growth-dependent chemical and mechanical properties of cuticular membranes from leaves of sonneratia...

Growth-dependent chemical and mechanical properties ofcuticular membranes from leaves of Sonneratia albapce_2482 1..10

YUKI TAKAHASHI1, SHUNTARO TSUBAKI1*, MASAHIRO SAKAMOTO1, SHIN WATANABE2 & JUN-ICHI AZUMA1

1Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, KitashirakawaOiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan and 2Iriomote Station, Tropical Biosphere Research Center, University ofRyukyus, 870, Uehara, Taketomi-cho, Yaeyama-gun, Okinawa 907-1541, Japan

ABSTRACT

Chemical and mechanical properties of the leaf cuticularmembranes (CMs) of a mangrove, Sonneratia alba J.Smith, were analysed at various leaf development stages toevaluate their tolerance to environmental stress. Our analy-ses demonstrate that the CMs from leaves of S. alba atdifferent growth stages are generally rich in wax (21.5–25.7%) and cutin (52.4–63.4%) which rapidly accu-mulate at the early stages of leaf growth, while cutan(4.3–10.3%) and polysaccharide (2.3–7.7%) continuouslyaccumulate throughout growth. Immature CMs are physi-cally weak and highly viscoelastic. However, CMs becomestrengthened and stiffened during leaf expansion and matu-ration (by factors of about 1.5 and 2.4, respectively) whiletheir flexibility decreases (68–83% decrease). Finally, theCMs lose their strength at the senescent stage (30–43%decreasement). Correlation analysis between chemicalcomposition and mechanical properties revealed that thecutin matrix is mainly responsible for the high viscoelasticproperties of CMs, while wax, cutan and polysaccharidecontributed to their elasticity. Wax also affected thestrength of the CMs, whereas cutan and polysaccharideshowed rigidizing effect. Rapid accumulation of wax andcutin in the CMs after bud burst followed by the mechanicalsupports of cutan and polysaccharide in an isolateralmanner contributed to the remarkable environmentaltolerance of S. alba.

Key-words: biomechanics; cuticular membrane; cutan; cutin;growth dependence; mangrove; polysaccharide; wax.

INTRODUCTION

Sonneratia alba, which belongs to a genus of mangroveplants found in the Indo-West Pacific region, is an ever-green tree with tall conical pneumatophores arising fromhorizontal roots. Its leaves are opposite arranged, ovalshaped, succulent and isolateral. They generally grow in the

outermost area of the intertidal zone, and often form theseaward fringe (Tomlinson 1986). Their most strikingfeature is high tolerance against severe environmentalgrowth conditions such as salinity, strong sunlight and saltywind (Wakushima, Kuraishi & Sakurai 1994). Highly devel-oped surface layers are considered to play an importantrole in S. alba’s durability.

Plant cuticular membranes (CMs) are continuous extra-cellular membranes located at the boundary of plant tissuesand their external environment, especially on the aerialsurface of leaves. CMs serve as the chemical and physicalbarrier against various biotic and abiotic impacts as well asmechanical support (Heredia 2003; Bargel et al. 2006; Stark& Tian 2006). In the case of S. alba, the primary role of theleaf CMs is providing physical protection against weather-ing, abrasions, leaching and water loss. CM is composed of astructural matrix called cutin, wax, polysaccharides andresidual materials called cutan. Waxes are embedded insidethe cutin and also cover the outer surface of CMs. Chemi-cally, waxes are composed of complex mixtures of longchain hydrocarbons (alkanes, alcohols, ketones, fatty acidsand esters), phenolics, terpenes and sterols (Tulloch 1976;Riederer & Markstädter 1996). Cutin, the major structuralpolymer of CMs, is an amorphous biopolyester formed byhydroxylated and/or epoxy-hydroxylated C16 and C18 fattyacids (Holloway 1980; Kolattukudy 1980; Jeffree 1996).Cutan is a structural polymer consisting of an aromaticfraction and/or an aliphatic part having polymethylenicchains (Domínguez, Heredia-Guerrero & Heredia 2011b)that is neither hydrolyzed with acid nor saponified withalkali (Nip et al. 1986; Jeffree 1996).

Mechanical properties of CMs have been extensivelystudied in tomato (Solanum lycopersicum L.) because oftheir importance in maintaining fruit quality (Bargel &Neinhuis 2005; Domínguez, Cuartero & Heredia 2011a).López-Casado et al. (2007) have reported the significantcontribution of cuticular polysaccharides and cutin toelastic and viscoelastic properties, respectively, of tomatofruit CMs. Accumulation of flavonoids also contributes tostiffening of the cutin network in relation with fruit matu-ration (Domínguez et al. 2009). On the other hand, hydra-tion of CMs increases their viscoelasticity and extensibility,suggesting a plasticizing effect of water on the cutin matrix(Petracek & Bukovac 1995; Wiedemann & Neinhuis 1998;

Correspondence: J. Azuma. Fax: +81 75 753 6471; e-mail: [email protected]

*Present address: Science Research Center, OceanographySection, Kochi University, Akebono-cho 2-5-1, Kochi-shi, Kochi,780-8520, Japan.

Plant, Cell and Environment (2012) doi: 10.1111/j.1365-3040.2012.02482.x

© 2012 Blackwell Publishing Ltd 1

Edelmann, Neinhuis & Bargel 2005). This kind of effectof water on cutin matrix was also obtained by atomicforce microscopy (AFM) and nuclear magnetic resonance(NMR) spectroscopy at microscopic scale (Round et al.2000).

