Illinois, Steve Long); 80 gal EtOH/ton
Energy from crop in liquid fuel: GJ/ha
After oil extraction, lignocellulose remains and is available for fermentation, burning, etc.
Making Oil Costs Energy but not Water
• Sunlight is rarely limiting in modern ag systems. Light can power an increase in biomass energy density without additional water
• Plants stop photosynthesis when water is limited; lack of water is the greatest limitation to plant productivity
• Converting fixed carbon from carbohydrate (CHOH)n to oil (CH2)n does not require water or additional N.
Advantages of Oil-rich Crops
No fermentation required
But oil is compatible with future cellulosic fermentation strategies
Comparatively easy extraction/recovery
Higher energy density is valuable for liquid fuel OR for burning
“Harvesting and transporting biomass to a bioenergy facility are collectively the most expensive part of feedstock supply.” Ceres website
How to increase 20 % oil in vegetative tissues?
Oil/Triacylglycerol (TAG)s biosynthesis in plants
Saturated fatty acid
Monounsaturated fatty acid
Polyunsaturated fatty acid
Sucrose Pyruvate TAG Fatty Acid Cytosol & Plastid Plastid ER
Plant/vegetable oils are triacylglycerols (TAGs) formed by esterification of glycerol with three fatty acids (FA)
Oil biosynthesis in non-seed tissues
Plant Oil palm Avocado Olive
Tissue Seed Mesocarp Seed Mesocarp Seed Mesocarp
% Oil (DW) 60 80-90 2 60-70 4 70-80
% Saturated 84 52 24 21 13 16
% Monounsaturated 14 39 29 65 70 75
% Polyunsaturated 2 9 47 14 17 9
Objective
1. To understand the expression of lipid genes in different oilseeds and non-seed tissues
2. Identify key steps that control fatty acid (FA) biosynthesis
Approach- comparative transcriptome analyses to understand storage of oil in seed and non-seed tissues
Persea americana
Elaeis guineensis
Phoenix dactylifera
Euonymus alatus
Ricinus communis
Tropaeolum majus
Brassica napus
Mesocarp - 70
Mesocarp - 90
Mesocarp - 1
Endosperm - 50 Aril - 40
Endosperm - 60
Embryo - 20
Embryo - 45
Values are ~ % Oil by DW
Seed tissues
Non-seed tissues
Cyperus esculentus Roots - 26
Collect seed & non-seed tissue samples at 4-5
developing stages
RNA extraction & cDNA library synthesis
Pyro (454) sequencing of 35 cDNA libraries
Assembly and annotation
Analysis of transcripts for biochemical pathways
Database for transcriptome analysis
Results expressed as # of EST/100,000 ESTs
Generated 35 seed and non-seed tissue libraries
Brassica Castor Euonymus Nasturtium
4 stages of Embryo
4 stages of Embryo
Embryo, 4 stages of Endosperm
Aril, embryo, & 4 stages of Endosperm
Leaf, 5 stages of Mesocarp
5 stages of Mesocarp
5 stages of Mesocarp
2.3 1.3 1.1 3.1 4.8 3.4 140
Million Expressed Sequenced Tags (ESTs)
Oil palm Date palm Avocado
Bourgis F, Kilaru A*, et al., 2011 PNAS USA. 108:12527-32. * Co-first author
Troncoso-Ponce MA, Kilaru A*, et al., 2011 Plant J. 68: 1014-1027.
Oilseed data is also available on: http://aralip.plantbiology.msu.edu/)
http://aralip.plantbiology.msu.edu/
Comparative transcriptomics
Expression profiles of seed and non-seed tissues were compared to identify
• The differences and similarities in gene expression associated with carbon partitioning
• Clues to key steps that control fatty acid (FA) and TAG biosynthesis
Transcriptome Analyses
Oil-rich seed tissues
Br, Ca, Na, Eu
Oil-rich Non-seed
tissues
Op, Avo
Non-oil tissues
Op leaf Dp mesocarp
At seedling 0
20
40
60
80
100
FA c
on
ten
t (%
DW
)
Oil palm
Date palm
Developing Stages
S1 S2 S3 S4 S5
Metabolite Analyses
80-90% Oil
80-90 % Sugar
Sugar Oil
Protein Fiber
mesocarp
mesocarp
Members of the same family show dramatic difference in oil content
What is responsible for this >100-fold difference in fatty acid biosynthesis?
