Plant Cuticle and Suberin.pdf

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Plant Cuticle and SuberinPE Kolattukudy, Ohio State University, Columbus, Ohio, USA

Plantcuticle comprises an insolublelipid-derived polymeric structural componentcutin

embedded in a complex mixture of soluble lipids (wax). When a plant needs to erect a

barrier to seal a wound or to generate an internal diffusion-resistant compartment, apolymeric cell wall accrustation suberin together with soluble waxes is deposited. Both

polymers can be degraded,analysed and characterized. The epidermal cellsand suberizing

cells have specialized enzymesthat convert thecommon cellular fatty acids into theunique

components of the cuticle and suberin.

Introduction

Living organisms are packaged in envelopes that consist ofpolymeric structural components. Animals use proteins orcarbohydratesas the structural component, whereas plantsuse lipid-derived polymers as the main component of the

outermost layer. In terrestrial organisms, this envelope ismade waterproof by association with a complex mixture ofsoluble waxes. The chemistry, biosynthesis and somefunctions of the cuticular components on aerial plantorgans and the barrier layer formed in the bark andunderground parts of plants are summarized in this article.

Composition of Cuticle

Cuticle is attached by a pectinaceous layer to the epidermalcell walls of the aerial parts of plants, such as fruits and

leaves (Figure 1). The cuticle is composed of an insolublelipid-derived polymeric structural component, cutin, thatis embedded in a complex mixture of soluble lipidscollectively called wax. Soluble waxes are also secreted tothe plant surface, where they are found in characteristiccrystalline forms that appear as powdery blooms on plantsurfaces such as those of cabbage and blue spruce. Thus,the plant cuticle is almost entirely derived from lipids(Kolattukudy, 1980, 1987).

Cuticular waxes can readily be obtained by mildextraction with organic solvents and can be analysed bythin-layer and gas chromatography together with mass

spectrometry (Kolattukudy and Walton, 1973; Tulloch1976). The most common components of cuticular waxeinclude nonisoprenoid hydrocarbons in the range C21 tC35, with C29 and C31 usually the dominant ones, and themid-chain hydroxylated and/or oxo derivatives. C25C3

2-methyl branched and C26C32 3-methyl branchehydrocarbons are also found in many waxes. The othemost common components are wax esters composed ofatty alcohols, with C26 and C28 as the most commodominant ones, esterified to n-C14 to n-C32 saturated fattacids, together with free fatty alcohols and free fatty acidsIn some plants the major wax components also includother classes of compounds such as b-diketones (mainlC31) and derivatives; terpenoids, dominated by triterpeneflavanoids; and fatty acid esters of sugars. There arnumerous minor components in most plant cuticulawaxes (Kolattukudy and Walton, 1973; Tulloch, 1976).

The cuticle that is released by disrupting the pectinac

eous layer can be thoroughly extracted to remove thsoluble waxes, and the insoluble polymer can be depolymerized by hydrolysis with alcoholic KOH, by transesterification with BF3 or sodium methoxide in methanol, or bexhaustive hydrogenolysis with LiAlH4. The monomerare derivatized, usually as trimethylsilyl ethers, ansubjected to combined gas chromatography and masspectrometry to determine the structure and compositioof the cutin monomers (Holloway, 1973; Kolattukudy anWalton, 1973).

Cutin is a polyester composed of hydroxy and hydroxyepoxy fatty acids derived from saturated C16 an

Article Contents

Secondary article

. Introduction

. Composition of Cuticle

. Formation of Cuticle

. Roles of Plant Cuticle in the Interaction with Microbe

. Role of Cuticle in Fertilization

. Composition of Suberin

. Formation of Suberin

. Transport of Cuticular Components

Cuticle

Pectin

Cell wall

Cell wall

Suberized wall

Plasma membrane

Cytoplasm

Vacuole

Figure 1 Schematic representation of the cuticle (left) and suberized cell wall (right).

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unsaturated C18 fatty acids (Figure 2). The most commonmajor components of the C16 group are 9,16- or 10,16-dihydroxypalmitic acid, their 9- or 10-oxo and/or 16-oxoderivatives, and 16-hydroxypalmitic acid. The commonmajor components of the C18 group are 18-hydroxy-9,10-

epoxy and 9,10,18-trihydroxy saturated and D12

-unsatu-rated C18 acids and the corresponding 18-hydroxy C18saturated and unsaturated acids. In some plants 9,10,18-trihydroxy-12,13-epoxy and 9,10,12,13,18-pentahydroxyC18 acids are also significant components. The monomercomposition of cutin can vary with the anatomicallocation. The C16 family monomers tend to dominate inthe most rapidly expanding plant organs, although in mostplant organs a combination of the C16 and C18 group ofmonomers characteristic of the species is found.