Biomechanics of CMs of matured leaves of Yucca aloifo-lia L., Clusia fluminensis Planch. & Triana, Nerium oleanderL. and Hedera helix L. were reported (Wiedemann & Nein-huis 1998; Edelmann et al. 2005). However, as pointed outby Domínguez et al. (2011a), little work has been done onthe variations of the chemical and mechanical properties ofCMs during growth. Maintaining the durability of the leafCMs by increasing their physical strength is important forS. alba which grows in severe environmental conditions atthe seaward fringe. Therefore, great efforts should befocused on understanding the process of biomechanicaldevelopment of the S. alba leaf CMs during growth. In thispaper, we describe the growth-dependent changes in chemi-cal and mechanical properties of the CMs from leaves ofS. alba, and discuss the chemical factors that contribute totheir durability by analysing five pairs of leaves at differentgrowth stages.

MATERIALS AND METHODS

Plant materials and isolation of CMs

Pairs of oppositely arranged isolateral leaves of S. alba J.Smith were collected from mature canopy trees which weresparsely distributed in the outer fringes of a mangroveforest in the Komi estuary (24°19′ N, 123°54′ E), IriomoteIsland, Okinawa, Japan in October 2008, May 2009 andFebruary 2010. Branches with five pairs of unfolded leaveswere selected and all pairs of leaves were consecutivelynumbered from the top to the bottom (L1-L5) according toLowman & Box (1983).The first pair of leaves (L1) from thebranch top was at the immature expanding stage just afterbud burst. The second leaf (L2) was also at the immaturestage but almost attaining full expansion.The third (L3) andthe fourth (L4) leaves were at the fully grown maturedstage. The fifth leaf (L5) was at senescent stage beforeabscission.Thickness of the leaf was measured at 4–6 pointsper sheet using a digital micrometer (DP-1 VR, MitutoyoCo., Tokyo, Japan). Surface areas of the leaves were calcu-lated using image analysis software (ImageJ, National Insti-tutes of Health, Bethesda, MD, USA).

Each pair of leaves was treated with an enzyme solutioncontaining fungal pectinase (2%, w/v, Aspergillus niger,Sigma-Aldrich, St Louis, MO, USA) and a commercial cel-lulase preparation (0.5%, w/v, Meicelase CEP 16710, Tri-choderma viride, carboxymethyl cellulase activity 2920 U,Meiji Seika Kaisha Ltd, Tokyo, Japan) in sodium acetatebuffer (5 mm, pH 5.0) for 72 h at 36 °C. CMs from bothadaxial (upper) and abaxial (lower) sides were separatelyisolated as thin films, washed with distilled water and dried.Surface of isolated CMs were morphologically character-ized using low-voltage scanning electron microscopy (LV-SEM, VE-8800, Keyence Co., Osaka, Japan), at 500-fold

magnification with 1.7 kV of accelerating voltage.Thicknessof the CMs was measured in the same way as those ofleaves.

Analyses of chemical constituents of CMs

The chemical components of the CMs were sequentiallyremoved according to the modified method of Domínguezet al. (2009). Cuticular waxes were extracted three timeswith a mixture of chloroform/methanol (2:1, v/v) at 50 °Cfor 2 h. Dewaxed CMs were saponified three times with 1%potassium hydroxide in methanol at 70 °C for 2 h to esti-mate the cutin content. Decutinized residues were thor-oughly washed with methanol after neutralization with 3%acetic acid in methanol, and then hydrolyzed according tothe method of Saeman, Bubl & Harris (1945). The samplewas mixed in 72% sulfuric acid, and kept at room tempera-ture for 1 h, and subsequently the mixture was diluted withdistilled water to 3% H2SO4, and autoclaved at 120 °Cfor 1 h. The final non-saponifiable and non-hydrolyzableresidue was defined as cutan as described by Chen et al.(2008). Composition of monosaccharides obtained by acidhydrolysis of dewaxed and decutinized residues was deter-mined by high performance anion-exchange chromato-graphy (HPAEC, DX-500, Dionex, Sunnyvale, CA, USA)equipped with a pulsed amperometric detector and acolumn of CarboPac PA-1 (column size: 4.0 ¥ 250 mm,Dionex) using 1.0 mm aqueous NaOH as an eluent at a flowrate of 1.0 mL min-1 as previously reported (Tsubaki et al.2008). The polysaccharide content was quantified by thephenol-sulfuric acid method (Dubois et al. 1956) using astandard solution containing arabinose, xylose, rhamnose,galactose, glucose and mannose at a ratio determined byHPAEC. Removal of CM components by sequential extrac-tions were evaluated by Fourier transform infrared (FT-IR)spectroscopy (FT/IR-4100, JASCO Co., Tokyo, Japan), at a2.0 cm-1 resolution, and analytical range of 400–4000 cm-1

by the thin-film method or the KBr disc method.

