1 running head: 2 localizing the cuticular transpiration ... · 72 percentages of alicyclic...

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RUNNING HEAD: 1

Localizing the cuticular transpiration barrier 2

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CORRESPONDING AUTHOR: 5

Reinhard Jetter 6

Department of Botany 7

University of British Columbia 8

6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada 9

Phone: (604) 822-2477 10

Fax: (604) 822-6089 11

Email: [email protected] 12

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TITLE: 15

Localization of the transpiration barrier in the epi- and intracuticular waxes of eight plant 16

species: water transport resistances are associated with fatty acyl rather than alicyclic 17

components1 18

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AUTHORS: 21

Reinhard Jetter* and Markus Riederer 22

Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, 23

BC, V6T 1Z4, Canada (R.J.) 24

Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, 25

Canada, V6T 1Z1, Canada (R.J.) 26

Universität Würzburg, Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 27

3, D-97082 Würzburg, Germany (M.R.) 28

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Plant Physiology Preview. Published on December 7, 2015, as DOI:10.1104/pp.15.01699

Copyright 2015 by the American Society of Plant Biologists

www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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ONE-SENTENCE SUMMARY: 31

The cuticular transpiration barrier is formed by very-long-chain acyls rather than alicyclic wax 32

compounds, primarily in the intracuticular layer and complemented by epicuticular wax in some 33

species. 34

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FOOTNOTES: 37 1 This work was supported by grants from the German Science Foundation DFG (M.R. and R.J.), 38

the Natural Sciences and Engineering Research Council of Canada NSERC (R.J.), the Canada 39

Foundation for Innovation (R.J.), the Canada Research Chairs Program (R.J.) and a Chinese 40

Academy of Sciences Visiting Professorship for Senior International Scientists (grant no. 41

2011T2S31; M.R.). 42

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*Corresponding author: Reinhard Jetter 44

Fax: (604) 822-6089 45

Email: [email protected] 46 47

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ABBREVIATIONS: 50

CM : Cuticular Membrane 51

MX : Polymer Matrix 52

VLCFA : Very-long-chain fatty acid 53

GC : Gas Chromatography 54

MS : Mass Spectrometry 55

SEM : Scanning Electron Microscopy 56

tr : traces 57

UV : Ultraviolet 58

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ABSTRACT 60

61

Plant cuticular waxes play a crucial role in limiting non-stomatal water loss. The goal of the 62

present study was to localize the transpiration barrier within the layered structure of cuticles of 63

eight selected plant species, and to put its physiological function into context with the chemical 64

composition of the intra- and epicuticular wax layers. Four plant species (Tetrastigma 65

voinierianum, Oreopanax guatemalensis, Monstera deliciosa and Schefflera elegantissima) 66

contained only very-long-chain fatty acid (VLCFA) derivatives such as alcohols, alkyl esters, 67

aldehydes and alkanes in their waxes. Even though the epi- and intracuticular waxes of these 68

species had very similar compositions, only the intracuticular wax was important for the 69

transpiration barrier. In contrast, four other species (Citrus aurantium, Euonymus japonica, 70

Clusia flava and Garcinia spicata) had waxes containing VLCFA derivatives, together with high 71

percentages of alicyclic compounds (triterpenoids, steroids or tocopherols) largely restricted to 72

the intracuticular wax layer. In these species, both the epi- and the intracuticular waxes 73

contributed equally to the cuticular transpiration barrier. We conclude that the cuticular 74

transpiration barrier is primarily formed by the intracuticular wax, but that the epicuticular wax 75

layer may also contribute to it depending on species-specific cuticle composition. The barrier is 76

associated mainly with VLCFA derivatives, and less (if at all) with alicyclic wax constituents. 77

The sealing properties of the epi- and intracuticular layers were not correlated with other 78

characteristics such as the absolute wax amounts and thicknesses of these layers. 79

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INTRODUCTION 81

82

The plant cuticle is one of the major adaptations of vascular plants for life in the 83

atmospheric environment. Accordingly, the primary function of cuticles is to limit non-stomatal 84

water loss, and thus to protect plants against drought stress (Burghardt and Riederer, 2006). 85

However, plant cuticles also play roles in minimizing the adhesion of dust, pollen and spores 86

(Barthlott and Neinhuis, 1997), protecting tissues from UV radiation (Krauss et al., 1997; 87

Solovchenko and Merzlyak, 2003), mediating biotic interactions with microbes (Carver and 88

Gurr, 2006; Leveau, 2006; Hansjakob et al., 2010; Hansjakob et al., 2011; Reisberg et al., 2012) 89

as well as insects (Eigenbrode and Espelie, 1995; Müller and Riederer, 2005), and preventing 90

deleterious fusions between different plant organs (Tanaka and Machida, 2013). 91

92

Cuticles are composite (non-bilayer) membranes consisting of an insoluble polymer 93

matrix and solvent-soluble waxes. The polymer matrix (MX) is mainly made of the hydroxy 94

fatty acid polyester cutin (Nawrath, 2006), and also contains polysaccharides and proteins 95

(Heredia, 2003). In contrast, cuticular waxes are complex mixtures of aliphatic compounds 96

derived from very-long-chain fatty acids (VLCFAs) with hydrocarbon chains of C20 and more 97

(Jetter et al., 2007). Wax quantities and compositions vary greatly between plant species, and in 98

many cases even between organs and developmental stages. Diverse VLCFA derivatives can be 99

present, including free fatty acids, aldehydes, ketones, primary and secondary alcohols, alkanes 100

and alkyl esters. Besides, the cuticular waxes of many plant species also contain cyclic 101

compounds such as triterpenoids and aromatics. 102

103

In order to characterize the physiological function of cuticular waxes, methods have been 104

developed for isolation of astomatous cuticles and measurement of transpiration rates under 105

exactly controlled conditions, so that well-defined physical transport parameters such as 106

permeances and resistances can be determined and compared across species and organs 107

(Schönherr and Lendzian, 1981; Kerstiens, 1996; Riederer and Schreiber, 2001; Lendzian, 108

2006). With these methods, it was demonstrated that the cuticular water permeance increases by 109

up to three orders of magnitude upon wax removal, thus showing the central role of waxes as 110

transpiration barrier (Schönherr, 1976). Permeances for water determined so far with astomatous 111



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isolated leaf cuticular membranes (CMs) or in situ leaf cuticles range over 2.5 orders of 112

magnitude from 3.63 · 10-7 m/s (Vanilla planifolia) to 7.70 · 10-5 m/s (Maianthemum bifolium) 113

(Riederer and Schreiber, 2001). 114

115

The species-dependent differences of both wax composition and permeance led to a 116

search for correlations between cuticle structure and function. If such a structure-function 117

relationship could be established, then it would become possible to select or alter wax 118

composition in order to improve cuticle performance for example in crop species (Kosma and 119

Jenks, 2007). However, all attempts to understand cuticle permeance based on cuticle 120

composition have failed so far: correlations between wax amounts and permeances could not be 121

established, contrary to the common assumption that thicker wax layers must provide better 122

protection against desiccation (Schreiber and Riederer, 1996; Riederer and Schreiber, 2001). 123

