Biosynthesis and functions of free and combined fatty alcohols associated with suberin

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1 Laboratoire Biosynthesis and functions of free and combined fatty alcohols associated with suberin Collaborating Laboratories: Dr. Owen Rowland Sollapura Vishwanath PhD Candidate Department of Biology and Institute of Biochemistry Carleton University Ottawa, CANADA. Dr. Frédéric Domergue (CNRS, Univ. of Bordeaux) Dr. Dylan Kosma (Dr. M. Pollard/Dr. J. Ohlrogge) B I O G E N E S E MEMBRANAIRE

2 Free and Combined Primary Fatty Alcohols R (H, OH, or O-CH 3 ) Cuticular waxes Seed Storage (jojoba) Tuber periderm waxes Root waxes

3 Synthesis of Primary Fatty Alcohols O Fatty Acyl Reductase (FAR) n fatty acyl-coa or -ACP O n unreleased fatty aldehyde intermediate S H CoA or ACP n FATTY ALCOHOLS OH Rowland and Domergue (2012) SURFACE LIPID POLYMERS Suberin (e.g. root, seed coat, wound-induced) Cutin Sporopollenin

4 Predicted Plastid- Localized FARs Predicted Endoplasmic Reticulum- Localized FARs Alcohol-Forming Fatty Acyl Reductases (FARs) GXXGXX(G/A) YXXXK +/- N-terminal Extension (~ a.a) NAD(P)H Binding Rossmann- Fold Domain Core Enzyme (~ a.a.) 35S:FAR1-GFP FAR_C Domain 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 100 Rice FAR2/DPW Brachypodium FAR2 Poplar FAR2 100 Arabidopsis FAR2/MS2 100 Brassica rapa MS2 Euglena FAR Rowland and Domergue (2012) 0.1

5 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

6 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)

7 Main Components of Suberin Monomer type 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) Abundance (%) 1-10% 11-43% 24-45% 0-30% 0-8% 1-10% 14-26% 0-10% (Pollard et al., 2008)

8 Eight Member Arabidopsis FAR Protein Family (Chen et al., 2011) (Doan et al., 2011)

9 Gene Expression Patterns of FAR1, FAR4, and FAR5 5 weeks old matured root

10 Single mutants of FAR1, FAR4, and FAR5 are each differentially affected in primary alcohol levels associated with Suberin Domergue et al., (2010)

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

12 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.

13 Suberin polyester monomers and lipids found in the soluble fraction of 4-weeks old Col-WT roots % Suberin polymer FA- Fatty Acids VLCFA- Very Long Chain Fatty Acids DCA- Dicarboxylic Acids % in Soluble Lipid Fraction woh- OmegaHydroxy Acids 2-OH- 2-hydroxy (VLC) fatty acids Fatty OH- Fatty Alcohols

14 mg/mg DR 0.25 Fatty Alcohols composition of root suberin in 4-weeks old far mutant plants C18:0-OH C20:0-OH C22:0-OH 0 * Denotes the lines with FAR5amiRNA

15 mg/mg DW Soluble Fatty Alcohols (Delipidation fraction) OH 20OH 22OH * Denotes the lines with FAR5amiRNA Soluble fraction from delipidation of roots from 4-weeks old Arabidopsis plants

16 mg/g FW Reduced levels of alkyl hydroxycinnamates (AHCs) in root waxes of double and triple far1/far4/far5 mutants AHCs: C18 C20 C22 C18 C20 C22 C18 C20 C22 C18 C20 C22 Alkan-1-ols Coumarates Ferulates Caffeates (R=H) (R=O-CH 3 ) (R=OH) ColWT far1far4 far1far5 far4far5* far1far4far5* Chloroform-soluble waxes from roots of 7-week old Arabidopsis plants.

17 mg/mg DR 0.3 Fatty Alcohols composition of seed coat Suberin in far mutants C18:0-OH C20:0-OH C22:0-OH 0 * Denotes the lines with FAR5amiRNA

18 Absorbance at 485 nm Alterations in Seed Coat Permeability Col-WT far1far4 far1far5 far4far5* far1far4far5* #REF! gpat 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.

19 Germination (%) 120 Alterations in seed germination in response to abscisic acid (ABA) MS+0mM ABA MS+0.5 mm ABA MS+1.0 mm ABA MS+10 mm ABA 0 * Denotes the lines with FAR5amiRNA measured 4 days after sowing.

20 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.

21 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?

22 Laboratoire Supervisor: Dr. Owen Rowland Acknowledgments Rowland Lab Ian Pulsifer Dr. Frédéric Domergue (CNRS, Univ. of Bordeaux) B I O G E N E S E MEMBRANAIRE Dr. Dylan Kosma (Dr. M. Pollard/Dr. J. Ohlrogge)