Compositional analyses of cutin monomer

Depolymerization of cutin was carried out by hydrogenoly-sis or deuteriolysis according to the method of Walton &Kolattukudy (1972). Dewaxed CMs were refluxed in tet-rahydrofuran with an excess amount (2.5 times by weight)of LiAlH4 or LiAlD4 (Tokyo Chemical Industry Co., Ltd,Tokyo, Japan) for 48 h at 70 °C. Reduced cutin monomerswere extracted by diethyl ether after acidification byhydrochloric acid, then dehydrated by anhydrous so-dium sulfate and evaporated to dryness under reducedpressure. Obtained samples were dissolved in excessN,O-bis(trimethylsilyl)-acetamide (Tokyo Chemical Indus-try Co., Ltd) and heated at 70 °C for 30 min to preparetrimethylsilyl (TMS) derivatives. Monomer composition ofcutin was analysed by GC/MS (GC/MS 2010/PURVUM 2,Shimazu Co., Kyoto, Japan) equipped with a DB-1 column(J&W Scientific, 0.25 mm ¥ 30 m, d.f. = 0.25 mm, AgilentTechnologies, Inc., Santa Clara, CA, USA) with a helium

2 Y. Takahashi et al.

© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment

carrier gas at a flow rate of 0.91 mL min-1.The column oventemperature was programmed from 195 to 240 °C at therate of 2 °C min-1 and held for 10 min, then heated up to300 °C at the rate of 10 °C min-1. The mass spectra wereobtained in the range of 40–650 m/z by scanning mode withelectron impact ionization at 70 eV.

Biomechanical analyses of CMs

Mechanical properties of the native and dewaxed CMswere measured by a tensile tester (Tack Tester TA-500,UBM Co., Kyoto, Japan), the equipment which is similar tothat used by Wiedemann & Neinhuis (1998). Rectangularuniform segments 5 ¥ 20 mm in size were cut out from theCM films, clipped with stainless clamps and deformed at aconstant rate (0.01 mm s-1) at room temperature (23 °C).Data of strain and tensile force were collected twice persecond. Elastic modulus (E, MPa) was obtained from alinear region at the initial part of the S-S curve. Breakingstress (smax, MPa) and maximum strain (emax, %) were deter-mined at the point of failure of the CM films.

Statistics

Multiple comparisons were performed by the Tukey–Kramer’s HSD test at 5% level of significance, afterone-way analysis of variance (anova). Confirmations of nor-mality by Shapiro–Wilk test and rejections of outlier werecarried out prior to data analyses. Correlation analyseswere performed by using Pearson’s product-moment corre-lation coefficient at 5 and 1% levels of significance.

RESULTS

Characterization of isolated CMs

Full leaves and enzymatically isolated CMs of S. alba werecompared in respect to CMs physical and morphologicalcharacteristics at each stage of leaf growth. CMs of S. alba

had similar surface morphologies on both sides of theleaves. As shown in Fig. 1, the CMs had a wrinkled surface,and stomata were observed in both adaxial and abaxialCMs. Table 1 shows summarized values of leaf area, weightper unit area and thickness of the leaves and isolated CMs.The values of average leaf area greatly increased withexpansion of leaves from L1 (4.65 cm2) to L2 (17.35 cm2).The leaves at maturation and senescence stage (L3–L5) didnot change in area size (P < 0.05). However, both valuesof the weight per unit area and thickness of the leavesstill increased even after attaining full area size from66.5 mg cm-2 (L1) to 133.3 mg cm-2 (L5) and from 530 mm(L1) to 1255 mm (L5), respectively.

In the case of the CMs, the weight per unit area increasedabout 1.5-fold from L1 to L2; however, the increase reacheda plateau during L3–L5. Rapid increase in the average thick-ness of the CMs was also observed from L1 to L2 (increment5–6 mm), showing that both expansion and thickening of theS. alba leaf CMs take place mainly during growth from L1 toL2. No significant difference was observed between theweights of both sides of CMs (P < 0.05), while adaxial CMstend to be slightly thicker than abaxial CMs.

FT-IR analysis of CM components

Validity of the solvent extractions for the sequentialremoval of the constituents of the CMs was spectroscopi-cally verified by FT-IR prior to the compositional analyses.All CMs at different growth stages gave similar spectra.Figure 2 shows the representative data given by the adaxialCM at L4. The spectrum of the native CM had strongabsorptions at 2923, 2851 and 1734 cm-1 assigned as asym-metric and symmetric stretching vibrations of methylenegroups and stretching vibration of esterified carbonylgroups. These intense absorption bands showed predomi-nance of cutin (Ramirez et al. 1992; Villena, Domínguez &Heredia 2000). Spectrum of the extracted waxes was char-acterized by a strong absorption at 1687 cm-1 and corre-sponded to the stretching vibrations of the carbonyl groups

Figure 1. Typical surface LV-SEM images of the cuticular membranes (CMs) isolated from L2 leaves of Sonneratia alba (left, adaxialside; right, abaxial side; ¥500; bar equals 20 mm).

Cuticular membranes from leaves of Sonneratia alba 3


in addition to the strong alkyl peaks and a weak hydroxylpeak at 3349 cm-1. Removal of waxes from CMs slightlysharpened absorptions due to ester carbonyl, methyleneand hydroxyl groups. Decutinization drastically weakenedabsorptions due to the methylene and ester carbonylgroups. Acid hydrolysis decreased the absorptions around1000–1200 cm-1 together with lowering of hydroxyl groupdue to effective removal of polysaccharides (Kacurákováet al. 2000; Villena et al. 2000). Reappearance of the alkylpeaks in the final residue confirmed the presence of highlyrecalcitrant aliphatic polymer, cutan, in the S. alba leaf CMs.Broad absorption bands around 1650 cm-1 were assigned toC=C bonds (Villena et al. 1999; Chen et al. 2008) and pres-ence of aromatic cores was indicated by an absorption at1517 cm-1 due to C-C bonds conjugated with C=C bonds(Chen et al. 2008; Järvinen et al. 2011).