Similarly, a correlation between wax quality, i.e. the relative portions of its constituents, and 124

permeance could also not be established to date (Burghardt and Riederer, 2006). It is not clear 125

how certain wax components contribute to the vital barrier function of the cuticle. 126

127

Previous attempts to establish wax structure-function relationships may have failed 128

because only bulk wax properties were studied and important effects of sub-structures were 129

averaged out. However, distinct compartments of wax exist within the cuticle, most prominently 130

as a layer of intracuticular wax embedded within the MX and a layer of epicuticular wax 131

deposited on the outer surface of the polymer (Jeffree, 2006). Over the last years, methods have 132

been developed that allow the selective removal of epicuticular wax by adhesive surface 133

stripping, followed by then equally selective extraction of intracuticular wax (Jetter et al., 2000; 134

Jetter and Schäffer, 2001). Chemical analyses showed that, for most plant species investigated to 135

date, both wax layers have distinct compositions (Buschhaus and Jetter, 2011). The most 136

pronounced differences between both layers were found for the triterpenoids, which were 137

localized predominantly (or even exclusively) in the intracuticular wax. These findings raised the 138

possibility that the chemically distinct wax layers might also have distinct functions, leading 139

back to the long-standing question whether the water barrier function is exerted by the 140

intracuticular and/or the epicuticular wax. There are only scant data to answer this question so 141

far, mainly because methods allowing a distinction between epi- and intracuticular waxes were 142



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established only recently. Using these sampling techniques, it was recently found that, for leaves 143

of Prunus laurocerasus, the epicuticular wax layer does not contribute to the transpiration barrier 144

(Zeisler and Schreiber, 2015). In contrast, it had been reported that removal of the epicuticular 145

wax layer from tomato fruit caused a circa two-fold increase in transpiration, suggesting that in 146

this species the epicuticular layer constitutes an important part of the barrier (Vogg et al., 2004). 147

Based on these conflicting reports, it is not clear in how far the intra- or the epicuticular waxes 148

contribute to the sealing function of the plant skin. 149

150

The goal of the present study was to localize the transpiration barrier within the cuticular 151

membrane of selected plant species, and to put the physiological function into context with the 152

chemical composition of both the epi- and intracuticular wax layers. To this end, we selected 153

eight species from which leaf cuticles could be isolated and methods for step-wise wax removal 154

could be applied without damaging the cuticle. Preliminary studies had shown that the adaxial 155

cuticles on leaves of Citrus aurantium (Rutaceae), Euonymus japonica (Celastraceae), Clusia 156

flava (Clusiaceae), Garcinia spicata (Clusiaceae), Tetrastigma voinierianum (Vitaceae), 157

Oreopanax guatemalensis (Araliaceae), Monstera deliciosa (Araceae) and Schefflera 158

elegantissima (Araliaceae) were astomateous and showed wide chemical diversity. These eight 159

species were therefore selected to address the questions: a) What are the amounts of epi- and 160

intracuticular waxes? b) Do compositional differences exist between both layers? c) Where are 161

the cuticular triterpenoids located? d) How much do the epi- and intracuticular waxes contribute 162

to the transpiration barrier? e) Is the barrier associated with certain components of the intra- or 163

epicuticular waxes? 164 165 166



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RESULTS 168 169

The present investigation aimed at comparing the chemical composition and 170

physiological properties of cuticular membranes between plant species. In particular, we studied 171

(i) cuticle thickness and weight per unit surface area, (ii) wax amount and composition, (iii) epi-172

and intracuticular location of wax constituents, and (iv) cuticular water permeance for eight 173

selected species. 174

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176

Cuticle thickness and gravimetrically determined coverages 177

178

In order to assess the overall membrane characteristics that might affect the transpiration 179

barrier, the thickness and weight of the adaxial leaf cuticle of all eight species were first 180

measured. Cuticular membranes (CMs) were isolated, cryo-fractured and viewed in cross section 181

by Scanning Electron Microscopy (SEM) (Fig. 1). All cuticles showed relatively straight outlines 182

on the outer surface, and pronounced ridges protruding on the opposite side. The network of the 183

latter resembled the outlines of leaf epidermal cells of each species and was, therefore, 184

interpreted as thicker cuticle zones that mirrored the indentations over anticlinal cell walls of the 185

epidermal cells. These ridges were 1 - 2 μm high and approximately 1 μm wide at their base, 186

thus adding little to the cuticle thickness and covering less than 5% of the average surface area of 187

300 - 500 μm2 per epidermis cell. The average thickness of the periclinal parts of the cuticles 188

varied widely, from 1 μm for Citrus aurantium to 7 μm for G. spicata and M. deliciosa (Fig. 2). 189

190

To further compare the physical properties of the cuticles of the eight species, their 191

weights per unit surface area were determined gravimetrically. To assess the coverages of waxes 192

and cutin separately, the isolated membranes were weighed both before and after exhaustive wax 193

removal using hot chloroform. From the difference in weight before and after extraction, the wax 194

coverages of the isolated membranes could be calculated. These gravimetrically determined wax 195

coverages ranged from 25 μg/cm2 for Citrus aurantium to ca. 50 μg/cm2 for S. elegantissima, E. 196

japonica, G. spicata and T. voinierianum, ca. 100 μg/cm2 for Clusia flava, and ca. 200 μg/cm2 197

for O. guatemalensis and M. deliciosa (Fig. 2). 198



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The coverages of the remaining cutin polymer matrices ranged from approximately 300 200

μg/cm2 for Citrus aurantium, via 400 μg/cm2 for T. voinierianum, 500 μg/cm2 for S. 201



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elegantissima, 600 μg/cm2 for Clusia flava and E. japonica, and 800 μg/cm2 for O. 202

guatemalensis and M. deliciosa up to 1,200 μg/cm2 for G. spicata (Fig. 2). For all eight species, 203

the MX thus largely dominated cuticle composition, with weight percentages ranging from ca. 204

75% in M. deliciosa and O. guatemalensis to more than 90% in Citrus aurantium, E. japonica 205

and G. spicata. Accordingly, waxes contributed between <10% and 25% to the mass of the 206

cuticles of these species. Values for total cuticle weights per unit area were roughly proportional 207

to cuticle thicknesses measured in the previous experiment (compare Fig. 2). 208



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Finally, the measured CM coverages were combined with the corresponding thickness 210

values to calculate coverage:thickness ratios representing apparent cuticle density for each 211

species. The resulting values varied from ca. 1 g/cm3 for M. deliciosa, O. guatemalensis and E. 212

japonica, to approximately 2 g/cm3 for Clusia flava, G. spicata, T. voinierianum and S. 213

elegantissima, and to almost 3 g/cm3 for Citrus aurantium. 214

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GC-determined wax coverages 217

218

To study the overall amounts and the relative compositions of the epi- and intracuticular 219

layers, waxes were sampled from the adaxial leaf surfaces for chemical analyses by GC-FID and 220

GC-MS. In a first experiment, gum arabic was used as an adhesive to mechanically remove 221

surface waxes (Fig. 1). For all species tested here, this glue could be administered three times to 222

the same area without damaging the leaves, and the resulting three wax samples were analyzed 223

separately. Wax yields for the first treatment ranged from 2 μg/cm2 for O. guatemalensis to 15 224

