MPS 587 - Advanced Plant Biochemistry Course

Fall Semester 2011

Lecture 11

Lipids III

9. Triacylglycerol synthesis 10. Engineering triacylglycerol fatty acid composition

Today’s topics on the Arabidopsis node map

2

Tissue and function of triacylglycerol accumulation in plants

1. Seed, storage oil – energy for germinating seedlings

1. Soybeans

2. Brassica napus (Canola)

2. Fruit, animal attractant?

1. Avocado mesocarp

2. Palm fruit

3. Leaf, intermediate in lipid breakdown during senescence

4. Anthers and pollen, may be used for energy in pollen tube growth

3

Lipids in oil bodies (oleosomes)

• Contain TAGs.

• Surrounded by protein-rich membrane; size 1-2 um.

• ER contains biosynthetic enzymes; oil bodies are spun off from ER microdomains.

• Oleosins: 15-25 kDa proteins; conserved hydrophobic core; amphipathic flanking sequences; present only in seed and pollen.

4

Plant triacylglycerol biosynthesis – overview

Malonyl-CoA

Acyl-ACP (16:0;18:0)

Acyl-ACP (18:1)

Acetyl-CoA

ACCase

FAS Desaturase

PC

Pyruvate

LACS Acyl-CoA

Thioesterase

FA

G3P

LPA

PA

DAG

PLASTID

PAP

ACYL EDITING LPC

ENDOPLASMIC RETICULUM

FA SYNTHESIS

LIP

ID A

SSEM

BLY

FA MODIFICATION • Lipid linked desaturases

• Acyl-CoA elongases

DAG

TAG

TAG

Oil body

TAG

DGAT

PDAT

GPAT

LPAT

5

Enzymes of triacylgycerol (TAG) synthesis

Acyl-CoA CoA

1,2-DAG

Acyl-CoA : Diacylglycerol Acyltransferase (DGAT)

Phospholipid : Diacylglycerol Acyltransferase (PDAT)

TAG

OH

OH

1,2-DAG

P-Headgroup P-Headgroup

OH

TAG Phospholipid 2-lyso-phospholipid

+ +

Diacylglycerol Transacylase

OH

1,2-DAG

OH

1,2-DAG TAG

OH

2-monoacylglycerol

+ + OH

Three classes of plant DGAT • DGAT1 and DGAT2 are found in many eukaryotes and greatly differ in structure. • DGAT3 is soluble and has only been characterized in peanut seeds.

Enzymatic activity demonstrated in safflower seeds. The enzyme has yet to be isolated.

Identified in plants and yeast, PC appears to be the preferred substrate in plants while PE is the preferred substrate in yeast

(Li-Beisson Y et. al. (2010) Acyl-Lipid Metabolism. The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133)

(Dahlqvist et al. (2000) Proc. Natl. Acad. Sci. USA 97: 6487 )

(Stobart et al. (1997) Planta 203: 58)

6

Schematic representations of murine DGAT1 and

DGAT2 proteins.

(Yen et al. (2008) Journal of Lipid Research 49: 2283)

DGAT1 and DGAT2 have different structures and may not have overlapping functions

Major DGAT classes involved in TAG synthesis in plants: • Arabidopsis: DGAT1 • Brassica napus: DGAT1 • Castor bean: DGAT2 • Tung Tree: DGAT2

• DGAT1 and DGAT2 localize to separate subdomains of the ER

7

Malonyl-CoA

Acyl-ACP (16:0;18:0)

Acyl-ACP (18:1)

Acetyl-CoA

ACCase

FAS Desaturase

PC

Pyruvate

LACS Acyl-CoA

Thioesterase

FA

G3P

LPA

PA

DAG

PLASTID

PAP

ACYL EDITING LPC

ENDOPLASMIC RETICULUM

FA SYNTHESIS

LIP

ID A

SSEM

BLY

FA MODIFICATION • Lipid linked desaturases

• Acyl-CoA elongases

DAG

TAG

TAG

Oil body

TAG

DGAT

PDAT

GPAT

LPAT

DGAT1 and PDAT1 are the only confirmed acyltransferases known in Arabidopsis TAG synthesis