Chemical constituents

The chemical composition of the isolated leaf CMs ofS. alba was gravimetrically analysed. The results are shown

in Table 2 and Fig. 3. The overall quantitative results indi-cate presence of two sets of components that show differentpatterns of increase in concentration; one set of compo-nents was wax and cutin, and the other set was cutan andpolysaccharide.The former were present at a low level in L1

and at a rather high levels throughout L2–L5 (Fig. 3a,b),while the latter gradually increased from L1 to L5 (Fig. 3c,d).The proportion of wax did not show any differencethroughout L1–L5 (Table 2); however, its amount apparentlyincreased from L1 (98–112 mg cm-2) to L2 (142–153 mg cm-2),with slight increase to L5 (156–168 mg cm-2): n.b. statisticallysignificant difference was barely observed because of largevariance (Fig. 3a). Cutin was the most abundant constituentfollowed by wax, and the sum of these two predominantcomponents amounted to 74.2–88.5% of total CMs(Table 2). Similar to the wax content, the amount of cutindrastically increased from L1 (245–256 mg cm-2) to L2 (373–379 mg cm-2) although the values reached a plateau after L2

(Fig. 3b). These patterns of content increase of cutin andwax were consistent with the increase in the weight andthickness of the CMs (Table 1) showing that the construc-tion of the main framework has completed during therapid leaf expanding stages (L1 to L2). On the contrary,the amounts of cutan and polysaccharides continuouslyincreased throughout the leaf expansion, maturation andaging stages (L1–L5). The content of cutan was in the rangeof 19–77 mg cm-2 (adaxial) and 23–66 mg cm-2 (abaxial)while the content of polysaccharides was in the range of14–60 mg cm-2 (adaxial) and 13–49 mg cm-2 (abaxial). Accu-mulation of cutan and polysaccharide was evident at thematured and old senescent stages (Fig. 3c,d).Their contentswere, however, significantly smaller than those of wax andcutin; 5.2–11.1% (cutan) and 3.3–7.5% (polysaccharides)(Table 2).

Polysaccharides contained in the CMs were hydrolyzedand their monosaccharide composition was determinedby HPAEC as summarized in Table 2. Both adaxial andabaxial CMs showed similar monosaccharide compositions.

Table 1. Average area, weight and thickness of Sonneratia alba leaves and cuticular membranes (CMs) from adaxial and abaxial sides ofleaves

Component

Leaf number

L1 L2 L3 L4 L5

LeavesArea (cm2)† 4.65 � 2.10a 17.35 � 9.14b 17.77 � 6.35b 20.86 � 7.58b 19.31 � 6.25b

Weight (mg cm-2)† 66.5 � 7.2a 89.9 � 10.1b 93.2 � 20.4b 118.7 � 22.4c 133.3 � 29.2c

Thickness (mm)‡ 530 � 51a 720 � 96b 840 � 268c 1002 � 340d 1255 � 408e

Adaxial CMsWeight (mg cm-2)† 417 � 109a 647 � 169b 741 � 37bc* 754 � 127bc 888 � 122c*Thickness (mm)‡ 3 � 1a 9 � 1b* 9 � 2b* 9 � 3b 10 � 3b*

Abaxial CMsWeight (mg cm-2)† 457 � 106a 682 � 206b 692 � 52b* 728 � 86b 703 � 131b*Thickness (mm)‡ 3 � 1a 8 � 2b* 7 � 2b* 8 � 2b 9 � 2b*

Data are presented as mean value � SD. Significant differences (P < 0.05) among growth stages are indicated by different superscript letters.Asterisk shows significant differences (P < 0.05) between adaxial and abaxial CMs.†n = 5–12, ‡n = 28–40.

Figure 2. FT-IR spectra of native, dewaxed, decutinized andhydrolyzed cuticular membranes (CMs) and extracted cuticularwaxes from Sonneratia alba leaves. The spectra of adaxial CMs ofL4 was shown as representative.



Glucose was the most abundant monosaccharide amount-ing to 41.2–58.0%, followed by arabinose 27.3–40.1% withsmaller contents of galactose (4.7–8.8%) and xylose(5.3–11.1%). The results presented indicate that the cuticu-lar polysaccharides in the S. alba leaf CMs were composedof mixtures of cellulose, pectic and hemicellulosicpolysaccharides.

Cutin monomer composition

Monomer composition of the cutin in the CMs was deter-mined by reductive depolymerization followed by GC/MSanalysis. As shown in Supporting Information Table S1 andSupporting Information Figs S1–S3, fragmentation patternsof the mass spectra (MS) were identified according toWalton & Kolattukudy (1972). All cutins from the S. albaleaf CMs were composed of at least 14 monomers of which11 were identified as listed in Table 3. Predominant compo-nents were hexadecan-3-ol and octadecan-3-ol, accountingfor about 20–25 and 30–55% of the total monomers, respec-tively. Combination analysis of the deuterium-labelledmonomers allowed us to conclude that these componentswere derived from 9(10),16-dihydroxyhexadecanoic acid,which appeared as a single peak with a mixture of twoisomers, and 9,10-epoxy-18-hydroxyoctadecanoic acid,respectively (Supporting Information Figs S2 & S3).Four monohydroxy alkans (hexadecan-1-ol, octadecan-1-ol, hexadecan-2-ol and heptadecan-3-ol) and three

monohydroxy alkens (octadecen-2-ol, tetradecen-3-ol andoctadecen-3-ol) were also detected as minor components,amounting to less than 5% in total. Both of adaxial andabaxial CMs had similar cutin monomer compositionthroughout the growth stages.