μg/cm2 for G. spicata (Tab. I). For all eight species, the wax amounts liberated by consecutive 225

gum arabic treatments declined steadily, reaching values of 0.1 μg/cm2 to 0.8 μg/cm2 in the third 226

round (Tab. I). The latter values represent 1 – 15% of the cumulative gum arabic yields for the 227

different species. Based on these results, it can be extrapolated that a fourth glue treatment would 228

not have yielded significant further wax amounts, and that the three sampling rounds had 229

therefore exhaustively removed the mechanically accessible wax. However, when the samples 230

that had been treated three times with gum arabic were then extracted with chloroform (instead 231

of a fourth glue treatment), the extraction yielded wax amounts ranging from 1 μg/cm2 for M. 232

deliciosa to 27 μg/cm2 for E. japonica (Tab. I). 233

234

In a separate experiment, the waxes on the adaxial leaf surfaces of the eight plant species 235

were extracted directly with chloroform (without prior gum arabic treatments). The resulting wax 236

yields varied widely, from 5 μg/cm2 for M. deliciosa to 30 μg/cm2 for E. japonica (Tab. I). In all 237

cases, the wax amounts released by direct extraction in a single treatment were not significantly 238



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different from the summed wax quantities released by four consecutive treatments in the 239

previous experiment (3 x gum arabic plus consecutive extraction) (Tab. I). 240

241

All combined, these results show that the gum arabic treatments were selectively 242

removing mechanically accessible material, and hence the three glue samples together give the 243

composition of the epicuticular wax layer for each species. As the repeated glue treatments 244

achieved an exhaustive removal of mechanically accessible material, these samples also give 245

quantitative information on the epicuticular wax amounts. Conversely, the waxes remaining after 246

three rounds of gum arabic treatments must have been localized (almost) entirely within the MX, 247

and analyses of the fourth, extractive treatment will reflect the composition of the intracuticular 248

wax layers of the species. This material was also removed exhaustively and can be used to 249

quantify wax amounts in this layer, as confirmed by the second experiment where direct 250

extraction of the total wax mixture yielded exactly the same amounts as the epi- and 251

intracuticular samples from the first experiment taken together. 252

253

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Compound class compositions 255

256

The total wax extracts from the second experiment were found to contain relative 257

amounts of compound classes widely differing between the eight species (Fig. 3, Suppl. Tab. I 258

and II). Typical wax constituents were identified in all samples, including VLCFAs and their 259

primary alcohol, alkyl ester, aldehyde and alkane derivatives. In addition, varying amounts of 260

pentacyclic triterpenoids, steroids and tocopherols were identified (Suppl. Tab. III and IV). The 261

wax mixtures of Citrus aurantium, G. spicata and S. elegantissima were dominated by single 262

VLCFA derivative classes, namely by primary alcohols in the first case and by alkanes in the 263

latter two. E. japonica wax also contained one predominant compound class, but here the 264

triterpenoids prevailed instead of VLCFA derivatives. In the remaining species, two or more 265

compound classes were found at equally high abundance, with both alkanes and triterpenoids 266

dominating for Clusia flava and various VLCFA derivatives in the wax mixtures of T. 267

voinierianum, O. guatemalensis and M. deliciosa. 268

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The compound classes described above for the direct wax extraction experiment were 270

also identified in the wax mixtures sampled separately either by gum arabic treatment or by the 271

following extraction step (Fig. 3, Suppl. Tab. I and II). For O. guatemalensis and M. deliciosa, 272



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both experiments gave not only qualitatively but also quantitatively matching results, with all 273

compound classes present at similar percentages in the gum arabic samples and the final extracts. 274

In contrast, the relative compositions differed between the epi- and intracuticular wax layers in 275

all other species (Fig. 3, Suppl. Tab. I and II). The cyclic compounds (triterpenoids, steroids and 276

tocopherols) exhibited drastic gradients, with percentages in the intracuticular waxes always 277

much higher than in the epicuticular layer. The relative amounts of cyclic compound classes in 278

the intracuticular wax mixtures varied from trace levels in O. guatemalensis and S. 279

elegantissima, over moderate levels (10 – 20%) in T. voinierianum and M. deliciosa, to large 280

percentages (>50%) in the other four species. The corresponding epicuticular waxes contained 281

less than 10% of cyclic compounds in all eight species. These gradients of cyclic wax 282

constituents were balanced by counter-gradients of VLCFA derivatives, with particularly high 283

primary alcohol concentrations in the epicuticular wax of Citrus aurantium and high alkane 284

percentages in the epicuticular layer of Clusia flava and G. spicata. 285

286

287

Chain length distribution of VLCFA derivatives 288

289

All VLCFA derivatives identified in the waxes of the eight species were found to have 290

unbranched and fully saturated hydrocarbon structures (Suppl. Tab. V and VI). The homologous 291

series of fatty acids, primary alcohols, alkyl esters and aldehydes showed very pronounced 292

predominance of compounds with even carbon numbers, while odd-numbered homologs 293

prevailed in the alkane series. One homolog was dominating the compound classes in most 294

cases, with few exceptions where bimodal chain length distributions were found (e.g. for E. 295

japonica epicuticular wax). Chain length distributions were relatively similar between compound 296

classes within the same species. In contrast, the chain length ranges and predominant chain 297

lengths differed substantially between species. 298

299

In the waxes of T. voinierianum, O. guatemalensis, M. deliciosa and S. elegantissima the 300

complete homologous series of VLCFA derivatives could be accurately quantified, and were 301

found to be very similar for epicuticular and intracuticular wax layers (Suppl. Tab. VI). These 302

chain length distributions were also confirmed by analyses of the corresponding total wax 303



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extracts generated in the second experiment. In the waxes of Citrus aurantium, E. japonica, 304

Clusia flava and G. spicata, where triterpenoids and steroids were present in high concentration, 305

a few VLCFA derivatives with C28 and C30 chain lengths could not be completely separated and 306

quantified (Suppl. Tab. V). As this affected mainly the chemical analyses of intracuticular wax 307

samples, their chain length distribution can only partially be compared with the corresponding 308

epicuticular wax composition of the same species, and also with the other species. 309

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Transpiration barrier properties 312

313

In order to characterize the transpiration barriers of the adaxial leaf cuticles of all eight 314

plant species, the water permeances were measured first for entire cuticles, then again after 315

removing the epicuticular wax layer, and finally after also removing the intracuticular wax. 316

Permeances of intact isolated cuticles ranged from 1.3 · 10-6 m/s for M. deliciosa to 1.4 · 10-5 m/s 317

for E. japonica (Tab. II). From these permeances, corresponding transpiration resistances could 318

be calculated as inverse values (R = 1/P). Corresponding water transport resistances of CMs 319

varied among the eight species from 9.8 · 104 s/m to 8.8 · 105 s/m (Fig. 4). 320

321

After removal of epicuticular waxes, the permeances of the eight species ranged from 1.8 322

