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Cutin and Suberin

Cutin and Suberin. Yan Liang PBIO691 Fall 2010. Schematic of cutin and suberin deposittion. Example of cutin deposition in a leaf Example of suberin deposition in the endodermis of a root EW: epicuticular waxes C: cuticle proper (cutin and intracuticular wax)

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Cutin and Suberin

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  1. Cutin and Suberin Yan Liang PBIO691 Fall 2010

  2. Schematic of cutin and suberin deposittion Example of cutin deposition in a leaf Example of suberin deposition in the endodermis of a root EW: epicuticular waxes C: cuticle proper (cutin and intracuticular wax) CL: the cuticular layer (cutin and polysaccharide) ML: middle lamellae PW: primary cell walls PM: plasma membrane Cy: cytoplasm V: vacuole SL: suberin lamellae SW: secondary walls Pollard et al. (2007)

  3. Representative monomers of cutin and suberin Modification of fatty acid: ω or mid-chain carbon hydroxylation ω or mid-chain carbon hydroxylation ω carbon carboxylation

  4. Hypothetical monomer connectivity patterns (b) (c) Dentrimer structures formed by ω-hydroxy fatty acid and glycerol -Cutin polyester? (d) A cross-linking structure formed by α,ω-dicarboxylic acid and glycerol -Suberin polyester ? -Arabidopsis cutins ? (e) Dentrimer structures formed by α,ω-dicarboxylic acid and glycerol -Cutin in Arabidopsis ?

  5. Techniques to study cutin and suberin structure • Cutin/suberin isolation solvent extraction and enzymatic removal of cellulose, pectins and hemicelluloses • Depolymerization: trans-esterification RCOOR’+CH3OH RCOOCH3+R’OH • Monomer analysis by GC-MS or thin layer chromatography • Microscopic techniquesTransmission electron microscopy (TEM) Scanning electron microscopy (SEM) Light microscropy Confocal laser scanning microscopy (CLSM) , 3D-CLSM

  6. Identification of candidate genes involved in cutin/suberin biosynthesis • Forward genetics Mutant identification and positional cloning • Reverse genetics Transcriptome analysis of epidermis Co-expression analysis Genes regulated by specific transcription factors

  7. Major steps of cutin/suberin/cuticle wax biosynthesis Cytosol Plastid Phenylalanine ER Fatty acid (C16:0-C18:0) synthesis • Fatty acid modification • Ester formation: acylglycerol esters • /alkylferulates • Fatty acid elongation to C24-C36 (VLCFAs) • Modification of VLCFAs p-coumaric acid Feruloyl-CoA Polymer assembly?? Cell Wall

  8. Fatty acid elongation and wax biosynthesis

  9. Suberin biosynthesis

  10. Questions remain in • The polymer assembly mechanism • The orders of the reactions • The transportation mechanism

  11. Intra- and ultra- cellular transportation LTPG

  12. Regulation of lipid polymer deposition • Developmentally regulated • Wound/stresses induced • Coordinately regulation of genes in the biosynthetic pathways • Transcription factor: WINclade • CER7: 3’-5’ exoribonuclease

  13. Potato is a model plant for periderm and suberin studies • The plant periderm is an external • barrier • Periderm lipids: • Suberin (96%) • Wax (4%) • “Skin set” • The phellogen stops dividing • The phellem adheres to phelloderm • The phellemaquires complete lipid • coverage and full water barrier • properties http://www.geochembio.com/biology/organisms/potato/ http://www.gov.pe.ca/af/agweb/index.php3?number=1000971

  14. The plant BAHD family • A acyl-CoA dependant acetyltransferase family • The name of BAHD is from BEAT, benzyl alcohol acetyltransferase AHCT, anthocyanin-O-hydroxycinnamoyltransferase HCBT, anthranilate-N-hydroxycinnamoyl/benzoyltransferase DAT, deacetylvindoline 4-O-acetyltransferase using hydroxycinnamoylCoA esters as acyl donors • Use a range of CoA-thioesterdonars • Use acceptors including shikimatephenylpropanoid, alkaloids, terpenoids, polyamines, short- or middle chain aliphatic alcohols