Mechanical properties

Biomechanical properties of the isolated CMs were mea-sured by the tensile test. Specimens from both adaxial andabaxial CMs gave similar profiles. Typical S-S curves, repre-sentative of both adaxial and abaxial CMs, are shown inFig. 4. A clear biphasic (two phase with different slope)behaviour was observed in S-S curves from young leaf CMs(L1-L3), whereas most CMs from the fully matured leaves(L4 and L5) usually showed almost monophasic behaviour.Table 4 shows summarized values of biomechanical param-eters of the isolated leaf CMs. The values in smax increasedwith the leaf growth, attaining a maximum at L4 (7.29–8.74 MPa), then decreasing to 4.14–6.10 MPa at L5. TheCMs isolated from immature leaves (L1) showed highextensibility with emax of 7.55–9.07%; however, this valuedecreases during the leaf growth to 1.54–2.42% at L5. TheE-values of the isolated CMs were below 200 MPa at L1 inboth adaxial and abaxial CMs, but increased with leafgrowth attaining values higher than 390 MPa in CMs fromthe fully matured leaves (L4 and L5). Additionally, both ofthe adaxial and abaxial CMs showed similar mechanical

Table 2. Chemical composition ofSonneratia alba leaf cuticular membranes(CMs) and relative neutral sugarcomposition of cuticular polysaccharides(%, w/w)

Component

Leaf number

L1 L2 L3 L4 L5

Adaxial CMSWaxa 25.7 � 8.2 23.1 � 7.5 22.9 � 3.9 24.1 � 3.7 23.4 � 4.4Cutina 63.4 � 10.1 60.1 � 3.6 59.2 � 6.4 57.5 � 2.9 54.4 � 4.3Cutanb 5.0 � 4.4 5.1 � 2.3 6.5 � 2.8 8.6 � 4.2 10.2 � 3.5Polysaccharideb 3.5 � 0.9 3.6 � 0.6 5.8 � 1.2 6.4 � 1.3 7.4 � 2.3Monosaccharide compositionc

Arabinose 27.4 � 2.4 31.9 � 0.2 39.3 � 1.7 37.1 � 1.0 37.9 � 2.5Rhamnose 0.7 � 0.4 0.6 � 0.1 0.9 � 0.6 0.1 � 0 0.6 � 0.2Galactose 7.2 � 0.5 4.7 � 0.8 6.9 � 0.8 7.5 � 0.5 6.7 � 0.7Glucose 58.0 � 4.5 54.1 � 1.2 45.7 � 1.9 45.8 � 1.3 43.5 � 2.5Xylose 5.6 � 1.7 5.8 � 0.5 5.6 � 1.6 6.8 � 0.3 7.8 � 0.8Mannose 1.2 � 0.3 2.8 � 0.8 1.6 � 0.6 2.6 � 0.6 3.6 � 0.1

Abaxial CMsWaxa 23.2 � 4.0 21.5 � 5.4 24.5 � 5.4 24.5 � 4.4 25.2 � 4.7Cutina 60.7 � 11.8 60.9 � 3.8 52.4 � 5.5 55.9 � 2.9 53.5 � 3.3Cutanb 4.3 � 1.3 6.4 � 2.4 8.4 � 2.7 8.4 � 2.7 10.3 � 3.5Polysaccharideb 2.3 � 0.6 3.3 � 0.4 4.4 � 0.9 6.5 � 1.3 7.7 � 3.1Monosaccharide compositionc

Arabinose 28.3 � 4.4 27.3 � 3.4 40.1 � 0.9 39.4 � 2.0 35.1 � 0.4Rhamnose 0.9 � 0.3 0.8 � 0.3 1.0 � 0.5 0.3 � 0.2 0.6 � 0.2Galactose 6.1 � 1.1 8.8 � 2.4 7.1 � 1.2 8.0 � 0.4 6.7 � 0.4Glucose 56.6 � 3.5 47.8 � 4.5 41.2 � 0.9 41.9 � 2.4 45.0 � 1.1Xylose 5.3 � 1.2 11.1 � 5.4 7.7 � 0.1 7.6 � 0.8 9.1 � 0.7Mannose 1.5 � 0.5 4.2 � 0.5 2.9 � 0 2.8 � 0.4 3.6 � 0.2

Data are presented as mean value � SD.an = 8–12, bn = 4–6 and cn = 3.



properties as expected from their similar morphologicaland chemical properties.

To investigate the role of wax in the mechanicalproperties of CMs, the tensile test was also applied todewaxed CMs from the mature leaves (Fig. 4). In theadaxial side, the dewaxed CM at L4 showed 4.82 MPaof smax, 5.43% of emax and 162 MPa of E. The cor-responding values of the abaxial side were 3.89 MPa,4.16% and 138 MPa, respectively. By removal of waxes,both values of physical strength and stiffness of maturedleaf CMs drastically decreased strongly, indicating thatthe accumulation of cuticular waxes in CMs contributesto the mechanical properties of CMs. After dewaxingand decutinization, all CMs became very brittle and didnot maintain mechanical strength enough to carry out thetensile test.