The cutin monomers are held together mainly byprimary alcohol ester linkages, leaving few if any freeprimary hydroxyl groups in the polymer (Figure 3) (Ko-

lattukudy, 2000). Almost one-half of the mid-chain(secondary) hydroxyl groups are in ester linkages, indicat-ing branching and/or crosslinks in the polymer. There areindications of peroxide bridges and ether linkages, and allof the hydrolytic or reductive depolymerization methodsleave a depolymerization-resistant polymethylenic corethat is held together by COC and CC bonds andcontains unsaturation and carboxyl groups (Kolattukudyand Walton, 1973; Kolattukudy, 2000). Cross-polariza-tion magic angle spinning (CPMAS) NMR of isolatedcutin shows the expected spectrum from an aliphaticpolyester of moderately flexible netting with motionalconstraints at crosslink sites. More than half of the

methylenes may be in the rigid category, with more thanone-third in the mobile category (Zlotnik-Mazori andStark, 1988).

Formation of Cuticle

Biosynthesis of the cuticle is a specialized function of theterminally differentiated epidermal cells (Kolattukudy,

1980, 1987, 1996). Excised epidermis from leaves caincorporate 14C-labelled acetate as well as fatty acids sucas palmitic acid into soluble waxes and the major cutimonomers. The presence of very long-chain molecules is hallmark of the waxes. The different chain length classes owax components, such as acyl portions of wax esters, freacids, free alcohols, alkanes and derivatives, are mo

probably producedby different elongating systems that arfunctionally (probably physically also) coupled to thenzymes responsible for further modifications. For example, one elongating system, which generates mainlyC26 anC28 acids, is coupled to an alcohol-generating reductasthat in turn is coupled to acyl-CoA:fatty alcohol transacylase that generates wax esters. Another elongatinsystem, which generates longer acids, mainly C30 anC32, is coupled to an aldehyde-generating reductase whicin turn is coupled to a decarbonylase that converts thaldehydes to alkanes and CO; the alkanes are thesubjected to mid-chain hydroxylation followed by oxidation to generate the corresponding secondary alcohols an

ketones that were once thought to be precursors of alkane(Kolattukudy, 1996). The membrane-localized elongatinenzyme systems have been solubilized and partiallpurified; the alcohol-generating and aldehyde-generatinreductases and the decarbonylase have been solubilizeand purified.

Incorporation of exogenous labelled precursors intcutin occurs only in the actively cutin-synthesizinepidermis-containing tissues from rapidly expandinorgans such as developing leaves and fruits (Kolattukudy1980, 1987, 1996, 2000). The classical metabolic experiments to study the incorporation of specifically labelleprecursors and labelled intermediates into cutin monomer

in such rapidly expanding tissues led to the postulation opathways for the biosynthesis of the major C16 and Cfamily of cutin monomers. The biochemical reactionpostulated to be involved in this process have beedemonstrated in cell-free preparations uniquely from thcutin-synthesizing tissues (Kolattukudy, 2000). o-Hydroxylation of C16 acid by a cytochrome P450-type enzymgenerates the o-hydroxy C16 acid that is hydroxylated athe 9 or 10 position by another cytochrome P450-typenzyme to generate the dihydroxy C16 acid, a major cutimonomer in most plants. After o-hydroxylation of oleacid, the D9 is epoxidized by a cytochrome P450 enzymthat probably uses the CoA ester as the substrate. Th

epoxide is hydrated to generate the vic-diol to yield 9,10,18trihydroxy C18 acid. All of these enzyme activities arfound in particulate preparations from the actively cutinsynthesizing tissues. Plant cytochrome P450s that cacatalyse o-hydroxylation and epoxidation of fatty acidhave been cloned and characterized. They have beesuggested to be involved in cutin biosynthesis (Salaun anHelvig, 1995), although no direct linkage of such enzymeto cutin biosynthesis is available. The CoA esters of thmonomers are the substrates for the enzyme that transfer

CH2(CH2)7CH CH(CH2)7COOH


C Family16

CH3(CH2)14COOH

OH

CH2(CH2)xCH (CH2)yCOOH

OH OH

(y= 8, 7, 6, or 5; x+ y= 13)

C18 Family*


OH

OH O

OH OHOH

CH2(CH2)14COOH


Figure2 Structureof themost commonmajormonomersof cutin.* D12-unsaturated analogues also occur.

