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/