Relationship between chemical andmechanical properties

The relationship between chemical and mechanical proper-ties of the CMs was statistically evaluated and the resultsare summarized in Table 5. In the wax content, significantcorrelations were observed with the smax value (R = 0.331)and the E-value (R = 0.433). The cutin content was posi-tively correlated with the emax value (R = 0.507) and nega-tively correlated with the E-value (R = -0.468). In contrast,the contents of cutan and polysaccharide showed goodnegative correlations with the emax value (R = -0.549 and-0.694) and relatively weak positive correlation with theE-value (R = 0.237 and 392), respectively. No significant cor-relation was observed in the compositions of cutin mono-mers and monosaccharides.

300(a)

250

200

150

Wax

(mg

cm

–2)

100

50

01

a a

b ab a b a b a

2 3Leaf number

4 5

600(b)

450

300

Cu

tin

(mg

cm

–2)

150

01

a a

b b b b b b b b

2 3Leaf number

4 5

120(c)

100

80

60

Cu

tan

(mg

cm

–2)

40

20

01

a a

ab ab ab

abc bc bc

cc

2 3Leaf number

4 5

90(d)

75

60

Po

lysa

cch

arid

e (m

g c

m–2

)

30

45

15

01

a a

ab* ab*

b*

ab*

bc

bc

c

c

2 3Leaf number

4 5

Figure 3. Amount of chemical constituents (a, wax; b, cutin; c, cutan; d, polysaccharide) of the cuticular membranes (CMs) from adaxialand abaxial sides of Sonneratia alba leaves. Data are presented as mean � SD (a and b, n = 8–12; c and d, n = 4–6). Solid and opencolumns show adaxial and abaxial CMs, respectively. Significant differences (P < 0.05) among growth stages were indicated by differentsuperscript letters. Asterisk shows significant differences (P < 0.05) between adaxial and abaxial CMs.



DISCUSSION

In the study presented here, we characterized the variationin chemical composition of the CMs from leaves of S. albaduring expansion, maturation and aging stages. The quanti-tative analyses of the chemical constituents revealed agrowth dependence (Fig. 3), consistent with previous devel-opmental models of leaf CMs; deposition of wax and cutinrapidly proceeds in the early stages of development,whereas the quantity of cutan appears to increase at laterstages (Jeffree 1996).

CMs of S. alba contained higher amount of wax(Table 2) than previously reported in H. helix, Olea euro-paea L., Agave americana L., Clivia miniata Reg. andCitrus limon Burm. f. These plants were shown to containapproximately 12, 26, 16, 7 and 8% of wax, respectively(Nip et al. 1986; Hauke & Schreiber1998; Johnson et al.2007). By comparison, the leaf CMs of S. alba are rich inwax (Table 2). The abundance of the cuticular wax shouldprovide higher water repellency to the leaves of S. albawhich are often submerged in sea water and subjected tosalty winds.

Figure 4. Typical stress–strain curves of isolated cuticularmembranes (CMs) and dewaxed CMs from Sonneratia albaleaves. The curves of adaxial CMs are shown as representatives.

Table 3. Composition (relative area %) of reduced cutin monomers obtained by hydrogenolysis of cutin in Sonneratia alba leaf cuticularmembranes (CMs)

Component

Leaf number

L1 L2 L3 L4 L5

Adaxial CMsHexadecan-1-ol (C16:0) 0.23 � 0.14 0.04 � 0 0.18 � 0.11 0.09 � 0.01 0.25 � 0.11Octadecan-1-ol (C18:0) 0.12 � 0.05 0.03 � 0.02 0.03 � 0.03 0.06 � 0.02 0.13 � 0.07Hexadecan-2-ol (C16:0) 0.76 � 0.09 0.57 � 0.02 0.77 � 0.17 0.72 � 0.06 1.10 � 0.23Octadecen-2-ol (C18:1) 0.54 � 0.18 0.71 � 0.22 1.85 � 0.20 1.22 � 0.08 2.99 � 0.10Hexadecan-3-ol (C16:0) 23.70 � 1.88 21.57 � 0.84 25.57 � 1.14 24.81 � 0.27 20.91 � 1.35Tetradecen-3-ol (C14:1) 0.70 � 0.08 0.48 � 0.07 2.91 � 0.18 1.70 � 0.07 10.73 � 0.56Heptadecan-3-ol (C17:0) 0.18 � 0.03 0.12 � 0.01 0.27 � 0.03 0.51 � 0.02 0.14 � 0.04Unknown 1 2.96 � 0.09 2.15 � 0.13 0.20 � 0.03 0.86 � 0.06 0.42 � 0.06Octadecen-3-ol (C18:1) 0.56 � 0.13 0.60 � 0.05 0.79 � 0.05 0.50 � 0.04 3.33 � 0.53Octadecan-3-ol (C18:0) 54.02 � 1.25 49.35 � 0.45 47.48 � 1.21 49.27 � 0.28 31.71 � 2.19Unknown 2 – 0.79 � 0.11 9.80 � 0.08 4.71 � 0.16 5.59 � 1.62Hexadecen-3-ol (C16:1) 0.90 � 0.15 1.08 � 0.09 3.29 � 0.68 3.15 � 0.10 11.75 � 1.76Octadecan-4-ol (C18:0) 11.57 � 0.40 15.38 � 0.60 2.75 � 0.01 10.22 � 0.07 3.38 � 0.11Unknown 3 3.76 � 0.23 7.13 � 0.69 4.11 � 0.22 2.20 � 0.07 7.56 � 0.72

