Plant Physiol. (1976) 58, 411-416

Synthesis of Suberin during Wound-healing in Jade Leaves,Tomato Fruit, and Bean Pods'

Received for publication March 25, 1976 and in revised form June 1, 1976

BILL BRYAN DEAN AND P. E. KOLATTUKUDY2Department of Agricultural Chemistry, Washington State University, Pullman, Washington 99163

ABSTRACT

The structure and composition of the aliphatic monomers of thepolymeric material deposited during wound-healing of tomato fruit,bean pods, and Jade leaves were examined. After removing the cuticle-containing layer of tissue, the wounds were healed for 14 days and theresulting surface layer was excised, lyophilized, solvent-extracted, anddepolymerized by hydrogenolysis with LiAIH4 or transesterified withBF3 in methanol. The products obtained by the chemical depolymeriza-tion were subjected to thin layer chromatography and combined gaschromatography and mass spectrometry. The major aliphatic compo-nents isolated from the hydrogenolysate of the wound polymer producedby tomato fruit were hexadecane-1,16-diol and octadec-9-ene-1,18-diol,which were shown to be derived from a 1:1 mixture of w-hydroxy anddicarboxylic acids of the appropriate chain length by LiAIH4 reduction.Also identified in the wound polymer were long chain (>C20) fatty acidsand alcohols. This monomer composition is typical of suberin polymersand is in sharp contrast with that of the cutin of tomato fruit whichcontains dihydroxy C16 add as the major aliphatic component. Thehydrogenolysis of the wound material from bean pods gave octadecene-1,18-diol as the major aliphatic component, and smaller amounts ofhexadecane-1,16-diol and long chain alcohols. Similar treatment of thenormal cuticular tissue of these pods gave hexadecane triol, as well as C16and C18 alcohols. Hydrogenolysis of wound material from the Jadeleaves gave octadecene-1,18-diol, C16 and C22 diols, as well as alcoholsfrom C16 to C26, whereas similar treatment of the cutin-containing tissuefrom these leaves gave C16 triol as the major aliphatic component. Thus,the major aliphatic monomers of the polymeric material deposited dur-ing the wound-healing of bean pods and Jade leaves are very similar tothose of suberin, although the natural protective polymer of these tissuesis cutin. From these results, it is concluded that suberization is a funda-mental process involved in wound-healing in plants, irrespective of thechemical nature of the natural protective polymer of the tissue.

In recent years, the composition of the monomers of cutin andsuberin, the lipid polymers which cover plants, has been studiedwith modern analytical techniques. The major components ofcutin are dihydroxy C16 fatty acids, 18-hydroxy-9,10-epoxy C18fatty acids, and trihydroxy C18 fatty acids. Also found are smalleramounts of w-hydroxy C16 and C18 fatty acids. The major ali-phatic components of suberin are w-hydroxy fatty acids anddicarboxylic acids from C16 to C28 and fatty acids and alcohols inwhich aliphatic chains longer than C20 often predominate (4, 6,7, 10, 13, 17). The composition of the aliphatic monomers of the

' This work was supported in part by National Science FoundationGrant BMS 74-09351. Scientific Paper No. 4581, Project 2001, Collegeof Agriculture Research Center, Washington State University, Pullman,Wash. 99163.

2 Author to whom inquiries should be made.

lipid polymer produced at the wound periderm of potato tubertissue is known to be identical to that of the suberin which coversthe intact tuber; in both cases, the major components are c-hydroxy and dicarboxylic acids and long chain acids and alcohols(10, 11). It has been suggested that when aerial portions of theplant such as leaves or fruit are wounded, they produce cutin orsuberin to seal off the wound. Several authors have concludedfrom histological studies that wounded leaves, stems, and fruitsproduce suberin (16, 18, 20), but others have suggested thatcutin was being resynthesized (2, 3). Bredemeijer and Heinen(3) assumed that cutin was being resynthesized when they usedwounded leaves of Gasteria for what was called biosyntheticstudies on cutin, and concluded "that experiments with detachedleaves reflect the actual events occurring during the resynthesisof cutin on the wound surface of an injured part of the plantmore clearly than those under the complex and less controlled insitu conditions" (3). However, the chemical nature of the poly-mer formed during the wound-healing of aerial parts of plantshas not been examined heretofore.