  15. Identification of the target gene • The EST encoding BAHD acyltransferase was isolated from a subtractive library tuber skin of potato vs. tuber parenchyma • The EST best matches TC169622 gene • Full length cDNA was amplified with TC169622 primers • Information from bioinformatic analysis 79% similarity to At5g41040 88-92% similarity to cork ESTs Contains HxxxD and DFGWG motifs No signal peptide or transmembrane domains

  16. FHT transcript is expressed in tuber periderm and in the root (Northern blot)

  17. FHT expression was down regulated by RNAi in potato tuber skin

  18. Phenotype of the tuber skin of the FHT RNAiline Wild Type FHT RNAi Tuber SEM micrograph of the skin surface Cross-sectional SEM microgram

  19. Phenotype of the tuber skin of the FHT RNAiline cultured in vitro WT FHT RNAi WT FHT RNAi

  20. Phenotype of the isolated phellem layer of the FHT RNAiline • The phellem layer was isolated by cellulase and pectinase treatment • Figures obtained by SEM

  21. Effect of FHT on suberin ultrastructure • FHT RNAi epidermis • Showed normal suberin lamellae structure • No changes in thickness or electron density of walls • Showed more vestiges of organellar structure

  22. FHT RNAilines lost more weight during storage and their epidermis showed higher water permeance

  23. Effects of FHT down-regulation on suberin composition • Total amount of suberin remained the same • Total amount of glycerol released by trans-esterification remained the same Decreased monomers in FHT-RNAi Increased monomers in FHT-RNAi

  24. Effects of FHT down-regulation on wax composition • Total amount of wax doubled in FHT RNAilines

  25. FHT biochemical activity FHT • Bacterial expression of FHT in fusion with N-terminal GST tag • The protein was purified by affinity chromatography and digested by thrombin • SDS-PAGE shown a single band of 55 kDa

  26. In vitro assay for FHT activity AcylDonerAcyl Acceptor (b) Hypothetical scheme of the FHT assay HPLC analysis of the reaction product (c) GC-MS/MS of the product peak shown in HPLC analysi (c) Negative mode [feruloyloxy-MeOH-H]- [feruloyloxy-H2O-H]- [M-MeOH-H]- Positive mode [feruloyl]+ [M-H2O+H]-

  27. FHT assay with other acyl acceptors Acyl acceptors tested Primary alcohol of various chain length (C7, C8, C12, C14, C16, C18, C20) Product peaks were shown for C16, C12 and C14 primary alcohols by HPLC.

  28. FHT silencing induced changes in soluble phenolics of periderm

  29. Discussion • FHT is a fatty alcohol/fatty ω-hydroxyacidhydroxycinnamoylacyltransferase involved in suberin biosynthesis • FHT down-regulation does not alter suberin lamellation • FHT deficiency and the control of vapour water loss • Effects of FHT deficiency on free and conjugated phenoliccompounds • FHT-deficient potatoes have russeted skin and impaired periderm maturation

  30. References • Alberscheim et al. (2011) Plant Cell Walls from Chemistry to Biology • Buda et al. (2009) Plant J 60, 378 • DoBono et al. (2009) Plant Cell 21, 1230 • Franke and Schreiber (2007) CurrOpin Plant Bio 10, 252 • Gou et al. (2009) PNAS 106, 18855 • Hooker et al. (2007) Plant Cell 19, 904 • Kunst and Samuels (2009) CurrOpin Plant Biol12, 721 • Pollard et al. (2008) Trends Plant Sci13, 236 • Serra et al. (2010) Plant J 62, 277 • http://aralip.plantbiology.msu.edu/pathways

  31. Thank You & questions ?

  32. 3-D models of tomato cuticle Back

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