Abaxial CMsHexadecan-1-ol (C16:0) 0.17 � 0.01 0.08 � 0.03 0.31 � 0.09 0.10 � 0.05 0.68 � 0.08Octadecan-1-ol (C18:0) 0.13 � 0.02 0.03 � 0.02 0.11 � 0.04 0.04 � 0.01 0.08 � 0.01Hexadecan-2-ol (C16:0) 0.62 � 0.10 0.76 � 0.06 0.84 � 0.06 0.75 � 0.11 1.08 � 0.13Octadecen-2-ol (C18:1) 0.86 � 0.21 2.39 � 0.17 1.73 � 0.15 2.47 � 0.15 4.61 � 0.14Hexadecan-3-ol (C16:0) 19.72 � 0.63 20.23 � 0.70 18.14 � 0.64 20.84 � 1.41 25.96 � 0.88Tetradecen-3-ol (C14:1) 3.08 � 0.27 4.60 � 0.50 3.31 � 0.18 3.25 � 0.33 4.28 � 0.41Heptadecan-3-ol (C17:0) 0.38 � 0.15 0.18 � 0.02 0.23 � 0.01 0.19 � 0.03 0.39 � 0.02Unknown 1 Tr 1.78 � 0.07 0.24 � 0.02 1.26 � 0.04 0.94 � 0.04Octadecen-3-ol (C18:1) 0.70 � 0.39 1.77 � 0.12 1.60 � 0.15 3.47 � 0.09 4.06 � 0.24Octadecan-3-ol (C18:0) 57.36 � 0.35 46.76 � 0.96 47.87 � 0.59 42.52 � 0.35 37.02 � 0.49Unknown 2 – 7.47 � 0.22 5.14 � 0.11 7.32 � 0.97 4.90 � 0.28Hexadecen-3-ol (C16:1) 5.70 � 0.39 5.49 � 0.19 7.84 � 0.24 6.45 � 0.80 6.21 � 0.25Octadecan-4-ol (C18:0) 3.90 � 0.78 3.58 � 0.14 4.12 � 0.05 5.70 � 0.19 5.94 � 0.10Unknown 3 6.96 � 0.14 4.88 � 0.26 8.52 � 0.43 5.63 � 0.37 3.85 � 0.27

Data are presented as mean value � SD (n = 3). Tr indicates trace amount.



Overall similarity of chemical composition in the CMsfrom adaxial and abaxial sides confirmed the unique isolat-erality of S. alba leaves (Tomlinson 1986). Both adaxial andabaxial CMs of S. alba leaves were found to have similarcutin monomer composition. The predominance of C18

monomers was in accord with monomer profiles of leafCMs of H. helix, C. miniata, Hordeum vulgare L. andCamellia sinensis (L.) Kuntze (Riederer & Schönherr 1988;Richardson et al. 2007; Tsubaki et al. 2008; Graça & Lamosa2010). Graça & Lamosa (2010) proposed that cutin hasdomains of linear structure when C18-epoxy monomers aredominant, whereas branched networks occur when the C16-dihydroxy monomers predominate. According to these cri-teria, linear structure might be predominated in the cutin ofS. alba leaves.

Mechanical tests quantitatively showed how the leaves ofS. alba acquired environmental tolerance. Physically weakand highly viscoelastic CMs at the immature stage werestrengthened and stiffened during leaf growth together withthe loss of high extensibility (Fig. 4; Table 4). However,physical strength of leaf CMs was finally lost at the senes-cent stage. Growth-dependent mechanical properties pre-sented in this study can be attributed to the variation of thechemical constituents. As previously reported (Petracek &

Bukovac 1995), removal of waxes resulted in decrease inthe values of smax and E, showing that wax has strengthen-ing and stiffening effects on the CMs (Fig. 4).

The CMs isolated from different species or organsshowed a wide diversity in chemical composition (Holloway1980), and mechanical properties of CMs depend on theirspecies of origin (Wiedemann & Neinhuis 1998). Yet mostof the studies on biomechanics of CMs address fruit growthand ripening, and no similar work has been conducted onthe alteration of mechanical properties of CMs during thegrowth of vegetative tissues (Domínguez et al. 2011a). Theresults on the growth-dependent chemical and mechanicalproperties presented here provide new information on thefunctional aspects of cuticular constituents in leaf epider-mal layers of S. alba.

In the present study, the chemical composition of theCMs was statistically correlated with its mechanical prop-erties (Table 5). Significant contribution of cuticular waxesto the CM biomechanics was in accord with previoussuggestion made by Petracek & Bukovac (1995). Intra-cuticular waxes can be regarded as fillers that reduce freevolume and restrict the segmental mobility of cutin mol-ecules within the matrix (Petracek & Bukovac 1995; Bargelet al. 2006). Contributions of cutin to the increase in exten-sibility and decrease in stiffness and inverse contributionsof polysaccharides demonstrated that the chemomechanicsystem, previously shown to operate in fruit CMs (López-Casado et al. 2007), also works in leaves. This stiffening andrigidizing effects of polysaccharides on the cutin matrixseemed to be reasonable as cellulose microfibrils probablystiffened cuticular matrix in the same way as in fibre-reinforced composite materials (Bargel et al. 2006; López-Casado et al. 2007). In addition, the accumulation of cutanduring the leaf growth statistically correlated with decreasein extensibility and increase in stiffness of the CMs. Previ-ously, cutan was suggested to be a factor that influenced therigid appearance of the CMs (Bargel et al. 2006). Cutanpresent in the leaf CMs of S. alba contains C = C bondsand/or aromatic rings which weakens the stacking ofmolecular chains as evidenced by the FT-IR spectra (Fig. 1).We can conclude that cutan contributes to the rigidity of the