It was the purpose of this study to determine if aerial planttissues produce, during wound-healing, cutin similar to theirnatural cuticular polymer or suberin as previously observed inthe wound-healing of potato tuber tissue. Here we show that thealiphatic components of the polymeric material deposited at thewound surface on aerial parts of three plants are typical suberincomponents and not similar to those of cutin, which is thenatural protective polymer of these tissues. Thus, we concludedthat suberization is the fundamental process involved in wound-healing irrespective of the nature of the natural protective poly-mer of the tissue.

MATERIALS AND METHODS

Preparation of the Wound-covering Material. Tomato fruit,Lycopersicon esculentum var. Bonny Best, was harvested at animmature green stage of growth. The fruit was divided into twogroups, and the cuticle-containing layer of tissue was removedwith a razor blade to a depth of approximately 1 to 2 mm. Thefirst group of peeled fruit was placed in wide mouth gallon jarsand aerated with humidified air at a rate of 0.6 I/hr. From thesecond group of peeled fruit, another layer of tissue, approxi-mately 1 to 2 mm deep, was excised immediately after removingthe cuticle-containing layer, and this sample served as controltissue to represent unhealed tissue. The cuticle-containing tissuelayer was removed in the above manner from another lot oftomato fruit, and it was then allowed to heal while attached tothe plant. After 14 days from wounding, the wound coveringsfrom both groups of fruit were excised with a razor blade to adepth of 1 to 2 mm. Any attached seeds and fleshy tissue wereremoved with a spatula from the fairly hard brown or greenmaterial covering the wound. The original cuticle-containingtissue layer, the control tissue from beneath the cuticle, and the

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DEAN AND KOLATFUKUDY

material covering the healed wound were all frozen at the time ofexcision and lyophilized.

Jade leaves (Crassula argentea) were chosen as experimentalmaterial because of the large cross-sectional area of these leaves.They were cut in cross-section with a razor blade at approxi-mately the middle of the leaf and were allowed to heal whileattached to the plant. Bean pods, Phaseolus vulgaris var. Bonus,were used to assess the ability of another genus to wound-heal.The cuticle-containing layer of the pods was removed with arazor blade to a depth of approximately 1 mm on about one-third of a side of the pod and allowed to heal while attached tothe plant. Both the Jade leaves and the bean pods were allowedto heal for 14 days, and the wound coverings were excised,avoiding any cuticular material adjacent to the wound. Anyadhering fleshy tissue or seeds were removed from these sampleswith a spatula, leaving thin sheets of transparent material foranalysis. The cuticle-containing layer of tissue and the wound-covering material were frozen and lyophilized.

Extraction of Soluble Lipids. All of the lyophilized sampleswere ground in a Wiley mill and passed through a 60 meshscreen or ground to a powder with a mortar and pestle. Theywere then Soxhlet-extracted for 24 hr with CHC13 to remove thesoluble lipids, and the remaining insoluble solid material ishereafter referred to as the cutin or wound polymers, althoughthese are admittedly crude mixtures.

Depolymerization with LiAIH4. Powdered cutin polymer,control tissue, and wound polymers from the tomato fruit, Jadeleaves, and bean pods were refluxed with an excess of LiAlH4 intetrahydrofuran for 24 to 48 hr. Excess reagent was then decom-posed by careful addition of the reaction mixture to 50 ml ofwater with vigorous stirring. The mixtures were acidified withconcentrated HCl and extracted with CHCl3 (3x 150 ml), andthe solvent was then removed with a rotary evaporator underreduced pressure.

Transesterification with BF3 in Methanol. The wound poly-mer, from the tomato fruit healed while attached to the plant,was also depolymerized by refluxing the finely powdered mate-rial with 14% BF3 in methanol for 24 hr. The reaction mixturewas added to water, the lipid products were extracted withCHCl3 (3 x 100 ml), and the solvent was evaporated underreduced pressure.Chromatography. TLC was done on 1-mm layers of Silica Gel

G (20 x 20 cm) activated overnight at 110 C. Components onthe thin layer chromatograms were visualized by viewing theplates under UV light after spraying them with a 0.1 % ethanolicsolution of 2',7'-dichlorofluorescein. The components were re-covered from the silica gel with a 2:1 mixture of chloroform andmethanol and absolute methanol. The hydrogenolysis (LiAlH4)products were analyzed with ethyl ether-hexane-methanol(8:2:1, v/v), and the methyl esters were analyzed with hexane-ethyl ether-formic acid (65:35:2, v/v) as the developing solvents.When the total hydrogenolysate was to be examined by com-bined gas-liquid chromatography and mass spectrometry (GLC-MS), a preliminary purification by TLC with the former solventsystem was performed. Since the purpose was to remove theintensely colored polar material from the aliphatic components,all of the products which migrated from the origin were re-covered by solvent extraction and the total products were sub-jected to GLC-MS.Gas chromatography was performed with a coiled glass col-

umn (199 x 0.31 cm o.d.) packed with 5% OV-1 on 80 to 100mesh Gas-chrom Q. Part of the effluent of the gas chromato-graph was passed into a Perkin Elmer-Hitachi RMU 6D massspectrometer with a Bieman separator interphase. Mass spectrawere recorded at the apex and ascending and descending por-tions of the gas chromatographic peak with 70 ev ionizing volt-age.