· 10-6 m/s to 2.2 · 10-5 m/s (Tab. II). The corresponding transpiration resistances may be 323

compared with the overall cuticular resistances of the same species. It can be assumed that the 324

epicuticular and the intracuticular resistances act in series and are thus additive. Consequently, 325

the differences in resistances before and after removal of epicuticular waxes corresponded to 326

water transport resistances of the epicuticular layer alone. Respective epicuticular resistances 327

ranged from 3.9 · 104 s/m for E. japonica to 1.8 · 105 s/m for Clusia flava (Fig. 4). In a control 328

experiment, the same CMs were tested after opening, closing and refilling the transpiration 329

chambers as in the experiment described above, but without wax removal. There was no 330

significant difference between the permeances before and after these manipulations (data not 331

shown), demonstrating that the effects described above were due to the removal of the 332

epicuticular waxes alone and not caused by mechanical damage during treatments or by the 333

ageing of the cuticle mounted in the transpiration chamber over the time of the experiments. 334



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335

After further removal of the intracuticular waxes, the permeances of the membranes of all 336

eight species increased drastically, to values between 3.6 · 10-4 m/s (M. deliciosa) and 1.7 · 10-3 337



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m/s (Citrus aurantium; Tab. II). On the one hand, the difference of permeances before and after 338

removing the intracuticular waxes can be used to calculate the water transport resistance of this 339

wax fraction. The results varied between relatively small values of approximately 6 · 104 s/m for 340

Citrus aurantium, E. japonica and Clusia flava to more than ten-fold higher intracuticular 341

resistances for M. deliciosa (Fig. 4). On the other hand, these residual permeances can be used to 342

assess the resistance of the MX devoid of waxes. Resulting values ranged from 5.9 · 103 s/m to 343

2.9 · 104 s/m (Fig. 4), and were thus approximately two orders of magnitude smaller than those 344

for corresponding CMs. 345

346

347

Tests for correlations between barrier properties and structure 348

349

In order to link cuticle barrier properties and structure, comprehensive correlation 350

analyses were carried out for combinations between the data described above. In particular, 351

structural parameters (thickness, matrix amount and wax coverage) of the isolated cuticular 352

membranes of the eight species were selected together with chemical parameters (amounts and 353

percentages of each compound class in either the epicuticular or the intracuticular wax layers) 354

and physiological parameters (transpiration resistances of the CM, the epicuticular wax, the 355

intracuticular wax and the cutin matrix). All possible combinations between these parameters 356

were tested in pairwise regression analyses (Suppl. Table VII, significance threshold Bonferroni-357

adjusted for 18 independent parameter families to P < 0.0028). In the following, the results for 358

the most important parameter pairs will be summarized. 359

360

Within the chemical subset of data, a few correlations were found between compound 361

classes either within a wax layer or between layers. Most notably, the epicuticular alkane 362

amounts (in μg/cm2) were proportional to the overall aliphatic contents of the epicuticular waxes 363

(r2 = 0.92) as well as the total epicuticular wax amounts (r2 = 0.92). The overall aliphatic 364

contents were strongly correlated with the amounts of epicuticular wax (r2 = 1.00). In particular, 365

the amounts of epicuticular and intracuticular esters were correlated, both when expressed in 366

μg/cm2 (r2 = 0.93) and as percentages within each layer (r2 = 0.90). Similarly, the percentages of 367



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acids in the epi- and intracuticular waxes were correlated (r2 = 0.91), as were aldehyde 368

percentages in both layers (r2 = 0.73). Finally, the amounts of total intracuticular waxes, of 369

intracuticular alicyclics and of epicuticular alicyclics were all correlated with each other (r2 = 370

0.75 – 0.95). 371

372

Total cuticular resistances of the eight species investigated here were found not to be 373

correlated either with overall cuticle thickness (r2 = 0.01; Fig. 5), with cuticle weight (r2 = 0.03), 374

or with total wax amounts (r2 = 0.17). Similarly, correlations did not exist between the weight 375

and the resistance of the MX (r2 = 0.21; Fig. 6), between the amounts and resistances of the 376

epicuticular waxes (r2 = 0.002), or between the amounts and resistances of the intracuticular 377

waxes (r2 = 0.24). However, the intracuticular resistances were found correlated with the total 378

resistances (r2 = 0.98). 379

380

Strong correlations between any of the compositional parameters and resistances could 381

not be found. As one notable exception, both the intracuticular and the total resistances were 382

somewhat correlated with the percentage of aliphatic compounds within the intracuticular wax 383

mixtures (r2 = 0.57). Conversely, relatively low contributions of the intracuticular resistance 384

were in our species associated with relatively high amounts of alicyclic compounds in the 385

intracuticular wax (Fig. 7), expressed as percentages either of the triterpenoids alone (r2 = 0.56) 386

or the sum of all three classes (r2 = 0.52; triterpenoids, steroids, tocopherols). The absolute 387

amounts (μg/cm2) of the alicyclic compounds were not proportional to the absolute resistances 388

(s/m) of the intracuticular compartments (r2 = 0.26). 389

390

There were also no correlations between the relative composition and absolute amounts 391

of VLCFA derivatives in the epicuticular wax mixture and the transpiration resistance of this 392

layer (data not shown). For example, G. spicata and S. elegantissima had very similar 393

composition and amounts of epicuticular waxes, but very different epicuticular barrier properties. 394

Conversely, Citrus aurantium and G. spicata had similar epicuticular barriers, but very different 395

epicuticular wax compositions and amounts. 396

397



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DISCUSSION 399

400

The principal goal of the present study was to quantify to which degree the intra- and 401

epicuticular waxes contribute to the barrier against non-stomatal water loss. To address this 402

question, water permeances from eight plant species were measured, and corresponding 403

transpiration resistances calculated. To further distinguish between the barrier properties of the 404

polymer matrix (MX), the intracuticular wax and the epicuticular layer within the cuticles of 405

each of these species, transpiration resistances of each part of the cuticle were determined. 406

407

408

Total wax composition and barrier properties 409

410

The amounts and compositions of total cuticular wax mixtures of the eight species 411

investigated here, comprising both epicuticular and intracuticular waxes together, fall well within 412

the range of compositions previously described for various plant waxes. Among the present 413

species, only three had been investigated before, with similar results. Most notably, a detailed 414

analysis of Citrus aurantium leaf wax had shown very similar compound class and chain length 415



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profiles (Riederer and Schneider, 1989). The overall amounts of C. aurantium leaf waxes, 416

extracted by short surface immersion, were also nearly identical with our results (see Tab. I). 417

However, much higher wax amounts could be extracted from isolated CMs with hot chloroform 418



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in a soxhlet apparatus (see Fig. 2), similar to previous reports for C. aurantium showing that wax 419

yields largely depend on extraction conditions (Riederer and Schneider, 1989, Schreiber and 420

Riederer, 1996). In particular, it had been reported that surface extraction with chloroform yields 421

wax mixtures with reproducible amounts and composition free of cellular lipids, but 422

discriminates against longer-chain compounds mainly due to limited solubility (Riederer and 423

Schneider, 1989). This may also, at least in part, explain the consistently higher wax amounts 424

released by prolonged hot chloroform extraction from isolated CMs of all species tested here 425

