Cutin and Suberin Yan Liang PBIO691 Fall 2010

Cutin and Suberin Yan Liang PBIO691 Fall 2010

Cutin and Suberin Yan Liang PBIO691 Fall 2010 Cutin Cuticle Wax Suberin/Suberin Wax Distribution in

plants Epidermal cells of aerial organs (leaves, stems, flowers, fruits and seeds) Epidermis of underground organs and stems, and cotton fibers Cork of woody plants Casparian bands of the root endodermal cell layer Bundle sheaths of grasses

Synthesized in response to wounding and stress Function Resisting mechanical damage Controling nonstomata water/gas exchange Involved in host-pathogen interaction Maintaining organ identity (cutin) Restrict the movement of water and

solutes in root prevent water loss from woody stems Pathogen resistance Stress resistance Ultrastructural organization Structural characteristics The cuticle layer deposits outside the primary cell wall. Cutin is

embedded with intracuticular waxes, which is covered by epicuticular waxes Deposit on the inner face of primary cell wall Form a lamellae structure Ester polymers Major monomers: Modified fatty acid (C16 and C18)

Glycerol Opaque layer: aliphatic polyester of modified fatty acid and glycerol; some fatty acid may have chain length >20 Translucent layer: Polyaromatic constituents mainly contains hydroxycinnamic acids. Ferulate esters may function to link the aliphatic domain to the polyaromatic

domain A mixure of very-long-chain (C20C36) fatty acid derivatives , including primary alcohols, alkanes, secondary alcohols, ketones, aldehyde, etc Schematic of cutin and suberin deposittion a) Example of cutin deposition in a leaf b) 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) Representative monomers of cutin and suberin Modification of fatty acid: or mid-chain carbon hydroxylation or mid-chain carbon hydroxylation carbon carboxylation

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

by ,-dicarboxylic acid and glycerol -Cutin in Arabidopsis ? 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+ROH

Monomer analysis by GC-MS or thin layer chromatography Microscopic techniques Transmission electron microscopy (TEM) Scanning electron microscopy (SEM) Light microscropy Confocal laser scanning microscopy (CLSM) , 3D-CLSM 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 Major steps of cutin/suberin/cuticle wax biosynthesis Cytosol Plastid Fatty acid (C16:0-C18:0)

synthesis Phenylalanine p-coumaric acid Feruloyl-CoA ER Fatty acid modification Ester formation: acylglycerol esters /alkylferulates Fatty acid elongation to C24-C36 (VLCFAs) Modification of VLCFAs

Polymer assembly?? Cell Wall Fatty acid elongation and wax biosynthesis Suberin biosynthesis Questions remain in The polymer assembly mechanism

The orders of the reactions The transportation mechanism Intra- and ultra- cellular transportation LTPG Regulation of lipid polymer deposition Developmentally regulated Wound/stresses induced Coordinately regulation of genes in the biosynthetic

pathways Transcription factor: WIN clade CER7: 3-5 exoribonuclease 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 phellem aquires 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

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 hydroxycinnamoyl CoA esters as acyl donors Use a range of CoA-thioester donars Use acceptors including shikimate phenylpropanoid, alkaloids,

terpenoids, polyamines, short- or middle chain aliphatic alcohols 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 FHT transcript is expressed in tuber periderm and in the root (Northern blot) FHT expression was down regulated by RNAi in potato tuber skin

Phenotype of the tuber skin of the FHT RNAi line Wild Type FHT RNAi Tuber SEM micrograph of the skin surface

Crosssectional SEM microgram Phenotype of the tuber skin of the FHT RNAi line cultured in vitro WT FHT RNAi WT

FHT RNAi Phenotype of the isolated phellem layer of the FHT RNAi line The phellem layer was isolated by cellulase and pectinase treatment Figures obtained by SEM 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 FHT RNAi lines lost more weight during storage and their epidermis showed higher water permeance

Effects of FHT down-regulation on suberin composition Total amount of suberin remained the same Total amount of glycerol released by transesterification remained the same Decreased monomers in FHT-RNAi

Increased monomers in FHT-RNAi Effects of FHT down-regulation on wax composition Total amount of wax doubled in FHT RNAi lines FHT biochemical activity

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 FHT

In vitro assay for FHT activity Acyl Doner Acyl Acceptor (b) Hypothetical scheme of the FHT assay (a) 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]- 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. FHT silencing induced changes in soluble phenolics of periderm Discussion

FHT is a fatty alcohol/fatty -hydroxyacid hydroxycinnamoyl acyltransferase 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 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) Curr Opin Plant Bio 10, 252 Gou et al. (2009) PNAS 106, 18855 Hooker et al. (2007) Plant Cell 19, 904 Kunst and Samuels (2009) Curr Opin Plant Biol 12, 721 Pollard et al. (2008) Trends Plant Sci 13, 236 Serra et al. (2010) Plant J 62, 277 http://aralip.plantbiology.msu.edu/pathways

THANK YOU & QUESTIONS ? 3-D models of tomato cuticle Back

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