Preparation of Derivatives. Trimethylsilyl ethers were pre-

pared by heating the materials with an excess (0.3 ml) of N, 0-bis(trimethylsilyl) acetamide at 90 to 100 C for 20 min. Excessreagent was removed with a stream of N2, and the product wasdissolved in a 2:1 mixture of chloroform and methanol for gaschromatography. Introduction of a vic-diol function at the dou-ble bond was done by treatment of the olefinic material with a0.1 % solution ofOSO4 in dioxane for 2 hr. The reaction mixturewas decomposed with aqueous-methanolic Na2SO3; the resultingprecipitate was removed by centrifugation, and washed oncewith methanol and centrifuged. Products were recovered byether extraction of combined supernatants.

RESULTS AND DISCUSSION

Identif'ication of the Aliphatic Components of Normal TomatoCutin. To compare the monomer composition of the naturalcuticular polymer with that deposited during wound-healing, aknowledge of the components of the normal cutin is necessary.TLC of the products from depolymerization of the normal cutinwith LiAlH4 showed a major component which had an RF identi-cal to that of hexadecane-1,7,16-triol and two minor compo-nents with RF values corresponding to those of alkane diol andC18 triol. Gas chromatography of the trimethylsilyl ethers of thehydrogenolysis products of cutin showed one major componentwith a retention time identical to that of authentic hexadecane-1,7,16-triol (Fig. la), and this component was identified by itsmass spectrum to be hexadecanetriol. The spectrum showed aweak parent ion at m/e 490 and fragment ions at m/e 475 (M+-CH3), m/e 400 (M+-HOSi[CH3]3), and m/e 385 (M+-CH3-HOSi[CH3]3), in addition to the intense a-cleavage ions at m/e275, m/e 317, and weaker ions at m/e 289 and m/e 303. The twosets of a-cleavage ions are indicative of the presence of posi-tional isomers in the dihydroxy C16 acid from which this triol wasderived. Comparison of this spectrum with that previously pub-lished (21) confirmed that the major component was hexadec-ane-1,7,16-triol, and thus, the major monomer of tomato cutin

5TIME (min.)

FIG. 1. Gas-liquid chromatogram of trimethylsilyl ethers of the hy-drogenolysate of (a) normal tomato peel; (b) control tomato tissue; and(c) wound-healed tomato tissue. In order to avoid interference from theextremely polar dark brown material contained in the hydrogenolysate,preliminary TLC was performed with ethyl ether-hexane-methanol(8:2:1, v/v) as the solvent, and the components which migrated off theorigin were eluted from the silica gel and subjected to GLC. Compo-nents less polar than fatty alcohols were excluded. Column temperaturewas 240 C and inlet pressure was 25 p.s.i. The identity of each compo-nent is discussed in the text.

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SUBERIZATION OF AERIAL PARTS OF PLANTS

was dihydroxy C16 acid. A minor component had a retentiontime identical to that of hexadecanediol (not large enough to beshown in Fig. la). The mass spectrum of this component showeda weak parent ion at m/e 402, fragment ions at m/e 387 (M+-CH3), m/e 371 (M+-CH3-CH4), m/e 312 (M+-HOSi[CH3]3), m/e297 (M+-CH3-HOSi[CH3]3), and m/e 281 (M+-CH4-CH3-HOSi[CH3]3), and a characteristic doubly charged ion at m/e186 (M+-30/2) and its first isotope peak at m/e 186.5 (15, 21).This spectrum confirmed that this component was hexadecane-1,16-diol, which was most probably derived from 16-hydroxy-hexadecanoic acid of the cutin polymer. Also detected on the gaschromatogram, although not large enough to be shown in Figurela, was octadecanetriol, as judged by the retention time and themass spectrum which showed very intense a-cleavage ions at m/e303 and 317. This triol most probably originated from 13-hydroxy-9,10-epoxy C18 acid, as reported for other cutin samples(21).