8

Determination of the enzymes responsible for triacylglycerol synthesis in Arabidopsis

• DGAT1 mutant has ~20% reduction in seed TAG

(Katavic et al. (1995) Plant Physiology 108: 399)

•DGAT2 cannot complement yeast DGAT knockouts as DGAT1 does • http://arabidopsisacyllipids.plantbiology.msu.edu/enzymes/105

•PDAT1 mutant has no effect on amounts of seed TAG

(Mhaske et al. (2005) Plant Physiology and Biochemistry 43: 413)

•DGAT1/PDAT1 double mutant is pollen lethal

•dgat1/dgat1 + PDAT1 RNAi or pdat1/pdat1 + DGAT1 RNAi has 70-80% reduction in seed oil content

(Zhang et al. (2009) The Plant Cell 21: 3885)

9

The source of DAG for TAG synthesis may differ between plants and effect the TAG fatty acid composition

Malonyl-CoA

Acyl-ACP (16:0;18:0)

Acyl-ACP (18:1)

Acetyl-CoA

ACCase

FAS Desaturase

PC

Pyruvate

LACS Acyl-CoA

Thioesterase

FA

G3P

LPA

PA

DAG

PLASTID

PAP

ACYL EDITING LPC

ENDOPLASMIC RETICULUM

FA SYNTHESIS

LIP

ID A

SSEM

BLY

FA MODIFICATION • Lipid linked desaturases

• Acyl-CoA elongases

DAG

TAG

TAG

Oil body

TAG

DGAT

PDAT

GPAT

LPAT

10

DAG source can affect TAG composition

P P P GPAT LPAAT PAP

G3P LPA PA de novo DAG

PC derived DAG

P

Cho PC

desaturation, hydroxylation,

acyl editing, etc.

Acyl-CoA pool

DGAT PDAT

TAG

DGAT PDAT

TAG

11

Reduced oleate desaturation mutnat (rod1) encodes an enzyme that affects the flux of DAG through PC into TAG

Phosphatidylcholine : Diacylglycerol CholinephosphoTransferase (PDCT)

P

Cho 1,2-DAG

P

Cho 1,2-DAG

+ +

(Lu et al. (2009) Proceedings of the National Academy of Sciences in the USA 106: 18837)

PC PC

12

Determine relative flux through competing TAG biosynthesis pathways with [14C]glycerol labeling

P P P GPAT LPAAT PAP

G3P LPA PA de novo DAG

DGAT PDAT

TAG

DGAT PDAT

PC derived DAG

TAG

P

Cho PC

CPT PDCT

PDCT

rCPT

PLC PLD/PAP

[14C]glycerol

13

P P P GPAT LPAAT PAP

G3P LPA PA de novo DAG

DGAT PDAT

TAG

DGAT PDAT

PC derived DAG

TAG

P

Cho PC

CPT PDCT

PDCT

rCPT

PLC PLD/PAP

[14C]glycerol

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35

DP

M/

tota

l lip

id F

AM

E

PC DAG

TAG

Flux of glycerol through TAG synthesis in WT Arabidopsis (Bates and Browse (2011) The Plant Journal, in press)

14

Flux of glycerol through TAG synthesis in WT Arabidopsis

P P P GPAT LPAAT PAP

G3P LPA PA de novo DAG

DGAT PDAT

TAG

DGAT PDAT

PC derived DAG

TAG

P

Cho PC

CPT PDCT

PDCT

rCPT

PLC PLD/PAP

[14C]glycerol

14x

1x

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35

DP

M/

tota

l lip

id F

AM

E

PC DAG

TAG

>90% of de novo DAG fluxes through PC prior to TAG synthesis

(Bates and Browse (2011) The Plant Journal, in press)

15

Flux of DAG through PC for TAG synthesis may depend on plant species

Seed Oil content (weight %)

Arabidopsis yes ~20% ~37%

Castor bean No ~90% ~50%

Use of PC-derived DAG

Non-membrane lipid FA in TAG

Soybean yes no ~20%

Plant

(Bafor et al. (1991) Biochemical Journal 280: 507)