(see Fig. 2), as compared to short surface dipping of corresponding leaf material (see Tab. I). 426

This conclusion is further supported by previous literature, since the wax yields reported before 427

for exhaustive extraction of E. japonica and M. deliciosa CMs also match our current findings 428

(Schreiber and Riederer, 1996). Finally, it should be noted that our results for Clusia flava also 429

match previous reports on amounts and compositions of wax mixtures extracted from other 430

Clusia species (Medina et al., 2004). 431

432

The permeances of intact CMs had been reported before for three of the species 433

investigated here, with similar results. In particular, twelve investigations using methods 434

comparable to ours showed P values for Citrus aurantium ranging by more than one order of 435

magnitude from 5.5 · 10-6 m/s to 1.7 · 10-4 m/s (Schönherr, 1976; Schönherr and Schmidt 1979; 436

Haas and Schönherr 1979; Schönherr and Lendzian 1981; Becker et al. 1986; Lendzian et al. 437

1986; Kerstiens and Lendzian 1989; Geyer and Schönherr 1990; Schreiber and Riederer 1996; 438

Riederer 2006; Ballmann et al. 2011), with a median of 3.2 · 10-5 m/s fairly similar to the value 439

1.2 · 10-5 m/s found here. Similarly, the permeance determined here for Monstera deliciosa and 440

Euonymus japonica (1.3 · 10-6 m/s and .4 · 10-5 m/s, respectively) deviated somewhat from the 441

literature (1.9 · 10-5 m/s and 1.6 · 10-4 m/s, respectively; Schreiber and Riederer 1996), the 442

differences being similar to those reported before for repeat permeance measurements of other 443

species. 444

445

446

Comparisons of epicuticular and intracuticular wax compositions 447

448



23

23

Among the eight species investigated here, the epicuticular wax amounts varied widely 449

from 3 μg/cm2 to 16 μg/cm2. Accordingly, and assuming a wax density of 0.8 – 1.0·106 g/m3 (Le 450

Roux, 1969; Small, 1984), the thickness of the epicuticular layer can be estimated to vary from 451

30 nm for O. guatemalenis to 200 nm for G. spicata. At the same time, the intracuticular wax 452

amounts ranged from 1 to 27 μg/cm2 in the species investigated here. Consequently, the ratios 453

between wax amounts in both layers varied from 5:1 to 1:7, further illustrating the diversity in 454

absolute and relative quantities of material in the different layers within the cuticles of these 455

species. In previous studies, epicuticular layers had been found to vary in thickness from ca. 10 456

nm on Arabidopsis thaliana leaves to 130 nm on mature Prunus laurocerasus leaves, while 457

ratios of epi- and intracuticular waxes ranged from 9:1 to 1:1 (Jetter et al., 2000; Buschhaus and 458

Jetter, 2012). The species investigated here thus had similar epi- and intracuticular wax amounts 459

and proportions as previously studied plants, but showed a wider range of layer dimensions. This 460

presented an opportunity to test for correlations between wax amounts and barrier properties. 461

However, such correlations could not be found. The permeances of the epi- and intracuticular 462

wax layers are independent of their thickness, and wax resistances must instead be determined by 463

more subtle effects including wax composition within both layers. 464

465

All eight species investigated here had epicuticular waxes containing large percentages of 466

VLCFA derivatives and only little or no alicyclic compounds (see Fig. 4). Depending on the 467

species, between one and four VLCFA compound classes were present at high concentrations in 468

this layer forming the true surface of the leaves. In many cases alkanes dominated, but primary 469

alcohols, fatty acids and aldehydes also accumulated to levels above 20% in some epicuticular 470

wax mixtures. More subtle differences among the species were found in the exact chain length 471

distributions within these epicuticular wax compound classes (Suppl. Tables V and VI). Overall, 472

these findings are similar to previous reports for the composition of the epicuticular wax on other 473

plant species that are also characterized by smooth surfaces lacking epicuticular wax crystals 474

(Buschhaus and Jetter, 2011). For example, tomato fruits and mature Prunus laurocerasus leaves 475

have epicuticular waxes dominated by alkanes (Jetter et al., 2000; Vogg et al., 2004), whereas 476

young P. laurocerasus leaves and Macaranga tanarius leaves have predominantly primary 477

alcohols in their surface waxes (Jetter and Schäffer, 2001; Guhling et al., 2005). 478



24

24

479

In the present study, four species (T. voinierianum, Oreopanax guatemalensis, M. 480

deliciosa and S. elegantissima) had relatively small differences between the composition of their 481

epi- and intracuticular wax layers. The bulk wax analyses closely matched those of both the 482

separate epi- and intracuticular wax samples, further confirming that no transversal gradients 483

existed within the cuticles of these species. It should be noted that the same species contained no 484

(or only very small amounts of) alicyclic wax compounds. In the cuticular wax mixtures of the 485

other species (Citrus aurantium, E. japonica, Clusia flava and G. spicata), triterpenoids, steroids 486

or tocopherols were present at relatively high concentrations. These alicyclic compounds were in 487

all cases largely (or entirely) restricted to the intracuticular wax layer and, consequently, drastic 488

compositional gradients existed within the cuticular waxes: alicyclic concentrations decreased 489

outward, whereas VLCFA derivative percentages increased in the same direction. These findings 490

are very similar to previous reports on plant species with smooth epicuticular wax surfaces (i.e. 491

lacking surface wax crystals) and alicyclic wax constituents (Buschhaus and Jetter, 2011). In five 492

cases, namely for tomato fruits and for leaves of Prunus laurocerasus, Rosa canina, Ligustrum 493

vulgare and Macaranga tanarius, had pentacyclic triterpenoids been found at high 494

concentrations in the intracuticular wax only (Jetter et al., 2000; Jetter and Schäffer, 2001; Vogg 495

et al., 2004; Guhling et al., 2005; Buschhaus et al., 2007a; Buschhaus et al., 2007b; Zeisler and 496

Schreiber, 2015). 497

498

499

Model underlying the analysis of cuticular resistances 500

501

In order to calculate values for the contribution of various structural components to the 502

overall cuticular resistance, it can be assumed that a) the epicuticular wax layer and the 503

intracuticular compartment (polymer matrix plus waxes) act as two resistances in series, and b) 504

the major resistance within the intracuticular layer is due to intracuticular waxes, whereas the 505

matrix contributes relatively little. The first part of this model is based on the finding that the 506

majority of water molecules are absorbed into and cross through the cuticular wax (as opposed to 507

larger, polar solutes that pass through a limited number of aqueous channels extending from the 508

epidermal cell wall to the tissue surface) (Riederer and Schreiber, 2001; Schönherr and 509



25

25

Schreiber, 2004; Beyer et al., 2005; Schreiber, 2005; Popp et al., 2005; Schönherr, 2006; 510

Weichert and Knoche, 2006; Arand et al., 2010). The epicuticular wax film must consequently 511

impose a resistance on water movement, and this resistance acts in series with the resistance(s) 512

imposed on inner pathway sections. 513

514

The second part of this model is based on previous results that the MX alone imposes 515

only a relatively small resistance (Schönherr, 1976). This is confirmed by our finding that, for all 516

eight species investigated here, cuticular water transport resistances increased by two orders of 517

magnitude when the intracuticular wax was removed from the cutin matrix. It is therefore likely 518

that the major part of the intracuticular resistance is due to wax, and not cutin. However, it must 519

be noted that the intracuticular wax is likely associated with the MX on a molecular scale 520