Identification of the Aliphatic Components of the TomatoWound Polymer. TLC of the hydrogenolysis (LiAIH4) productsof the wound polymer showed two major components with RFvalues identical to those of fatty alcohols and alkane diols. Thegas chromatogram of these products (Fig. lc) showed a consid-erably different pattern than that obtained with normal cutin.Components 1-4, 6, and 9 were identified by their retentiontimes and mass spectra to be a homologous series of aliphaticalcohols. The characteristic ions used for identification by mass

spectra were a weak parent ion (M+), an intense fragment ioncorresponding to M+-CH3, and a large fragment ion at m/e 103corresponding to +CH2OSi(CH3)3 (19, 21). Using these ions andthe gas chromatographic retention times, the components wereassigned the following structures: peak 1, hexadecanol; peak 2,heptadecanol; peak 3, a mixture of saturated and unsaturatedC18 alcohols; peak 4, nonadecanol; peak 6, eicosanol; and peak9, docosanol. Tetracosanol was also detected when a largeramount of the material was injected. The alcohol componentsmay be present in the polymer as such, or as fatty acids whichwould be reduced to alcohols upon treatment with LiAlH4.Peak 5 was identified to be hexadecane-1,16-diol by its reten-

tion time and the mass spectrum as discussed above. Othersaturated (C22 and C24) diols were detectable if larger amountswere injected, and they were identified by their retention timesand the mass spectra as discussed for the C16 diol. The diolcomponent of greatest significance is found in peak 7. Theretention time and mass spectrum of this component showed thatit was octadecene-1,18-diol. The mass spectrum showed a weakparent ion at m/e 428 and cleavage ions at m/e 413 (M+-CH3),m/e 397 (M+-CH3-CH4), m/e 338 (M+-HOSi[CH3]3), m/e 323(M+-CH3-HOSi[CH3]3), together with the diagnostic doublycharged ion at m/e 199 (M+-30/2) and the first isotope ion at m/e199.5 (21). Since the position of the double bond cannot bedetermined by the mass spectrum, the diol fraction was treatedwith OS04, which introduces a vic-diol function at the doublebond. This treatment gave rise to a C18 tetraol, whereas the otherdiols were not affected, consistent with the conclusion that the.other diols are saturated. The mass spectrum of this tetraol (astrimethylsilyl ether) showed a weak parent ion at m/e 606,fragment ions at m/e 591 (M+-CH3), m/e 516 (M+-HOSi[CH3]3), and m/e 501 (M+-CH3-HOSi[CH3]3), and a veryintense a-cleavage ion at m/e 303. This fragmentation pattern,as well as the other ions in the spectrum, showed that thiscompound was octadecane-1,9,10,18-tetraol, as discussed previ-ously (21). Thus, the C18 diol from which the tetraol was derivedwas identified to be octadec-9-ene-1,18-diol. This diol and thesaturated diols discussed above were probably derived fromeither the corresponding &-hydroxy acids or dicarboxylic acids,both of which would give rise to diols upon LiAIH4 reduction.Peak 8 was identified to be hexadecanetriol by its retention

time and mass spectrum, as discussed above. Also detectable in

trace amounts in the hydrogenolysis products from this woundpolymer was octadecanetriol.

Quantitation of the hydrogenolysis (LiAlH4) products fromthe wound polymer by triangulation of the gas chromatographicpeaks gave the following results. Alcohols comprised 37.7% ofthe total lipid monomers, and among the alcohols identifiedwere C16 (9.7%), C17 (2.9%), C18:0 + C,8:u (18.6%), C19(1.5%), C20 (0.9%), and C22 (3.6%). The occurrence of longchain alcohols (C20 and C22) is indicative of suberin polymers,and they are not usually large constituents of cutin polymers (9).The diol components accounted for 52.2% of the total, and inthis fraction, the major components were C16 (24.4%) and C18:1(27.8%) diols; C16 triol made up the balance of 10.7%. If thealiphatic components obtained from the wound polymer were, infact, synthesized during the wound-healing process, the corre-sponding tissue, which was excised immediately after the re-moval of the cuticle-containing layer (1-2 mm) and prior to anywound-healing, should not contain these compounds. Hydro-genolysis (LiAlH4) followed by analysis of the lipid products bycombined GLC-MS showed that this tissue gave rise to a smallamount of C16 triol and a much smaller amount of C16 diol, butnone of the other components identified in the wound polymercould be detected (Fig. lb). Thus, most of the aliphatic compo-nents identified in the wound polymer are indeed producedduring the wound-healing process. The small amount of C16 triolidentified in the wound polymer could have been derived fromthe cuticular material which apparently penetrated into the tis-sue, as suggested by the finding that C16 triol was obtained fromthe internal tissue excised prior to wound-healing. It is apparentthat the major aliphatic components produced during wound-healing gave rise to alkane diols and alkanols upon treatmentwith LiAlH4. The composition of these components is in sharpcontrast to that of cutin, and it closely resembles that of suberinfrom several species of plants which have been examined thus far(4, 10, 13, 17).