(Bates & Browse (2011) The Plant Journal, in press)

(Bates et al. (2009) Plant Physiology 150: 55)

16

World vegetable oil production: Four major crops

0

10

20

30

40

50

60

70

16:0 18:0 18:1 18:2(n-6) 18:3(n-3)

Pe

rce

nt

fatt

y ac

id Palm

0

10

20

30

40

50

60

70

16:0 18:0 18:1 18:2(n-6) 18:3(n-3)

Pce

cen

t fa

tty

acid

Soybean

0

10

20

30

40

50

60

70

16:0 18:0 18:1 18:2(n-6) 18:3(n-3)

Pe

rce

nt

fatt

y ac

id

Canola

0

10

20

30

40

50

60

70

16:0 18:0 18:1 18:2(n-6) 18:3(n-3)

Pe

rce

nt

fatt

y ac

id Sunflower

(http://lipidlibrary.aocs.org/market/fourmain.htm) (http://www.scientificpsychic.com/fitness/fattyacids1.html)

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(http://www.soystats.com/)

World vegetable oil consumption 2010 146.9 Million Metric Tons

U.S. Fats & Oils Edible Consumption 2010 9.64 Million Metric Tons

U.S. Soybean Oil Consumption 2010 7.54 Million Metric Tons

~ US petroleum based Fuel consumption 2009 770 Million Metric Tons

Vegetable oil consumption

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http://oilreset.com/blog/?p=159

Vegetable oil can replace petroleum for the chemical industry

19

Uses of vegetable oil and reasons for engineering fatty acid content

Food oils, “Healthy” oils

•Low in saturated fatty acids •High in ω-3 polyunsaturated fatty acids •Fish type oils

•EPA and DHA

Good cooking properties •Low polyunsaturated fatty acids •High in 18:1

Industrial applications •Medium-chain fatty acids (12:0): soaps, detergents, surfactants •Long-chain (22:1): Lubricants, slip agents •Epoxy fatty acids: Plasticizers, coatings, paints, •Hydroxy fatty acids: Lubricants, polymers

Engineering Strategies

• Adjust proportions of endogenous fatty acids in crop plants •Introduce new fatty acids into crop plants

20

Breeding changed rapeseed (Brassica napus) into Canola

CANOLA = CANadian Oil Low Acid

Natural isolates of a β–ketoacyl-CoA synthase used to remove Erucic acid (22:1) content

LEAR Low Erucic Acid Rapeseed HEAR High Erucic Acid Rapeseed

21

https://www.vistivegold.com/Pages/About.aspx

New GM soybeans with improved cooking oil properties from Monsanto about to hit the market

Vistive® Gold soybeans have advanced to Phase IV (pre-launch), the final step within the R&D pipeline. Monsanto has recently completed submissions to the USDA and FDA in support of the trait. The U.S. Food and Drug Administration has issued a positive response letter to Monsanto’s Generally Recognized as Safe notification. The FDA’s letter supports the use of Vistive® Gold under its intended uses.

• Higher oxidative stability • Reduced polymerization during frying

• No need for hydrogenation • Therefore no trans-fats

• Less saturated fat

22

Production of low saturate high oleic soybeans by Monsanto

Malonyl-CoA

Acyl-ACP (16:0;18:0)

Acyl-ACP (18:1)

Acetyl-CoA

ACCase

FAS Desaturase

PC

Pyruvate

LACS Acyl-CoA

Thioesterase

FA

G3P

LPA

PA

DAG

PAP

ACYL EDITING LPC

ENDOPLASMIC RETICULUM

FA SYNTHESIS

LIP

ID A

SSEM

BLY

DAG

TAG

TAG

Oil body

TAG

DGAT

PDAT

GPAT

LPAT

18:1-PC

18:2-PC

18:3-PC

FAD2

FAD3

FA MODIFICATION • Lipid linked desaturases

FATb

•Conventional breeding FAD3 mutant • FAD3: desaturase 18:2 to 18:3

•RNAi FAD2 • FAD2: 18:1 to 18:2 •Only wanted a semi KD therefore specifically targeted 2 of 4 FAD2 isoforms