Plant Cuticle and Suberin

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the hydroxyacyl moieties from CoA to the free hydroxylgroups in the growing polymer. This polymer-generatingenzyme is physically associated with the cutin primer at theextracellular location where the polymer is deposited(Kolattukudy, 1980).

Roles of Plant Cuticle in the Interactionwith Microbes

Nutrient source for microbes on the aerialplant surfaces

Both the soluble cuticular waxes and the polymer mayprovide nutrients for the growth of microbes (Kolattuku-dy, 2000).Bacteria thatcan fix atmospheric N2 may inhabitthe plant surface, and cohabiting bacteria may provide thecarbon source by degrading cutin. A bacterial cutinase thathydrolyses cutin has been purified and cloned from

Pseudomonas mendocina, a cohabiting bacterium obtainedwith an apparently nitrogen-fixing bacterium from a plantsurface (Kolattukudy, 1999).

Relief of self-inhibition

Fungal conidia contain lipophilic molecules that inhibittheir germination and differentiation until they reach afavourable environment such as a plant surface. Plantcuticle can relieve this inhibition by extraction of these self-

inhibitors into the lipid-derived cuticle by diffusio(Kolattukudy, 1984a, 1999; Kolattukudy et al., 1998).

Signal for germination and differentiation

The soluble wax components can function as host-specifisignals to induce conidial germination and differentiatioof the infective structure called the appressorium (Salau

and Helvig, 1995; Kolattukudy et al., 2000). Avocadcuticular wax and components isolated from it (mainlvery long-chain fatty alcohols), but not other plant waxesinduce such processes in conidia of an avocado pathogeColletotrichum gloeosporioides, but not other species ofungalpathogens(Kolattukudy, 1984a;Kolattukudy etal1998). Cutin monomers released by the cutinase carried bthe conidia can also trigger appressorium formation isome pathogens.

Cuticular penetration

Fungal pathogens that infect intact aerial organs of plant

without requiring wounds often penetrate directly througthe cuticle. Cutin is the major barrier to such direct entrthrough the cuticle and therefore this process wapostulated to involve an extracellular fungal enzymcutinase. Such enzymes were first purified in the 1970(Kolattukudy et al., 1981; Kolattukudy, 1984b) and thcDNA and gene were cloned in the 1980s (Kolattukudy1980, 1987). Fungal cutinases (also bacterial) use an activserine catalytic triad involving Asp and His to preferentially cleave primary alcohol ester linkages, generatin

CH

OH

CH3O

O

CHCH

2C

O

CH

CH3O

CH

O

CH

O

CH

CH3O C

OO

CH

CH2O

C

CH2O

CO

O

CH

OCH3

CH

C

O

O

CH

CH

C

HO

CH

CH2

C

OO

O

NH

OH

Wax

OC

O

CH CH OH

OHO

OH

OO

O

O

OH

OCH2

CH2CO

O

CH O C

O

CH2

O C

OO

OH

Cell wall

O

C O CH2(CH2)5 CH(CH2)8 CO

O

CH2

(CH2)5

CH (CH2)14CH3O C

O

(CH2)8C O

OCH2 (CH2)14CH2O C

O

O

C O

(CH2)8

CH OH

(CH2)5CH2

(CH2)8

CH

(CH2)5

OH

O

C

O

(CH2)8 CH (CH2)5CH2O

O

C O

CH

CH

OH

(CH2)8CH(CH2)5CH2C

O OO

n

Cell wall Phenolics Aliphatics

Dark Light

O

Figure 3 Models showing the type of structures present in the polymers cutin (left) and suberin (right).




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acyl-CoA reduction and hydrocarbon synthesis duringsuberization (Kolattukudy, 1980, 1984b, 1987, 2000). o-Hydroxy fatty acid formation and oxidation of the o-hydroxy acid to the dicarboxylic acid are also inducedduring the few days of wound healing. This oxidationinvolves o-hydroxy fatty acid dehydrogenase that isinduced for suberization and the constitutively expressed

o-oxo acid dehydrogenase. The o-hydroxy acid dehydro-genase has been purified to homogeneity as a dimer of30 kDa monomers from wound-healing potato discs. Thisdehydrogenase has a unique substrate-binding pocket thatcan readily accommodate o-hydroxy acids 5 C20 andionically (using thee-amino group of Lys) interact with thedistal carboxyl group of the substrate. The aromaticcomponents are generated from cinnamic acid producedby phenylalanine ammonia-lyase, using the enzymesinvolved in the hydroxylation and methylation reactionsused in lignin biosynthesis. The CoA esters of the phenolicacids esterify to fatty alcohol and glycerol or form amidelinkages with tyramine via the appropriate transferases,

and these conjugates are polymerized onto the cell wall byperoxidase(s) that is induced during suberization. TheCoA esters of the dicarboxylic acids and fatty acids areprobably esterified to the free hydroxyl groups in thepolymer.