Table 4. Biomechanical parameters(breaking stress smax, maximum strain emax,elastic modulus E) of Sonneratia alba leafcuticular membranes (CMs)

Component

Leaf number

L1 L2 L3 L4 L5

Adaxial CMssmax (MPa) 5.20 � 1.31ac 6.51 � 1.31ab 6.47 � 1.99ab 7.29 � 0.98b 4.14 � 0.56c*emax (%) 9.07 � 1.65a* 6.63 � 1.22b 4.34 � 1.96c 3.52 � 1.06cd 1.54 � 0.29d*E (MPa) 169 � 63a 279 � 14b 303 � 43b 398 � 86c 403 � 43c

Abaxial CMssmax (MPa) 5.60 � 1.52a 6.41 � 0.80a 7.34 � 1.37ab 8.74 � 2.60b 6.10 � 1.32a*emax (%) 7.55 � 1.07a* 6.38 � 0.95a 4.50 � 1.55b 4.48 � 1.17b 2.42 � 0.46c*E (MPa) 174 � 66a 287 � 71b 364 � 48bc 441 � 92c 410 � 67c

Data are presented as mean � SD (n = 6–10). Significant differences (P < 0.05) amonggrowth stages are indicated by different superscript letters. Asterisk shows significant differ-ences (P < 0.05) between adaxial and abaxial CMs.

Table 5. Pearson’s product-moment coefficient of correlation(R) between biomechanical parameters (breaking stress smax,maximum strain emax, elastic modulus E) and content of chemicalconstituents (wax, cutin, cutan and polysaccharide) of Sonneratiaalba leaf cuticular membranes (CMs)

Component

Biomechanical parameters

smax emax E

Wax 0.331** -0.033 0.433**Cutin 0.023 0.507** -0.468**Cutan -0.163 -0.549** 0.237*Polysaccharide -0.103 -0.694** 0.392**

The R-values were evaluated at relative composition basis.Asteriskshows significance of the R-values (*P < 0.05, **P < 0.01).



CMs. These contributions of cuticular components maycause the modifications of the shape of the S-S curves(Fig. 4). The change of the S-S curves from biphasic tomonophasic was in accord with leaf age. This might be dueto uniform distribution of constituents, especially polysac-charides and cutan molecules in the CMs from maturedleaves after formation of a rigid network structure; in turn,flexibility remains in the CMs from immature leaves wheredeposition of these constituents is still working.

The substantial weakening of CMs at L5 was not ascribedexclusively to the variations of the quantity of eachconstituent. As decomposition of constituents may occurduring progress of senescence, the quality of the constitu-ents may contribute to their declining.

In conclusion, the present study characterized growth-dependent chemical and mechanical properties of the CMsfrom S. alba leaves and further elucidated the chemicalfactors which influenced the biomechanics of the CMs.Although the amounts of wax and cutin achieved plateauat an early stage of leaf growth, deposition of cutan andpolysaccharide continued throughout the growth. Physicalstrength and stiffness of the CMs increased with leaf expan-sion and maturation,but the strength weakened at the senes-cent stage. Flexibility of the CMs gradually decreased alongwith the leaf growth.The viscoelastic nature of the immatureCMs was ascribed to cutin whereas the stiffening of the cutinmatrix was owned by the other components, wax, cutan andpolysaccharides. In addition, wax strengthens the cutinmatrix. Cutan and polysaccharides showed a rigidizingeffect. Isolateral development of CMs of S. alba leaves andrapid accumulation of wax and cutin in the CMs after budburst followed by the mechanical supports of cutan andpolysaccharides contribute to its remarkable environmentaltolerance to allow growth at the seaward fringe.

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Received 16 July 2011; accepted for publication 1 January 2012

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Table S1. Mass spectral data for identification of reducedcutin monomers.Figure S1. Typical total ion chromatogram (TIC) ofreduced cutin monomers obtained by hydrogenolysis ofcutin in leaves of S. alba. The TIC from adaxial CM of L4

leaf is shown as representative. The peak numbers werecited as listed in Supporting Information Table S1.Figure S2. Mass spectra of the TMS ethers of thehexadecane-3-ols (cited as Peak 5 in Supporting Informa-tion Table S1 and Supporting Information Fig. S1) derivedfrom cutin hydrogenolysate (bottom) and deuteriolysate(top). Embedded schemes show diagnostic fragmentions produced by a-cleavages. This fragmentation patternshowed that the corresponding cutin monomer was9(10),16-dihydroxyhexadecanoic acid.Figure S3. Mass spectra of the TMS ethers of theoctadecane-3-ols (cited as Peak 9 in Supporting Informa-tion Table S1 and Supporting Information Fig. S1) derivedfrom hydrogenolysate (bottom) and deuteriolysate (top).Embedded schemes show diagnostic fragment ions pro-duced by a-cleavages. This fragmentation pattern showedthat the corresponding cutin monomer was 9,10-epoxy-18-hydroxyoctadecanoic acid.Appendix S1. Mass identification procedure.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials sup-plied by the authors. Any queries (other than missing mate-rial) should be directed to the corresponding author for thearticle.



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