(Nawrath, 2006), and therefore cutin will have an indirect effect on the barrier properties of the 521

wax. While this indirect contribution of the MX cannot be assessed at present, we assume that it 522

will be relatively small in comparison with that of the intracuticular wax and can be neglected in 523

a first approximation. 524

525

526

Correlations between cuticle structure and barrier localization 527

528

High levels of intracuticular alicyclics were found for four of the species investigated 529

here, and in the same species the intracuticular wax layers formed only ca. 50% of the cuticular 530

transpiration barrier (Fig. 7). The second half of the overall barrier was contributed by the 531

epicuticular wax in these species, consisting entirely of VLCFA derivatives. This is in contrast to 532

the other four species, with predominantly fatty acid derivatives in their intracuticular waxes 533

(and lacking cyclic compounds), where the barrier was localized entirely within the intracuticular 534

layer. It should be noted that, among the species tested here, the presence of intracuticular 535

alicyclics coincided not only with combined epi- and intracuticular barriers, but also with 536

relatively low overall transpiration resistances. In summary, the transpiration barrier may be 537

formed either by the intracuticular wax alone or by both the intra- and epicuticular wax layers 538

together, depending on the species and cuticular wax composition. 539

540



26

26

Our findings can be corroborated by the few previous studies in which permeances were 541

measured separately for epi- and intracuticular wax layers. In one example, the permeances of 542

wax layers on cherry fruits were reported, and the epicuticular contribution to the wax resistance 543

can be inferred to be approximately 50% (Knoche et al., 2000). However, cellulose acetate, an 544

adhesive typically applied in organic solution, was used to remove the epicuticular film in this 545

study, and the solvent may have disturbed the intracuticular layer and consequently caused an 546

overestimation of the epicuticular resistance (Jetter et al., 2000; Zeisler and Schreiber, 2015). 547

Furthermore, it seems plausible that the intracuticular wax of the cherry fruit contains high 548

concentrations of triterpenoids and only low amounts of fatty acid derivatives, but quantitative 549

data on its composition are missing to date. Overall, the current information on cherry fruit 550

matches our present findings qualitatively, but will need to be backed up by more quantitative 551

data. 552

553

In a second study, gum arabic had been used to distinguish the permeances of epi- and 554

intracuticular layers in tomato fruits, and transpiration resistances of 1.9·10-6 s/m and 2.5·10-6 555

s/m can be calculated, respectively (Vogg et al., 2004; Leide et al., 2007). With this roughly 556

equal split of resistances between both layers, the tomato fruit is another system where the 557

epicuticular wax contributes substantially to the transpiration barrier. The tomato fruit cuticle 558

was shown to contain a large percentage of intracuticular triterpenoids (Vogg et al., 2004), thus 559

further confirming the conclusion that these alicyclics are not directly involved in forming the 560

cuticular transpiration barrier. 561

562

Very recently, the composition and barrier properties of the epi- and intracuticular wax 563

layers on leaves of Prunus laurocerasus were investigated (Zeisler and Schreiber, 2015). Three 564

different methods for removal of epicuticular wax were compared, along with control 565

experiments testing the effects of the solvents involved in each of them. Two of these methods, 566

employing gum arabic or collodion (applied in water or 1:1 ether/ethanol, respectively), were 567

shown to remove epicuticular wax without releasing triterpenoids known to be intracuticular 568

components in this species (Jetter et al., 2000; Jetter and Schäffer, 2001). Removal of the 569

epicuticular material with either of these methods had no significant effect on the transport of 570

water through the isolated CM, leading the authors to conclude that, on P. laurocerasus leaves, 571



27

27

the epicuticular wax is not contributing to the transpiration barrier (Zeisler and Schreiber, 2015). 572

It should be noted that the methods used for assessing water transport in P. laurocerasus (short-573

term kinetics of radioactive water diffusion instead of gravimetry) and for data analysis 574

(calculation of relative effects instead of serial resistances) were fairly different from those 575

employed in our study, even though the protocols for epicuticular wax removal were similar. 576

Therefore, the results of both studies may not be comparable directly across methods and species 577

until they are corroborated in future studies employing the same methods on identical plant 578

material. However, it is interesting that the intracuticular wax mixtures extracted from P. 579

laurocerasus leaves after treatments with gum arabic or collodion contained substantial amounts 580

(ca. 6 μg/cm2 and 15 μg/cm2, respectively) of fatty acid derivatives (Zeisler and Schreiber, 581

2015). In comparison, the four species in our study resembling P. laurocerasus in their 582

substantial amounts of intracuticular alicyclics had much lower coverages of VLCFA 583

compounds (ca. 1 μg/cm2 for Citrus aurantium, Clusia flava and Garcinia spicata, and ca. 2.5 584

μg/cm2 for Euonymos japonica). These differences in intracuticular wax composition might, at 585

least in part, account for the different barrier qualities of the intracuticular wax layers in P. 586

laurocerasus and the species investigated here. 587

588

Only two studies so far have tested the effect of specific wax components on cuticular 589

transpiration by manipulating wax composition within a single species, rather than comparing 590

between different compositions across species. Each of these studies compared two plant lines 591

differing in the concentration of intracuticular triterpenoids. In one case, a tomato mutant 592

deficient in VLCFA derivatives was found to have strongly reduced transpiration resistance, 593

even though the amounts of (intracuticular) triterpenoids were significantly increased over wild-594

type levels (Vogg et al., 2004). In a second study, the ectopic expression of a triterpenoid 595

synthase gene led to accumulation of small percentages of the triterpenoid β-amyrin in leaves of 596

Arabidopsis thaliana, an organ devoid of cuticular triterpenoids in wild-type lines (Buschhaus 597

and Jetter, 2012). The triterpenoid was found only in the intracuticular wax, and this change in 598

composition led to a decrease of the intracuticular transpiration resistance. 599