Analysis of the Aliphatic Components of the Tomato WoundPolymer Depolymerized with BF3-Methanol. Dicarboxylic acidsare major components of suberin, while they are minor compo-nents of cutin (3, 9, 14). Since the depolymerization by LiAlH4converts both the dicarboxylic acids and o-hydroxy acids intodiols, it was necessary to use a technique which allows theseparation of these two major classes of suberin components, aswell as alcohols and acids whose identities also become obscuredafter LiAlH4 reduction. Depolymerization by transesterificationwith BF3 in methanol is a suitable technique for this purpose(10-12). TLC of the lipid products obtained by such a treatmentof the wound polymer showed an intense band corresponding tomethyl ester, two bands of nearly equal intensity correspondingto w-hydroxy acid methyl ester and dicarboxylic acid dimethylester, and a less intense band corresponding to fatty alcohol. Gaschromatography and mass spectrometry of this sample showedthat the fatty acid methyl esters constituted the largest compo-nent (67.5%). Each fatty acid was identified by its retentiontime and mass spectrum. The five fatty acids thus identified fromthe wound polymer were C16, C18, C18l, C20, and C22 (Table I).The large amount of C16 and C18 fatty acids could be a result ofincomplete extraction of internal tissue lipids.

Fatty alcohols were identified as their trimethylsilyl ethers bycombined GLC-MS using the diagnostic ions mentioned in theprevious section. The fatty alcohols identified from this prepara-tion were C16, Cl8, C18:1, C20, and C22 (Table I). The dicarboxylicacid fraction when subjected to combined GLC-MS revealedC16, C18, C18:1, C20, and C22 acids (Table II); these diesters wereidentified by their retention times and mass spectra. The diag-nostic ions used for identifying the saturated components wereM+, M+-31, M+-73, M+-92, M+-105, and M+-123, whereas theunsaturated component showed M+, M+-32, M+-50, M+-64, andM+-92 (5, 10). Analysis of methyl esters ofco-hydroxy fatty acids

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DEAN AND KOLAT17UKUDY

Table I. Composition of Fatty Acids and Fatty Alcohols from theWound-healed Tomato Tissue

Chain Fatty Fatty-Length Acid Alcohol

Ci16 50.5 Trace

C18 14.5 16.6

C18:u 21.8 3.1

C20 6.7 33.7

C22 6.5 46.6

Monomer composition was determined by thin layer chromatographyand gas-liquid chromatography of the wound material after depolymeri-zation with BF3 in methanol. Column temperatures were 230 C and240 C for the fatty acids and fatty alcohols, respectively, and inletpressure was 25 p.s.i. Values are expressed as a per cent of each fraction.

Table II. Composition of Dicarboxylic Acids and e-Hydroxy Acidsfrom the Wound-healed Tomato Tissue

Chain Dicarboxylic w-Hydroxy AcidLength Acid

C16 52.7 37.8

C18 8.6 Trace

Cl8:, 35.8 22.0

C20 0.8 11.6

C22 2.0 25.4

C24 ... 3.3

Monomers were obtained as in Table I. Column temperatures were230 C and 220 C for dicarboxylic acids and w-hydroxy acids, respec-tively, and inlet pressure was 25 p.s.i. Values are expressed as a per centof each fraction.

as trimethylsilyl ethers showed that this fraction contained C16 toC24 (Table II), and in this case, the major diagnostic ions usedwere MI, M+-15, M+-31, M+-47, and the metastable ion repre-senting the transition (M+-15) -* (M+-47) (5, 10). Also detectedwas dihydroxyhexadecanoic acid, as determined by its retentiontime and its mass spectrum, which showed a weak parent ion atm/e 446 and fragment ions M+-15, M+-31, M+-47-32, M+-15-90, and M+-31-90. Three pairs of a-cleavage ions at m/e 245,259, 273, 275, 289, and 303 indicated the presence of positionalisomers in this fraction (5, 8, 10). From the relative intensities ofthe a-cleavage ions, it was apparent that 10,16-dihydroxy-C16acid was the major component, whereas the 9,16- and 8,16-dihydroxy-C16 acids were minor components.The results discussed above show that the major aliphatic

