Transcript Cutin and Suberin

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
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
Structural
characteristics
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:
ω or mid-chain carbon hydroxylation
ω or mid-chain carbon hydroxylation
ω 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 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 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?n
umber=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 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
3-D models of tomato cuticle
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