Family: Arecaceae
Oil Palm
Date Palm
0 400 800 1200 1600 2000
Sucrose degradation
Glycolysis
OPPP
Plastid transporters
Fatty Acid Synthesis
PL & TAG Synthesis
Oil Palm
Date Palm
# ESTs/100,000 ESTs
Comparison of EST levels in oil and date palm for selected metabolic pathways
A >15-fold difference in fatty acid gene expression achieves a >100-fold difference in FA content?
(Data are average ESTs for five stages, in a pathway)
Sucrose
Pyruvate
Fatty acid
TAG
Generation of pyruvate – precursor for FA synthesis
• ESTs for some genes involved in providing plastid pyruvate were higher in oil palm than date palm
Plas dCytosol
G6P
F6P
F1,6P
1,3PG
3PGA
2PGA
PEP
Pyruvate
PFK
Aldolase
Enolase
PK
6.3
3
3.2
4
d-PGM
GAPC
PGK
3.2
1.5
3.2
1.3
G6P
F6P
F1,6P
DHAP
1,3PG
3PGA
2PGA
PEP
PYR
PFK
Enolase
PK
3.3
0.9
0.9
GAPC
GAP
0.6
0.8TPI
DHAP
GAP
TPI
GPT2
8.9
PPT13.7
DenovoFAMetabolism
Glycerolbackbonefor
TAGs
TPT
4.5
GPT1
0.7
Glu/FruGLT1
1.8
Sucrose
PFP
1.0
Glu
HK/FK
1.2HK
0.6
TCACycleinMitochondria
OPPPOPPP
StarchMetabolism
i-PGM
PGK
0.7
0.9
Sucrose Pyruvate Plastid
Cytosol
Oil palm ESTs/Date palm ESTs
• Upregulation of plastid transporters suggest that diversion of carbon to plastid to provide PEP/pyruvate may be crucial for high FA and TAG synthesis
Proportion of transcripts for plastid vs cytosol enzymes of glycolysis: Oil palm vs. Date palm
Higher proportion of gene expression in plastids of oil palm but not date palm suggests higher precursors for FA synthesis
Oil Palm Mesocarp
Date Palm Mesocarp
# o
f ES
Ts/1
00
,00
0 E
STS
0
100
200
300
PFK
PFP
FBA
TPI
Gap
C
PG
K
PG
M
ENO PK
0
100
200
300
400
500
PFK
PFP
FBA
TPI
Gap
C
PG
K
PG
M
ENO PK
PlastidCytosol
Glucose Pyruvate
Proportion of transcripts for plastid vs cytosol enzymes of glycolysis: Oil palm vs. Brassica
Pattern of EST distribution between the plastid and cytosol was similar between oil palm and brassica
Oil Palm Mesocarp
Brassica Embryo
# o
f ES
Ts/1
00
,00
0 E
STS
0
100
200
300
PFK
PFP
FBA
TPI
Gap
C
PG
K
PG
M
ENO PK
PlastidCytosol
0
60
120
180
PFK
PFP
FBA
TPI
Gap
C
PG
K
PG
M
ENO PK
Glucose Pyruvate
! "
ER
GPDH 1.0
Acetyl-CoA
KASIII
DHAP
Gly3P
LPA
PA
DAG
TAG
GPAT9
LPAAT
PAP
DGAT
Acyl-CoA Pool
PDCT
CPT
18:1-PC
18:2-PC
FAD2
PC
PC Pool
PDAT
LPC
FA
LPC
Malonyl-CoA ACCase
MMCT
Malonyl-ACP
16:0-ACP 18:0-ACP KASII
FATB
18:1-ACP SAD
FATA
LACS9
KAR
HD
ENR
ACP
6 cycles
KASI
LACS4 ?