600



28

28

Thus, the two manipulative investigations confirm the present finding that intracuticular 601

triterpenoids do not contribute directly to the transpiration barrier, but instead in their presence 602

the barrier is split between the intra- and epicuticular layer. The current study puts this 603

conclusion into a broader context, by analyzing multiple species with a wider range of 604

triterpenoid concentrations. All data taken together, we conclude that, at least when both VLCFA 605

derivatives and aliphatics together form the cuticular wax mixture, the former compounds alone 606

form the cuticular transpiration barrier and the (intracuticular) triterpenoids do not contribute 607

directly to the physiological function. This establishes a first, albeit crude, structure-function 608

relationship for the composition and barrier properties of cuticular waxes. 609

610

The structure-function relationship thus far is in accordance with a long-standing model 611

for the spatial arrangement of wax molecules and the resulting transport resistances (Riederer 612

and Schreiber, 2001). The model stipulates crystalline domains of VLCFA derivatives with 613

narrow chain length distribution, surrounded by amorphous zones containing alicyclic 614

compounds and wide arrays of VLCFA homologs. The tight packing of the crystalline domains 615

excludes water molecules, effectively forcing water molecules to follow a tortuous path through 616

the amorphous domains and thus establishing the transpiration barrier. Consequently, this model 617

predicts that the transpiration barrier is associated with the VLCFA wax components rather than 618

the alicyclics. The current experiments for the first time test this hypothesis, and our results are 619

in good overall accordance with the domain model. Conversely, we predict that in species with 620

intracuticular wax rich in VLCFA derivatives the overall transpiration barrier will be located 621

largely in the intracuticular layer alone, whereas in species with lower concentrations of 622

intracuticular fatty acid derivatives the barrier will be formed by the epi- and intracuticular layers 623

together. 624

625

The importance of individual VLCFA compounds cannot be assessed in more detail at 626

this point. In this context it should be noted that for some of the species investigated here the epi- 627

and intracuticular layers had similar VLCFA compositions, and yet the two layers showed 628

dramatically different resistances. It is currently not clear how similar compositions can cause 629

different physiological properties in these cases. Low intracuticular water diffusion resistances 630

were not only correlated with high intracuticular alicyclic concentrations, but also with a low 631



29

29

overall cuticular transpiration barrier. This second effect cannot be explained by a small 632

resistance contribution from the triterpenoids alone, but must be due to other factors. It seems 633

unlikely that differences in the relative or absolute composition of VLCFA derivatives caused 634

the low overall resistances in some species, as the VLCFA amounts were not correlated with 635

barrier properties in species comparisons or between wax layers within species. Instead, the 636

physical structure of the waxes may be a more important factor determining barrier properties. 637

The link between low overall resistances and relatively small intracuticular barrier contributions 638

suggests that the physical structure of the intracuticular wax is of central importance, and that the 639

epicuticular layer adds to the barrier function only where the intracuticular structure is imperfect. 640

641



30

30

CONCLUSIONS 642

643

We have shown that, depending on the plant species, the cuticular transpiration barrier 644

may be located entirely in the intracuticular wax or distributed up to 1:1 between the epi- and 645

intracuticular wax layers. In species where the intracuticular wax does not constitute the entire 646

barrier, high concentrations of intracuticular triterpenoids or other alicyclic compounds were 647

detected, and the overall transpiration resistance was found to be relatively low. Therefore, we 648

conclude that the majority of the resistance against cuticular transpiration is formed by the 649

VLCFA derivative compounds in the waxes, either entirely in the intracuticular wax or by a 650

combination of epi- and intracuticular compartments. Overall, these findings for the first time 651

link cuticle substructures and their composition with the physiological function of water 652

transport control. Our results are compatible with previous models describing cuticular structure 653

and transpiration, now enabling the deduction of more detailed models for the causal 654

relationships between wax composition and physiological function. These models may be tested 655

using the methods established here, providing quantitative information on the composition and 656

barrier properties of the intra- and epicuticular waxes separately. It will be very interesting to 657

thus compare further plant species selected for subtle differences in alicyclic composition or 658

VLCFA profiles, or to investigate series of transgenics with systematically varying wax 659

composition. 660

661

662 663 664 665 666 667 668 669

670



31

31

MATERIALS AND METHODS 671 672

Plant Material 673

674

Small individuals of Citrus aurantium L., E. japonicus Thunb., Clusia flava Jacq., G. 675

spicata Hook.f., T. voinierianum (Baltet) Gagnep., O. guatemalensis Decne. & Planch., M. 676

deliciosa Liebm. and S. elegantissima (Veitch ex Mast.) Lowry & Frodin were continuously 677

grown in pots in the greenhouses of the Botanical Garden of the University of Würzburg, 678

Germany. Mature leaves were harvested randomly from three individual plants and pooled 679

before using them either for isolation of adaxial cuticular membranes (CMs) or for wax removal 680

and analysis. 681

682

In order to isolate CMs, the method of Schönherr and Riederer (1986) was adapted to the 683

the current species. Circular discs with 20 mm diameter were cut from harvested leaves of the 684

eight species, marked with felt pen on the abaxial side, and then vacuum-infiltrated with a 685

sodium citrate buffer containing 2% (w/w) pectinase (Roth, Karlsruhe, Germany), 2% (w/w) 686

cellulase (Roth, Karlsruhe, Germany), and 1 mM sodium azide (NaN3) to prevent bacterial 687

growth. The enzyme solution was kept at room temperature and changed every 2 – 3 d until the 688

tissue was largely dissolved or at least the CMs completely removed from it. Adaxial CMs were 689

identified by the absence of pen marks, spread out onto Teflon discs, washed thoroughly with 690

distilled water, air-dried and transferred into glass vials for storage. Isolated CMs were either cut 691

with a hand-microtome for cross-sectional viewing by SEM or treated with gum arabic to 692

remove epicuticular waxes. 693

694

695

Scanning electron microscopy and determination of cuticle thickness and weight 696

697

Samples were mounted on aluminum stubs using double-sided adhesive tape, coated with 698

5 nm of Au using a Cressington Sputter Coater 208HR (Ted Pella, California, USA) and 699

investigated with a Hitachi S4700 field emission SEM (Nissei Sangyo America, California, 700

USA) using a 1 kV accelerating voltage and a 12 mm working distance. 701



32

32

702

In order to determine cutin and wax amounts in isolated cuticular membranes, a specified 703

number of circular CMs (diameter 20 mm) were weighed before and after extraction of cuticular 704

waxes. Exhaustive wax removal was achieved by extraction with chloroform for at least 4 h in a 705

Soxhlet apparatus. 706

707

708

Wax sampling 709

710

A polymer film of gum arabic was employed for the selective preparation and analysis of 711

epicuticular waxes. Commercial gum arabic (Roth, Karlsruhe, Germany) was extracted 712

exhaustively with hot chloroform to remove any soluble lipids, and residual organic solvent was 713

allowed to evaporate completely. Approximately 0.1 mL of a 90% (w/w) aqueous solution of de-714

lipidated gum was applied per cm² of leaf surface using a small paintbrush. After 1 – 2 h, a dry 715

and stable polymer film could be broken off in pieces. These were collected and transferred into 716

a vial containing 7 mL each of chloroform and water. The polymer films from five 3.1 cm² discs 717

were pooled into the same two-phase system. Tetracosane was added as internal standard, after 718

vigorous agitation and phase separation the organic solution was removed, and the solvent was 719

evaporated under reduced pressure. After treatment with gum arabic, the leaf discs were still 720

physically intact and could be used in repeated mechanical removal experiments or in a final 721

solvent extraction step to remove the remaining cuticular waxes. 722

723 Selective superficial extraction of intracuticular waxes or total cuticular waxes from the 724

adaxial surface was achieved by placing the intact leaf onto a flexible rubber mat, gently 725

pressing a glass cylinder with 10 mm diameter onto the exposed surface and filling the cylinder 726

with approximately 1.5 mL of chloroform. The solvent was agitated for 30 s by pumping with a 727

Pasteur pipette and removed. This procedure was repeated once, and both extracts were 728

combined. When any solvent leaked between cylinder and leaf surface, the sample was 729

discarded. Extracts from ca. 10 individual leaves were pooled for further analysis. Tetracosane 730



