Sollapura Vishwanath PhD Candidate Department of Biology and Institute of Biochemistry Carleton University Ottawa, CANADA.

Biosynthesis and functions of free and combined fatty alcohols

associated with suberin

Dr. Frédéric Domergue (CNRS, Univ. of Bordeaux)

Dr. Dylan Kosma (Dr. M. Pollard/Dr. J. Ohlrogge)

Dr. Owen Rowland

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Collaborating Laboratories:

Free and Combined Primary Fatty Alcohols

R (H, OH, or O-CH3)

• Seed Storage

(jojoba)

• Cuticular waxes • Tuber periderm waxes

• Root waxes

Synthesis of Primary Fatty Alcohols

SURFACE LIPID POLYMERS

• Suberin (e.g. root, seed coat, wound-induced)

• Cutin

• Sporopollenin

O

S CoA or ACP

O

H

n

n

OH n

fatty acyl-CoA or -ACP

unreleased fatty aldehyde intermediate

FATTY ALCOHOLS

Fatty Acyl

Reductase

(FAR)

Rowland and Domergue (2012)

Alcohol-Forming Fatty Acyl Reductases (FARs)

NAD(P)H Binding Rossmann-

Fold Domain

FAR_C

Domain

GXXGXX(G/A) YXXXK

Core Enzyme (~490-500 a.a.)

+/-

N-terminal

Extension

(~50-120 a.a)

Pre

dic

ted

Pla

stid-

Lo

ca

lize

d F

AR

s

Pre

dic

ted

En

do

pla

sm

ic R

eticulu

m-

Lo

ca

lize

d F

AR

s

Jojoba FAR

Poplar FAR3

Arabidopsis FAR3/CER4

Soybean FAR1

Artemisia GFAR1

Arabidopsis FAR7

Arabidopsis FAR1

Arabidopsis FAR4

Arabidopsis FAR5

Arabidopsis FAR8

Rice FAR3

Rice FAR4

Wheat TAA1a

Rice FAR1

Arabidopsis FAR6

Poplar FAR1

Physcomitrella FAR1

Rice FAR2/DPW

Brachypodium FAR2

Poplar FAR2

Arabidopsis FAR2/MS2

Brassica rapa MS2

Euglena FAR

100

100

100

100

61

100

51

100

26

99

99

95

78

9975

88

34

99

32

19

0.1Rowland and Domergue (2012)

35S:FAR1-GFP

Three Types of Extracellular Biopolymers in Plants

•Endodermis & Peridermis of roots. • Aerial plant surfaces. • Exine of pollen grains.

1. Cuticle (Cutin+Waxes) 2. Sporopollenin 3. Suberin

Franke et al., 2005

Protective barriers against water loss, pathogens and abiotic stresses

Sites of Suberin Deposition in Plants

1. Endodermis in roots

2. Peridermis of underground storage tubers

3. Phellogen cambium in bark tissue

4. Chalazae plug of seeds and seed coat integuments

5. Wound sites

CYP86A1/horst-1 (Hofer et al., 2008)

Wound site

Main Components of Suberin

Monomer type Abundance

(%)

Unsubstituted fatty acids (C18:0 to C24:0)

w-Hydroxy fatty acids (C18:1, C16:0 to C26:0)

a,w- Dicarboxylic acids (C18:1, C18:2, C16:0)

Epoxy-fatty acids (C18:0, C18:1)

Polyhydroxy- a,w- Dicarboxylic acids (C18:0)

Fatty Alcohols (C18:0 to C22:0)

Glycerol

Phenolics (Ferulates, Coumarates, Caffeates)

1-10%

11-43%

24-45%

0-30%

0-8%

1-10%

14-26%

0-10%

(Pollard et al., 2008)

Eight Member Arabidopsis FAR Protein Family

(Doan et al., 2011)

(Chen et al., 2011)

Gene Expression Patterns of FAR1, FAR4, and FAR5

5 weeks old matured root

Single mutants of FAR1, FAR4, and FAR5 are each differentially affected in primary alcohol levels associated with Suberin

Domergue et al., (2010)

Arabidopsis FAR1, FAR4, and FAR5: Suberin-Associated FARs

Fatty Alcohol Distribution in Roots

Fatty Alcohol Composition

Root Waxes Soluble Lipids (delipidation)

Suberin Polymer

Root waxes- Compounds extracted by rapid dipping of roots in solvent for 30 sec.