Transport of Cuticular Components

How the cuticular components are transported to theextracellular location of their ultimate deposition is notclearly understood. From circumstantial evidence, it hasbeen suggested that lipid transfer proteins (LTP) may beinvolved in transporting cuticular components and theirprecursors (Kolattukudy, 1996; Clark and Bohnert, 1999).Expression of some of these proteins at the sites of cutinand suberin synthesis, such as the epidermis and cottonfibre cells, respectively, is consistent with such a function,as are the findings that LTP is the major protein in thecuticle and most of the LTP can be found to be associatedwith the cuticle (Kolattukudy, 1996).

References

Clark AMand Bohnert HJ (1999)Cell-specific expression of genes of the

lipid transfer protein family from Arabidopsis thaliana. Plant Cell

Physiology 40: 6976.

Holloway PJ (1973)The chemical constitution of plant cutins. In:Cut

DF, Alvin KL and Price CE (eds) The Plant Cuticle, pp. 458

London: Academic Press.

Kolattukudy PE (1980) Cutin, suberin and waxes. In: Stumpf PK (ed

Comprehensive Biochemistry of Plants, vol. IV, pp. 571645. Londo

Academic Press.

Kolattukudy PE (1984a) Biochemistry and function of cutin a

suberin. Canadian Journal of Botany 62: 29182933.

Kolattukudy PE(1984b) Cutinasesfrom fungiand pollen. In: BorgstroB and Brockman H (eds) Lipases, pp. 471504. Amsterdam: Elsevi

Science.

Kolattukudy PE (1987)Lipidderived defensivepolymersand waxes a

their role in plantmicrobe interaction. In: Stumpf PK (ed.) Th

Biochemistry of Plants, vol. 9, pp. 291314. London: Academic Pres

Kolattukudy PE (1996) Biosynthetic pathways of cutin and waxes, an

their sensitivity to environmental stresses. In: Kerstiens G (ed.) Pla

Cuticles}An Integrated Functional Approach, pp. 83108. Oxfor

BIOS Scientific Publishers.

Kolattukudy PE (2000) In vitro biosynthesis of polyesters. In: Babel W

and Steinbu chel A (eds) Advances in Biochemical Engineering

Biotechnology: Biopolyesters. Heidelberg, Germany: Springer.

Kolattukudy PE and Espelie KE (1989) Chemistry, biochemistry an

function of suberin and associated waxes. In: Rowe J (ed.) Natur

Products of WoodyPlants,ChemicalsExtraneous to the Lignocellulos

Cell Wall, pp. 304367. Heidelberg, Germany: Springer.

Kolattukudy PE and Walton TJ (1973) The biochemistry of pla

cuticular lipids. Progress in the Chemistry of Fats and Other Lipids

121175.

Kolattukudy PE, Purdy RE and Maiti IB (1981) Cutinases from fun

and pollen. Methods in Enzymology 71: 652664.

Kolattukudy PE, Kim Y, Li D, Liu ZM and Rogers L (2000) Ear

molecular communication between Colletotrichum gloeosporioid

and its host. In: Host Specificity, Pathology and Host Pathoge

Interaction of Colletotrichum. St. Paul, MN: APS Press.

SalaunJP andHelvig C (1995)Cytochrome P450-dependent oxidation

fatty acids. Drug Metabolism and Drug Interaction 12: 261283.

Tulloch, AP (1976) Biochemistry of plant waxes. In: Kolattukudy P

(ed.) The Chemistry and Biochemistry of Natural Waxes, pp. 23627

New York: Elsevier/North Holland Press.

Zlotnik-Mazori T and Stark RE (1988) Nuclear magnetic resonan

studies of cutin, an insoluble plant polyester. Macromolecules 2

24122417.

Further Reading

Kolattukudy PE (1981) Structure, biosynthesis and biodegradation

cutin and suberin. Annual Review of Plant Physiology 32: 539567.

Kolattukudy PE (1985) Enzymatic penetration of the plant cuticle

fungal pathogens. Annual Review of Phytopathology 23: 223250.

Kolattukudy PE, Rogers LM, Li D, Hwang C-S and Flaishman M

(1995) Surface signaling in pathogenesis. Proceedings of the Nation

Academy of Sciences of the USA 92: 40804087.



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