•RNAi FATb •FATb: 16:0-ACP thioesterase

• Reduces 16:0 and 18:0 fatty acid release from fatty acid synthesis, and promotes production of more 18:1

23

Vernolic acid

Ricinoleic acid

Eleostearic acid

Uses

• Laxatives • Chemotherapy carrier • Lubricants • Polymers • Fuel additives

• Plastics • Adhesives • Paints

• Drying oils • Varnishes • Paints

Ricinus communis (Castor bean)

Vernonia galamensis (Iornweed)

Vernicia fordii (Tung tree)

Industrially useful fatty acids are found throughout the plant kingdom but not always in suitable crop plants

Agronomic features

•~50% oil by weight in seeds •Highly toxic co-product Ricin •Grows in the sub-tropics

Fatty acid

% unusual FA in oil

~90%

~80% •~40% oil by weight in seeds • only grows near the equator

~82%

•Slow growing tree •Only grows in the sub-tropics •Susceptible to hurricanes and condominium plantations

24

Ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid)

Engineering of industrial oils in Arabidopsis: Castor oil a case study

P

Cho

P

Cho

RcFAH12 ~ 90% ricinoleic acid

Phosphatidylcholine (PC)

TAG

RcFAH12 expressed in develoing Arabidopsis seeds, ~17% hydroxy fatty acids (HFA) mostly at sn-2 (Broun and Somerville (1997) Plant Physiology 113: 922) (Smith et al. (2003) Planta 217: 507) Lu et al. (2006) The Plant Journal 45: 847)

25

Malonyl-CoA

Acyl-ACP (16:0;18:0)

Acyl-ACP (18:1)

Acetyl-CoA

ACCase

FAS Desaturase

Pyruvate

LACS Acyl-CoA

Thioesterase

FA

G3P

LPA

PA

DAG

PAP

ACYL EDITING LPC

FA SYNTHESIS

LIP

ID A

SSEM

BLY

RcFAH12 •Hydryoxylation of sn-2 PC

DAG

TAG

TAG

Oil body

TAG

RcDGAT

RcPDAT

GPAT

LPAT

HFA specific TAG biosynthetic increase accumulation of HFA in Arabidopsis TAG and gave clues on HFA metabolism in Arabidopsis (Burgal et al. (2008) Plant Biotechnology Journal 6: 819)

(van Erp et al. (2011) Plant Physiology 155: 683)

PC-HFA •Co-expression of RcFAH12 with Castor DGAT or PDAT increased seed HFA content from 17% to 25-28% • Both RcDGAT and RcPDAT lower the amount of HFA that accumulate in PC

• HFA still mostly at sn-2 + sn-3 26

P P P GPAT LPAAT PAP

G3P LPA PA de novo HFA-DAG

DGAT PDAT

TAG PC derived HFA-DAG

P

Cho PC

CPT PDCT

PDCT

rCPT

PLC PLD/PAP

RcFAH12 sn-2

hydroxylation

HFA-CoA F.A.S.

1. RcFAH12 produces sn-2 HFA PC which can be converted to sn-2 HFA TAG 2. HFA can also be utilized for de novo DAG synthesis

1. HFA-de novo DAG is turned over 3. In efficient utilization of HFA leads to inhibition of FA synthesis and reduced oil 4. HFA specific sn-3 acyltransferases allow HFA-TAG accumulation

Flux analysis to determine bottlenecks in HFA TAG synthesis

[14C]glycerol

(Bates and Browse (2011) The Plant Journal, in press)

27

1. Inhibit enzymes that turnover HFA-containing de novo DAG 2. Allow de novo DAG to flux directly to TAG

P P P GPAT LPAAT PAP

G3P LPA PA de novo HFA-DAG

DGAT PDAT

TAG PC derived HFA-DAG

P

Cho PC

CPT PDCT

PDCT

rCPT

PLC PLD/PAP

RcFAH12 sn-2

hydroxylation

HFA-CoA

Future HFA engineering strategies

28