Soluble Lipids- Exhaustive solvent extractions of all the soluble lipids except the ones that are part of suberin polymer.

Suberin polymer- Poly-aliphatic and poly-aromatic compounds remaining in the delipidated roots.

FA- Fatty Acids

VLCFA- Very Long Chain Fatty Acids

DCA- Dicarboxylic Acids

wOH- OmegaHydroxy Acids 2-OH- 2-hydroxy (VLC) fatty acids Fatty OH- Fatty Alcohols

Suberin polyester monomers and lipids found in the soluble fraction of 4-weeks old Col-WT roots

0.0020.0040.0060.0080.00

100.00

% Suberin polymer

0.0020.0040.0060.0080.00

100.00

% in Soluble Lipid Fraction

Fatty Alcohols composition of root suberin in 4-weeks old far mutant plants

mg

/mg

DR

* Denotes the lines with FAR5amiRNA

0

0.05

0.1

0.15

0.2

0.25

C18:0-OH

C20:0-OH

C22:0-OH

Soluble Fatty Alcohols (Delipidation fraction)

mg

/mg

DW

* Denotes the lines with FAR5amiRNA

Soluble fraction from delipidation of roots from 4-weeks old Arabidopsis plants

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

18OH

20OH

22OH

Reduced levels of alkyl hydroxycinnamates (AHCs) in root

waxes of double and triple far1/far4/far5 mutants

Chloroform-soluble waxes from roots of 7-week old Arabidopsis plants.

0

10

20

30

40

50

60

70

80

90

C18 C20 C22 C18 C20 C22 C18 C20 C22 C18 C20 C22

Alkan-1-ols Coumarates Ferulates Caffeates

ColWT

far1far4

far1far5

far4far5*

far1far4far5*

mg

/g F

W

AHCs:

(R=H) (R=O-CH3) (R=OH)

Fatty Alcohols composition of seed coat Suberin in far mutants

mg

/mg

DR

* Denotes the lines with FAR5amiRNA

0

0.05

0.1

0.15

0.2

0.25

0.3

C18:0-OH

C20:0-OH

C22:0-OH

Alterations in Seed Coat Permeability

Time course of formazans produced by reduction of tetrazolium salts by the embryos of Arabidopsis

wild-type (WT) and mutants, measured by absorbance at 485 nm.

Incubation Time (hrs)

Ab

sorb

ance

at

48

5 n

m

0.000

1.000

2.000

3.000

4.000

5.000

6.000

4 hrs 24 hrs 48 hrs 72 hrs 120 hrs

Col-WT

far1far4

far1far5

far4far5*

far1far4far5*

#REF!

gpat5

Alterations in seed germination in response to

abscisic acid (ABA)

measured 4 days after sowing. * Denotes the lines with FAR5amiRNA

Ge

rmin

atio

n (

%)

mM ABA

mM ABA

mM ABA

mM ABA

0

20

40

60

80

100

120

MS+0

MS+0.5

MS+1.0

MS+10

Summary

• Arabidopsis FAR1, 4 and 5 generate C18:0-OH - C22:0-OH fatty alcohols associated with Suberin.

• Small proportion of C18-C22 fatty alcohol are part of suberin polymer in Arabidopsis.

• The triple mutant far1 far4 far5 has reduced levels of all three C18:0 to C22:0 primary fatty alcohols in the soluble fraction and the root and seed coat suberin.

• The triple mutants have reduced levels of C18-C22 AHCs in the suberin associated root waxes.

• The triple mutant seeds were found to have increased seed coat permeability to tetrazolium salts and had increased sensitivity to abscisic acid (ABA) hormone during germination.

Questions

How are bound fatty alcohols associated / integrated within the suberin polymer?

Are the unbound fatty alcohols and AHCs associated with the suberin polymer?

What roles do suberin-bound fatty alcohols, free fatty alcohols and alkyl hydroxycinnamates have in protection against biotic and abiotic stresses?

How are suberin-associated FAR genes regulated?

Acknowledgments

Ian Pulsifer

Rowland Lab

Dr. Frédéric Domergue (CNRS, Univ. of Bordeaux)

Dr. Dylan Kosma (Dr. M. Pollard/Dr. J. Ohlrogge)

Supervisor: Dr. Owen Rowland

Lab

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B I O G E N E S E MEMBRANAIRELab

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B I O G E N E S E MEMBRANAIRE