components produced during wound-healing of tomato fruits aredicarboxylic acids and w-hydroxy acids. These two classes ofcompounds are the major aliphatic components characteristic ofsuberin (4, 6, 7, 9, 10, 13, 17). In addition, C20 and C22constituted more than 80% of the fatty alcohol fraction, and inthe fatty acid fraction, similar very long carbon chains werefound. These alcohols and acids are also characteristic compo-nents of suberin. Dihydroxy C16 acid, which is a major compo-nent of the cutin of this plant, is only a minor component of thewound polymer, and as pointed out above, it is doubtful whethereven this small amount of the dihydroxy acid is synthesizedduring wound-healing. It is quite clear that the wound polymer is

a suberin-type material, and, therefore, the wound-healing proc-ess involved suberization rather than resynthesis of cutin, whichis the natural protective covering of this tissue.

Identification of the Aliphatic Components from the Cuticleand Wound Polymers of Bean Pods. The lipid monomer fractionobtained by LiAlH4 treatment of the cutin-containing outertissue layer, excised from bean pods, was subjected to TLC. Twomajor components with RF values corresponding to fatty alcoholand C16 triol were observed, together with a minor componentwith an RF identical to alkane-a,o.-diol. Combined GLC-MS wasused to identify the hydrogenolysis products (Fig. 2a), and ineach case, both retention time and the mass spectrum were usedfor this purpose, as indicated in the previous section. Among thefatty alcohols, C16 (peak 1) and C18 (peak 3) were the majorcomponents, whereas C17 (peak 2), C19 (peak 4), and C20 (peak6) were minor components. Some of the fatty alcohols, particu-larly C16 and C18 alcohols, might have resulted from the LiAlH4reduction of residual tissue lipids which might not have beencompletely removed by the extraction techniques used prior tohydrogenolysis. The major cutin components in this tissue wereC16 diol (peak 5) and C16 triol (peak 8). Even though detailedanalysis of the positional isomer composition of the dihydroxyC16 acid fraction of this cutin was not performed, the observationthat the sum of relative intensities of the a-cleavage ions at m/e289 and m/e 303 constituted about one-fourth of the sum of theintensities of the other pair at m/e 275 and m/e 317 clearly showsthat this cutin did indeed contain a mixture of positional isomersof dihydroxy C16 acid. In any case, cutin of bean pods appears tobe quite similar in composition to that of several other plants (9,12, 14).TLC of the hydrogenolysis products from the wound material

produced by the bean pods showed two components, one with anRF corresponding to alkane-a,cw-diol, and the other to fattyalcohol. Combined gas chromatography and mass spectrometryshowed (Fig. 2b) a homologous series of alcohols with retentiontimes of C16 (peak 1), C17 (peak 2), C18:0 + C18:u (peak 3), Cl9(peak 4), C20 (peak 6), and C22 (peak 10). The major compo-nents were found to be hexadecane-1,16-diol (peak 5) and

a3

4 28 z

0576(i)

0b

LU

8 2

4

10 5 0TIME (min.)

FIG. 2. Gas-liquid chromatogram of trimethylsilyl ethers of the hy-drogenolysate from (a) cutin-containing tissue; and (b) wound polymerfrom bean pods. Monomers were isolated by TLC as in Figure 1. Otherexperimental conditions are described in Figure 1 legend. Peak numbersdo not correspond to the same compound on separate chromatograms.

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octadecene-1,18-diol (peak 8). Although the retention time ofpeak 9 corresponds to C16 triol, the structure of this componentcould not be readily determined by its mass spectrum. Thecomposition of the wound material produced by the bean pods ischaracteristic of suberin polymers from other sources, as indi-cated in a previous section. Thus, it can be concluded that thebean pods did not resynthesize the cutin polymer similar to thatwhich had been removed, but instead synthesized a suberin-typepolymer during wound-healing.

