S5 O6 Biosynthesis and functions of free and combined fatty

Biosynthesis and functions of free
and combined fatty alcohols
associated with suberin
Sollapura Vishwanath
PhD Candidate
Department of Biology and Institute of Biochemistry
Carleton University
Ottawa, CANADA.
Collaborating Laboratories:
Dr. Frédéric Domergue
(CNRS, Univ. of Bordeaux)
Laboratoire
Dr. Owen Rowland
BIO
GENESE
MEMBRANAIRE
Dr. Dylan Kosma
(Dr. M. Pollard/Dr. J. Ohlrogge)
Free and Combined Primary Fatty Alcohols
R (H, OH, or O-CH3)
• Cuticular waxes
• Seed Storage
(jojoba)
• Tuber periderm waxes
• Root waxes
Synthesis of Primary Fatty Alcohols
O
S
n
fatty acyl-CoA or -ACP
O
Fatty Acyl
Reductase
(FAR)
n
H
unreleased fatty aldehyde intermediate
n
FATTY ALCOHOLS
Rowland and Domergue (2012)
CoA or ACP
OH
SURFACE LIPID POLYMERS
• Suberin (e.g. root, seed coat, wound-induced)
• Cutin
• Sporopollenin
Alcohol-Forming Fatty Acyl Reductases (FARs)
N-terminal
Extension
(~50-120 a.a)
NAD(P)H Binding RossmannFold Domain
Jojoba FAR
Poplar FAR3
32
Arabidopsis FAR3/CER4
99
Soybean FAR1
Artemisia GFAR1
88
Arabidopsis FAR7
Arabidopsis FAR1
99
99
Arabidopsis FAR4
75
78
Arabidopsis FAR5
100
Arabidopsis FAR8
Rice FAR3
Rice FAR4
95
Wheat TAA1a
100
61
Rice FAR1
Arabidopsis FAR6
26
Poplar FAR1
Physcomitrella FAR1
99
Rice FAR2/DPW
100
51
Brachypodium FAR2
Poplar FAR2
100
Arabidopsis FAR2/MS2
100
100
Brassica rapa MS2
Euglena FAR
34
19
FAR_C
Domain
Core Enzyme (~490-500 a.a.)
35S:FAR1-GFP
Rowland and Domergue (2012)
0.1
Predicted Endoplasmic ReticulumLocalized FARs
+/-
YXXXK
Predicted PlastidLocalized FARs
GXXGXX(G/A)
Three Types of Extracellular Biopolymers in Plants
1. Cuticle (Cutin+Waxes)
2. Sporopollenin
3. Suberin
Franke et al., 2005
• Aerial plant surfaces.
• Exine of pollen grains.
•Endodermis & Peridermis of roots.
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
Wound site
CYP86A1/horst-1 (Hofer et al., 2008)
Main Components of Suberin
Monomer type
Abundance
(%)
Unsubstituted fatty acids (C18:0 to C24:0)
1-10%
w-Hydroxy fatty acids (C18:1, C16:0 to C26:0)
11-43%
a,w- Dicarboxylic acids (C18:1, C18:2, C16:0)
24-45%
Epoxy-fatty acids (C18:0, C18:1)
0-30%
Polyhydroxy- a,w- Dicarboxylic acids (C18:0)
0-8%
Fatty Alcohols (C18:0 to C22:0)
1-10%
Glycerol
14-26%
Phenolics (Ferulates, Coumarates, Caffeates)
0-10%
(Pollard et al., 2008)
Eight Member Arabidopsis FAR Protein Family
(Chen et al., 2011)
(Doan 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.
Suberin polyester monomers and lipids found in the soluble
fraction of 4-weeks old Col-WT roots
% Suberin polymer
FA- Fatty Acids
100.00
80.00
60.00
40.00
20.00
0.00
VLCFA- Very Long Chain
Fatty Acids
DCA- Dicarboxylic Acids
% in Soluble Lipid Fraction
100.00
80.00
60.00
40.00
20.00
0.00
wOH- OmegaHydroxy
Acids
2-OH- 2-hydroxy (VLC)
fatty acids
Fatty OH- Fatty Alcohols
Fatty Alcohols composition of root suberin in
4-weeks old far mutant plants
0.25
mg/mg DR
0.2
0.15
C18:0-OH
0.1
C20:0-OH
C22:0-OH
0.05
0
* Denotes the lines with FAR5amiRNA
Soluble Fatty Alcohols (Delipidation fraction)
4.00
3.50
3.00
mg/mg DW
2.50
2.00
18OH
1.50
20OH
1.00
22OH
0.50
0.00
* Denotes the lines with FAR5amiRNA
Soluble fraction from delipidation of roots from 4-weeks old Arabidopsis plants
Reduced levels of alkyl hydroxycinnamates (AHCs) in root
waxes of double and triple far1/far4/far5 mutants
90
80
70
AHCs:
mg/g FW
60
ColWT
50
far1far4
40
far1far5
30
far4far5*
20
far1far4far5*
10
0
C18 C20 C22
Alkan-1-ols
C18 C20 C22
Coumarates
(R=H)
C18 C20 C22
C18 C20 C22
Ferulates
(R=O-CH3)
Caffeates
(R=OH)
Chloroform-soluble waxes from roots of 7-week old Arabidopsis plants.
Fatty Alcohols composition of seed coat Suberin in
far mutants
0.3
0.25
mg/mg DR
0.2
0.15
C18:0-OH
C20:0-OH
0.1
C22:0-OH
0.05
0
* Denotes the lines with FAR5amiRNA
Alterations in Seed Coat Permeability
6.000
Absorbance at 485 nm
5.000
Col-WT
4.000
far1far4
far1far5
3.000
far4far5*
far1far4far5*
2.000
#REF!
gpat5
1.000
0.000
4 hrs
24 hrs
48 hrs
72 hrs
120 hrs
Incubation Time (hrs)
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.
Alterations in seed germination in response to
abscisic acid (ABA)
120
100
Germination (%)
80
60
MS+0 mM ABA
MS+0.5 mM ABA
40
MS+1.0 mM ABA
MS+10 mM ABA
20
0
* Denotes the lines with FAR5amiRNA
measured 4 days after sowing.
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
Supervisor: Dr. Owen Rowland
Rowland Lab
Ian Pulsifer
Laboratoire
Dr. Frédéric Domergue (CNRS, Univ. of Bordeaux)
BIO
GENESE
MEMBRANAIRE
Dr. Dylan Kosma (Dr. M. Pollard/Dr. J. Ohlrogge)