Plastid
0.8
1.3
0.5
2.0 4.9
130
1.4 3.7
5 17
29 4
17
43
72
6
7
17
15
BCCP1
11
0.6
PLA2
3
Pyruvate PDC
42
LPCAT
7 cycles
Acyl -ACP
0.1
#$%&"&'"( ) *+",- ./"0$/1 "2"3$45"0$/1 6"
ESTs for all fatty acid synthesis proteins were up-regulated in oil palm, relative to date palm
ESTs for plastid FAS proteins were avg. 15-fold higher in oil palm than date palm, at last 2 stages
0
15
30
45
S1 S2 S3 S4 S5
% o
f FA
EST
s Mesocarp Stages
And increased by ~ 7-fold during 5 stages oil palm development
Oil palm ESTs/Date palm ESTs
A
ER
GPDH 1.0
DHAP
Gly3P
LPA
PA
DAG
TAG
GPAT9
LPAAT
PAP
DGAT
Acyl-CoAPool
PDCT
CPTPCPool
PDAT
LPC
LACS9
0.8
1.3
0.5
2.0
1.43.7
0.6
3
LPCAT
0.1
Fa yAcid
Ra oofESTs(Oilpalm:Datepalm)
Unexpectedly, ESTs for most glycerolipid synthesis genes differed
by < 2-fold between the palms
0
20
40
60
80
100
120
ESTs
/En
zym
e
Fatty acidSynthesis
High oil accumulation in oil-rich tissues is associated with higher EST levels for proteins of pyruvate generation, FA synthesis, and perhaps DGAT.
Adapted from S. Baud, L. Lepiniec, Progress in Lipid Research (2010)
Sucrose Pyruvate Fatty Acid Cytosol & Plastid Plastid
Transcriptional regulation of FA biosynthesis in seeds
Transcriptional Regulation of Fatty Acid Synthesis – in Seeds
WRINKLED1 (WRI1)-like gene in oil palm
WRI1-like was > 50-fold higher in oil palm than in date palm
Several WRI1-associated transcripts are expressed higher in oil palm than date palm . For example: pPK, PDH, KAS1, ACCase etc.
At WRI1
Oil Palm WRI-Like
Gene (bp) 3944 3490
Protein (aa) 430 337
# Exons 7 7
Oil Palm WRI1-Like
Developing stages of mesocarp
% O
il co
nte
nt
Ge
ne
exp
ress
ion
0
10
20
30
0
25
50
75
100
DP- % Oil
DP-WRI1
EgWRI1 complements the “wrinkled” feature of wri1-1 seeds
Ma et al., unpublished
Regulation of oil biosynthesis in non-seed tissues
Seed Oil Non-Seed Oil No Oil
Gene Family Annotation Br Em Ca En OP Me Avo Me DP Me
AT3G54320 AP2 WRI (WRINKLED 1) 15 61 15 21 0
Gene expression in Plastid > Cytosol >10-fold ~2-fold
Source
Sucrose Pyruvate TAG Fatty Acid
Sink
Transporters >3-fold
WRI-like >50-fold
??? LEC-like
TFs X
Summary High oil accumulation in seed and non-seed tissues is
associated with higher EST levels for proteins of FAS, plastid transporters and plastid glycolysis
Except for DGAT2, ESTs for enzymes of glycerolipid synthesis, OPPP, cytosolic glycolysis were similar in oil and non-oil rich tissues
Higher EST levels for WRI1 in most oil-rich tissues suggests WRI1 as a major control for oil production
Absence of LEC1, LEC2, FUS3 and other seed transcription factors in non-seed tissues suggests alternate regulators for WRI1
Monocot WRI1 complements the function of a dicot WRI1
On going work
• Characterization of WRI1 gene regulation in non-seed tissues
• Characterization and validation of additional transcription factors
• Elucidation of regulation of acyltransferases in non-seed tissues
• Understanding coordinated lipid accumulation and fruit development (avocado)
Acknowledgements
Support team @ • Joint Genome Institute, CA
• Research Technology Support Facility, MSU, MI
• Keithanne Mockaitis, Indiana University
Vincent Arondel CNRS-Bordeaux
John Ohlrogge MSU-East Lansing
Xia Cao MSU-East Lansing
Project members • Fabienne Bourgis, CNRS, France – Oil palm
• Georges-Frank Ngando-Ebongue, CEREPAH, Cameroon – Oil palm
• Noureddine Drira, Laboratoire de Biotechnologie Vegetale, Tunisia – Date palm
• Adrian Troncoso-Ponce, Centre for Research in Agricultural Genomics, Spain – Oilseeds
• Tim Durrett, Kansas State University, USA – Oilseeds
• Mary Lu Arpaia, Kearney Ag. Center, Parlier, CA – Avocado fruits
• Wei Ma, Michigan State University, USA – EgWRI 1 analysis
http://www.etsu.edu/