Identification of the Aliphatic Components of the Cuticle andWound Polymers from Jade Leaves. TLC of the hydrogenolysisproducts from the cutin-containing tissue of the Jade leavesshowed one major band with an RF corresponding to C16 trioland minor bands with RF values identical to those of fattyalcohol, alkane-a,o-diol, and C18 triol. Identification of thecomponents from these various fractions was achieved by GLC-MS (Fig. 3a). Octadecanol was the major alcohol identified fromthis sample (peak 1). Hexadecane-1,16-diol (peak 2) was pres-ent in significant amounts, as was C18 triol (peak 4) and C18tetraol (peak 5). The major aliphatic component from this tissuewas shown to be hexadecanetriol (peak 3). C18 diol was detectedonly as a minor component by mass spectrometry. Thus, thecutin of Jade leaves is similar in composition to cutins fromseveral other plants (9, 12, 14).TLC of the products of hydrogenolysis of the wound material

from Jade leaves showed bands with RF values corresponding tothose of fatty alcohol, alkane-a,-diol, and C18 triol. CombinedGLC-MS showed a homologous series of alcohols (Fig. 3b) withretention times and mass spectra identical to those of C16 (peak1), C17 (peak 2), C18:0 + C18g: (peak 3), C19 (peak 4), C20 (peak6), C22 (peak 8), C24 (peak 10, which also contained C18 tetraol),and C26 (peak 12) alcohols. The major component from thismaterial was identified as octadecene-1,18-diol (peak 7). Alsopresent were hexadecane-1,16-diol (peak 5) and docosane-1,22-diol (peak 11). C16 triol was detected by mass spectrometry, aswas C18 triol (peak 9) and C18 tetraol (peak 10). In the cutin-containing layer of tissue, dihydroxy C16 acid (measured as C16triol) constituted nearly 60% of the aliphatic monomers,whereas in the wound polymer, o-hydroxy acids and dicarbox-ylic acids (measured together as diols) accounted for nearly 60%of the aliphatic monomers. The composition of the aliphaticcomponents of the polymeric material deposited during wound-healing strongly suggests that in this leaf tissue, wound-healinginvolved suberization rather than resynthesis of the natural poly-mer, cutin.

TIME (min.)FIG. 3. Gas-liquid chromatogram of trimethylsilyl ether of the hy-

drogenolysate from (a) cutin-containing tissue; and (b) wound-healedJade leaf sections. Monomers were isolated by TLC as in Figure 1. Otherexperimental conditions were as in Figure 1.

CONCLUSIONS

Based on the monomer composition of cutin and suberin froma variety of plants, we had previously proposed a guideline forthe classification of phytopolymers as cutin and suberin (9, 10).The major distinction between these two polymers is that theformer contains polar in-chain hydroxylated and/or epoxy acidsas major components, whereas the latter contains o-hydroxyacids and dicarboxylic acids as the major aliphatic components.The results presented here show that the major aliphatic compo-nents of the polymeric material, deposited during wound-healingof tomato fruit, bean pods, and Jade leaves, are clearly thosewhich are characteristic of suberin. Even though the naturalprotective polymer of these tissues is shown to be cutin, wound-healing of these tissues involves deposition of suberin rather thanresynthesis of the natural polymeric cover. Therefore, it appearsthat suberization might be a fundamental process involved inwound-healing of all plant tissues. Thus, previous attempts touse wound-healing tissues as an experimental system for investi-gating cutin biosynthesis do not appear valid.

It is not known how wounding induces the synthesis of sub-erin, although it would appear that some component formed as adirect or indirect consequence of wounding triggers the induc-tion of the enzymes involved in suberin biosynthesis. Since o-hydroxy acids and dicarboxylic acids are two major aliphaticcomponents of suberin, &-hydroxylating enzyme and w-hydroxyacid dehydrogenase should be two of the key enzymes induced asa result of wounding. Even though the former enzyme has notbeen heretofore obtained from wound-healing tissues, the latterenzyme has been found in cell-free preparations obtained fromwound-healing potato tissue (1). With the use of cyclohexamideand actinomycin D, it was demonstrated that the synthesis ofRNA and protein, involved in the production of the o-hydroxyacid dehydrogenase, occurred between 72 and 96 hr after thewounding of this tissue (V. P. Agrawal and P. E. Kolattukudy,unpublished results). The previously reported time course ofdeposition of suberin monomers (11) was consistent with thisobservation, as was the time course of incorporation of [1-14C]acetate into suberin monomers in potato slices (B. B. Deanand P. E. Kolattukudy, unpublished results). Thus, all experi-mental evidence shows that the induction of the enzymes in-volved in the biosynthesis of the aliphatic components of suberinoccurs a considerable period of time after wounding and, there-fore, it appears likely that the chemical events which occur at thewound trigger suberization only indirectly, presumably througha series of biochemical changes which occur during the first 72hr. However, since the time course of suberization is not knownin any other tissue, the above generalization should be consid-ered tentative.

Acknowedgment-We thank C. Oldenburg for raising the plants.

