Cutin and Suberin

Yan Liang PBIO691 Fall 2010

Cutin Cuticle Wax Suberin/Suberin WaxDistribution in plants

Epidermal cells of aerial organs (leaves, stems, flowers, fruits and seeds)

Epidermis of underground organs and stems, and cotton fibersCork of woody plantsCasparian bands of the root endodermal cell layerBundle sheaths of grassesSynthesized in response to wounding and stress

Function Resisting mechanical damageControling nonstomata water/gas exchangeInvolved in host-pathogen interactionMaintaining organ identity (cutin)

Restrict the movement of water and solutes in rootprevent water loss from woody stems Pathogen resistanceStress resistance

Ultra-structuralorganization

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 wallForm a lamellae structure

Structural characteristics

Ester polymersMajor monomers:Modified fatty acid (C16 and C18) Glycerol

A mixure of very-long-chain (C20-C36) fatty acid derivatives , including primary alcohols, alkanes, secondary alcohols, ketones, aldehyde, etc

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

Pollard et al. (2007)

a) Example of cutin deposition in a leafb) Example of suberin deposition in the

endodermis of a root

EW: epicuticular waxesC: cuticle proper (cutin and intracuticular wax)CL: the cuticular layer (cutin and

polysaccharide)ML: middle lamellaePW: primary cell wallsPM: plasma membrane Cy: cytoplasmV: vacuole

SL: suberin lamellae SW: secondary walls

Schematic of cutin and suberin deposittion

Representative monomers of cutin and suberin

Modification of fatty acid: ω or mid-chain carbon hydroxylationω or mid-chain carbon hydroxylationω carbon carboxylation

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 ?

Techniques to study cutin and suberin structure

• Cutin/suberin isolationsolvent extraction and enzymatic removal of cellulose, pectins and hemicelluloses

• Depolymerization: trans-esterificationRCOOR’+CH3OH RCOOCH3+R’OH

• 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 geneticsMutant identification and positional cloning

• Reverse geneticsTranscriptome analysis of epidermisCo-expression analysisGenes regulated by specific transcription factors

Major steps of cutin/suberin/cuticle wax biosynthesis

Fatty acid (C16:0-C18:0) synthesis

PlastidCytosol

ER•Fatty acid modification •Ester formation: acylglycerol esters

/alkylferulates •Fatty acid elongation to C24-C36 (VLCFAs)•Modification of VLCFAs

Phenylalanine

p-coumaric acid

Feruloyl-CoA

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

http://www.geochembio.com/biology/organisms/potato/http://www.gov.pe.ca/af/agweb/index.php3?number=1000971

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

The plant BAHD family

• A acyl-CoA dependant acetyltransferase family• The name of BAHD is from

BEAT, benzyl alcohol acetyltransferaseAHCT, anthocyanin-O-hydroxycinnamoyltransferaseHCBT, anthranilate-N-hydroxycinnamoyl/benzoyltransferaseDAT, 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 ESTsContains HxxxD and DFGWG motifsNo 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

Wild Type FHT RNAi

Tuber

SEM micrograph of the skin surface

Cross-sectional SEM microgram

Phenotype of the tuber skin of the FHT RNAi line

WT FHT RNAi WT FHT RNAi

Phenotype of the tuber skin of the FHT RNAi line cultured in vitro

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 trans-esterification 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 activityAcyl Doner Acyl Acceptor

(c)

(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

Negative mode

Positive mode

[M-MeOH-H]-

[M-H2O+H]-

[feruloyloxy-H2O-H]-

[feruloyloxy-MeOH-H]-

[feruloyl]+

FHT assay with other acyl acceptors

Acyl acceptors testedPrimary 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 ω-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

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