The Lipid Underground: Dissecting the Alkyl Hydroxycinnamate Pathway

Transcription

1 The Lipid Underground: Dissecting the Hydroxycinnamate Pathway Dylan K Kosma, Adam Rice, Isabel Molina, Owen Rowland, Frédéric Domergue, John Ohlrogge, Mike Pollard Department of Plant Biology, Michigan State University, East Lansing, Michigan USA Department of Biology, Algoma University, Sault Ste. Marie, Ontario, Canada Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada Laboratoire de Biogenèse Membranaire, Université Victor Ségalen Bordeaux 2, CNRS, Bordeaux cedex, France July 10,

2 What are Hydroxycinnamates? OCH3 O OH O Fatty Alcohol: VLCFA pathway Phenylpropanoid pathway Junction of two distinct biochemical pathways: Phenylpropanoid derived from Phenylalanine VLCFA ER-based elongation of Fatty Acyl-CoAs 2

3 What are Hydroxycinnamates? O O Docosyl p-coumarate Docosyl caffeate Docosyl ferulate O O O O OH OH OH OCH 3 OH 3

4 Where do we find Hydroxycinnamates? hydroxycinnamates are found in root and periderm waxes Thought to be associated with the surfaces of mature taproots (i.e. periderm) Extracted by rapid chloroform dipping of roots Represent a poorly understood aspect of root biology Role in protecting sink tissue? Höfer et al. (2008) Bush (2008) 4

5 Objectives To identify and characterize genes involved in alkyl hydroxycinnamate synthesis Explore the phylogenetic distribution of alkyl hydroxycinnamates in the plant kingdom 5

6 Identification of Candidate Genes Gene Name GPAT5 CYP86A1 ASFT CYP86B1 r 2 r 2 r 2 r 2 Σr 2 /n ASFT FACT DCF

7 outgroups CHAT A (V) B Phylogenetic Tree of Arabidopsis BAHD HXXXD Acyltransferases C (III) D HCT E (II) F (V) AT5MAT AACT1 CER2 G (I) DCF FACT ASFT J I H Molina et al (2009) FACT = Fatty Alcohol:Caffeoyl CoA Caffeoyl Transferase 7

8 fact Mutant Root Waxes are Deficient in Caffeates 8

9 Characterization of BAHD2 Activity Recombinant FACT Enzyme Assay Coupled Enzyme Assay HO O + CoASH OH OH ATP AMP + CoAS PPi O + OH OH OH FACT OH O O OH CoASH 9

10 Recombinant FACT Activity CL Specificity nmols product Caffeate Coumarate Ferulate % Acitivity Caffeate Coumarate Ferulate 10

11 SH

12 far1 Mutant Root Waxes are Deficient C22 Hydroxycinnamates 12

13 far4 Mutant Root Waxes are Deficient in C20 Hydroxycinnamates 13

14 far5 Mutant Root Waxes are Deficient C18 Hydroxycinnamates 14

15 C16:0-ACP Plastid 16:0 C16:0-CoA C18:0-ACP 18:0 ER Coumaroyl-CoA BAHD??? C18:0 Coumarate C20:0 Coumarate C22:0 Coumarate C18:0-CoA C20:0-CoA FAR5 FAR5 FAR1 FAR4 C18:0 1-Alcohol C20:0 1-Alcohol Caffeoyl-CoA Feruloyl-CoA FACT C18:0 Caffeate C20:0 Caffeate C22:0 Caffeate FAR4 FAR1 C22:0 1-Alcohol BAHD??? C18:0 Ferulate C20:0 Ferulate C22:0-CoA C22:0 Ferulate 15

16 Hydroxycinnmate Occurrence in Plant Kingdom Compound Class Organism Tissue Alcohol Chain Length Citation Caffeates Ferulates Coumarate Ferulates Caffeates Ferulates Coumarates Ferulates Coumarates Arabidopsis thaliana Solanum tuberosum Acacia sp. Root Surface (periderm) Periderm & Wound Periderm Periderm (bark) Musa textilis Leaf fibers C18 C22 C18 C31 Li et al (2007) Molina et al (2009) Bernard et al (1992) Schreiber et al (2004) Serra et al (2009) C16 C28 Freire et al (2007) C20 C28 Del Rio et al (2004) 16

17 Maize Beet Rice What s up doc? Carrot Root Wax Hydroxycinnamate Profiling Sweet Potato Tobacco Garden Pea Thellungiella Camelina Arabidopsis Radish Daikon Rapeseed 17 Rutabaga

18 100% % of Total Hydroxycinnamates 80% 60% 40% 20% 0% Caffeate Ferulate Coumarate 18

19 100% % of Total Hydroxycinnamates 80% 60% 40% 20% 0% Caffeate Ferulate Coumarate 19

20 100% % of Total Hydroxycinnamates 80% 60% 40% 20% 0% Caffeate Ferulate Coumarate 20

21 100% % of Total Hydroxycinnamates 80% 60% 40% 20% 0% Caffeate Ferulate Coumarate 21

22 100% % of Total Hydroxycinnamates 80% 60% 40% 20% 0% Caffeate Ferulate Coumarate 22

23 100% % of Total Hydroxycinnamates 80% 60% 40% 20% 0% Caffeate Ferulate Coumarate 23

24 Conclusions We have identified a new acyltransferase gene required for the synthesis of alkyl hydroxycinnamates. FACT encodes a caffeoyl transferase for alkyl caffeate synthesis FAR1,4,5 fatty acyl-coa reductases provide fatty alcohols for alkyl hydroxycinnamate synthesis We have demonstrated biosynthetic overlap between suberin and root waxes We found that root wax alkyl hydroxycinnamates are widely distributed throughout plant kingdom The ability to synthesize AHCs likely originated >150 mya in a common ancestor of monocots and dicots Argues for important biological / physiological function 24

25 Future Directions What are the physiological functions of alkyl hydroxycinnamates? Antimicrobials? Feeding deterrents? Synergistic with allelochemicals? fact and other alkyl hydroxycinnamate deficient mutants can now be used as tools to explore alkyl hydroxycinnamates function Understanding the biosynthesis and physiology of these compounds is necessary to exploit their industrial & medicinal value FAR / FACT interaction How to deliver a hydrophobic substrate to a soluble protein? Does the phylogentic distribution of FACT explain lack of alkyl caffeates in some species? 25

26 Acknowledgements Mike Pollard Adam Rice John Ohlrogge Isabel Molina Owen Rowland National Research Initiative of the USDA CSREES, grant number Fréd Domergue Merissa Strawsine Grant # MCB

27 Questions? 27