LITERATURE CIED

1. AGRAWAL, V. P. AND P. E. KOLATrUKUDY. 1974. c-Hydroxy acid dehydrogenase enzymefrom suberized potato slices. Abstract 202, 29th Annual Northwest Regional Meeting ofAmerican Chemical Society.

2. BLACKMAN, F., G. FRosr, AND L. C. MATnRAE. 1901. On the reaction of leaves totraumatic stimulation. Ann. Bot. 15: 533-546.

3. BREDEMEUER, G. AND W. HFINEN. 1968. I. Free fatty acid movement during cutinsynthesis in injured Gasteria verrucosa leaves. Acta Bot. Neerl. 17: 15-25.

4. BmaEsKoRN, C. H. AND P. H. BINNEMANN. 1975. Carbonsiuren und Alkanole des Cutinsund Suberins von Solanum tuberosum. Phytochemistry 14: 1363-1367.

5. EGLINTON, G. AND D. H. HUNNEMAN. 1968. Gas chromatographic-mass spectrometricstudies of long chain hydroxy acids. I. The constituent cutin acids of apple cuticle.Phytochemistry 7: 313-322.

6. HOLLOWAY, P. J. 1972. The composition of suberin from the corks of Quercus suber L. andBetula pendula Roth. Chem. Phys. Lipids 9: 158-170.

7. HOLLOWAY, P. J. 1972. The suberin composition of the cork layers from some Ribes species.Chem. Phys. Lipids 9: 171-178.

8. HOLLOWAY, P. J. AND A. H. B. DEAs. 1971. Occurrence of positional isomers of dihydroxy-hexadecanoic acid in plant cutins and suberins. Phytochemistry 10: 2781-2785.

9. KoLArruKuDY, P. E. 1975. Biochemistry of cutin, suberin, and waxes, the lipid barriers on

415Plant Physiol. Vol. 58, 1976

www.plantphysiol.orgon July 13, 2018 - Published by Downloaded from Copyright © 1976 American Society of Plant Biologists. All rights reserved.

DEAN AND KOLATFUKUDY

plants. In: T. Galliard and E. I. Mercer, eds., Recent Progress in the Chemistry andBiochemistry of Lipids. Academic Press, London pp. 203-246.

10. KOLATrUKUDY, P. E. AND V. P. AGRAWAL. 1974. Structure and composition of aliphaticconstituents of potato tuber skin (suberin). Lipids 9: 682-691.

11. KoLATruKUDY, P. E. AND B. B. DEAN. 1974. Structure, gas chromatographic measure-

ment, and function of suberin synthesized by potato tuber tissue slices. Plant Physiol. 54:116-121.

12. KOLATTUKUDY, P. E. AND T. J. WALTON. 1973. The biochemistry of plant cuticular lipids.Prog. Chem. Fats Other Lipids 13: 121-175.

13. KoLAtruKUDY, P. E., K. KRONMAN, AND A. J. POULOSE. 1975. Determination of structureand composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet,and sweet potato by combined gas-liquid chromatography and mass spectrometry. PlantPhysiol. 55: 567-573.

14. MARTIN, J. T. AND B. E. JUNIPER. 1970. The Cuticles of Plants. St. Martins Press, Inc.,New York.

Plant Physiol. Vol. 58, 1976

15. McCLosKY, J. A., R. N. SnLLwELL, AND A. M. LAWSON. 1968. Use of deuterium-labeledtrimethylsilyl derivatives in mass spectrometry. Anal. Chem. 40: 233.

16. PRimsrLY, J. H. AND L. M. WOFFNDEN. 1932. Physiological studies in plant anatomy. PartV. Causal factors in cork formation. New Phytol. 21: 252-268.

17. RoDouQuEz-MIouENs, B. AND I. RiBAS-MARQUES. 1972. Investiquciones quimicas sobre elcorcho de Solanum tuberosum L. (Patata). Quimica 68: 302-308.

18. SCHNEIDER-ORELLI, 0. 1911. Versuche fiber Wundung und Wundverschluss an Pflanzenor-ganen. Centr. Bakt. 2 Abt. 30: 420-429.

19. SHARKEY, A. G., R. A. FRIEDEL, AND S. H. LANGER. 1957. Mass spectra of trimethylsilylderivatives. Anal. Chem. 29: 770.

20. WACHTER, W. 1905. Wundverschluss bei Hippuris vulgaris L. Beih. Zum. Bot. Centr. 18:1.

21. WALTON, T. J. AND P. E. KoLArruKUDY. 1972. Determination of the structures of cutinmonomers by a novel depolymerization procedure and combined gas chromatography andmass spectrometry. Biochemistry 11: 1885-1897.

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