33

33

was immediately added to all the extracts of cuticular waxes as internal standard, and the solvent 731

was removed under reduced pressure. 732

733

734

Chemical characterization 735

736

Prior to analysis by gas chromatography (GC), hydroxyl-containing compounds in all 737

samples were transformed to the corresponding trimethylsilyl derivatives by reaction with bis-738

N,O-trimethylsilyltrifluoroacetamide (Macherey-Nagel, Düren, Germany) in pyridine (30 min at 739

70°C). Wax mixtures were analyzed using a capillary GC (5890N, Agilent; column 30 m HP-1, 740

0.32 mm i.d., df = 0.1 μm, Agilent) with He carrier gas inlet pressure programmed for constant 741

flow of 1.4 mL min-1 with a mass spectrometric detector (5973N, Agilent). GC was carried out 742

with temperature-programmed on-column injection and oven temperature set at 50˚C for 2 min, 743

raised by 40˚C min-1 to 200˚C, held for 2 min at 200˚C, raised by 3˚C min-1 to 320˚C, and held 744

for 30 min at 320˚C. Individual wax components were identified by comparing their mass 745

spectra with those of authentic standards and literature data. Quantitative analysis of wax 746

mixtures was carried out using capillary GC with flame ionization detector (FID) under the same 747

conditions as above, but with H2 carrier gas inlet pressure regulated for constant flow of 2 mL 748

min-1. Wax loads were determined by comparing GC-FID peak areas against internal standard, 749

and dividing by the leaf surface area from which the corresponding sample had been taken. 750

These surface areas were calculated for the direct wax extraction steps of both experiments 751

(“final extraction” in the first experiment and “total extraction” in the second experiment) using 752

the inner diameter of the glass cylinders and assuming circular geometry. The gum arabic 753

sampling steps were also restricted to circular areas with known diameter, in this case marked 754

prior to glue application and wax removal. 755

756

757

Cuticular transpiration measurements 758

759

Isolated CMs (20 mm in diameter) were used in gravimetric transpiration measurements 760

using methods adapted from Schönherr and Lendzian (1981). All CMs were carefully handled 761



34

34

with blunt spring steel tweezers, and had been stored for at least six weeks after isolation. Three 762

consecutive experiments were performed as described below on each individual CM to measure 763

paired values of permeances before and after wax removal steps. 764

765

First, intact CMs were attached to stainless steel transpiration chambers by vacuum 766

grease (Wacker, Munich, Germany), a ring-like lid was placed on top and fastened by adhesive 767

tape. The chambers were filled with de-ionized water through a small opening in the bottom of 768

the chambers, which was then sealed with two layers of adhesive tape. The chambers were 769

incubated upside down at 25 ± 1°C over dried silica beads in a closed plastic container. After 770

overnight pre-incubation, the water loss from the chambers was determined at five to eight 771

defined time points. After the transpiration measurements had been carried out on the intact 772

CMs, the chamber lids were carefully removed and a gum arabic solution (1 g/mL water) was 773

applied by a soft brush onto the exposed CM surfaces as described above for chemical analyses, 774

the gum arabic film was dried for at least one hour and then stripped off in order to remove 775

adhering epicuticular waxes. The transpiration chambers were then re-assembled by installing 776

the lids, refilling the water reservoirs and sealing with adhesive tape. Using this setup, 777

transpiration rates were determined for the cuticles without epicuticular waxes by gravimetrically 778

determining water loss at five defined time points. Finally, the water was removed from the 779

chambers, the setup was dried for several hours, and the remaining cuticular waxes were 780

removed by immersing the chambers together with the still mounted cuticles in CHCl3. The 781

chambers were then refilled with water, and transpiration rates of the MX (i.e. dewaxed cuticular 782

matrices) were determined gravimetrically (five time points). In a control experiment, CMs were 783

measured twice, once in the original setup and a second time after disassembling the chambers 784

and refilling them, but omitting the wax removal steps in between. 785

786

The transpiration rates were used to calculate permeances according to the equation: 787 788 P = F/A·Δc 789 790

where F is the measured flux of water in g/s, A is the transpiring cuticle surface area in m2, and 791

Δc is the difference between the (vapor-based) water concentration on the inner (Ci = 23.074 792



35

35

g/m3) and outer side of the cuticle (Co = 0 g/m3; silica beads adsorb all moisture in the container 793

around the transpiration chambers). From the three consecutive experiments (see above), 794

permeances resulted for the untreated CM (Pu), for cuticles from which epicuticular waxes had 795

been stripped off with gum arabic (Pg), and for dewaxed cuticles (Pd). Permeance values 796

deviating from the sample average by more than three times the standard deviation were 797

removed as outliers. Data were tested for normal or log-normal distribution (Kolmogoroff-798

Smirnov) and for significant differences (t-test) using Statistica software (6.0; StatSoft, Tulsa, 799

USA). 800

801 Resistances of the three different types of samples against water transport (Ru, Rg and Rd) 802

were calculated as inverse permeances (resistance R = 1/P). By correcting for the additional 803

resistance imposed by water surface tension and diffusion within the container (determined in 804

control experiments with open transpiration chambers, R0 = 3.37·10-6 s/m), the total resistance of 805

the intact membrane (CM) and the resistance of the MX could be calculated: 806 807

RCM = Ru – R0 808

RMX = Rd – R0 809 810

Finally, the resistance of individual membranes after epicuticular wax had been stripped off 811

(RES) was calculated for each sample by correcting the values after gum arabic treatment for the 812

control resistance (RES = Rg – R0). The resulting resistances were compared with those before the 813

initial gum arabic treatment (RCM) and after the ensuing wax extraction (RMX), to assess the 814

contributions of the epicuticular and the intracuticular wax, respectively: 815 816 Repi = RCM – RES 817

Rintra = RES – RMX 818 819

Resistance values for individual specimens were then averaged for each compartment within the 820

cuticle. In all experiments, permeance and resistance data showed normal distribution. Multiple 821

regression analysis were performed using SPSS (13.0; SPSS, Chicago, USA) with default 822

settings. In the overall dataset 18 independent families of parameters were recognized, and 823

according Bonferroni adjustments were used for reporting significant correlations (Suppl. Table 824

VII). 825

826



36

36

SUPPLEMENTAL MATERIAL 827

828 The following materials are available in the online version of this article. 829 830 Supplementary Table I. Compound class composition of the cuticular wax from adaxial leaf 831 sides of Citrus aurantium, Euonymus japonica, Clusia flava and Garcinia spicata. Percentages 832 within individual compound classes are given as mean values ± standard deviation (n = 5). 833 834 Supplementary Table II. Compound class composition of the cuticular waxes from adaxial leaf 835 sides of Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera deliciosa and Schefflera 836 elegantissima. Percentages of individual compound classes within respective wax mixtures are 837 given as mean values ± standard deviation (n = 5). 838 839 Supplementary Table III. Relative composition of the alicyclic compounds in the cuticular 840 waxes from adaxial leaf sides of Citrus aurantium, Euonymus japonica, Clusia flava and 841 Garcinia spicata. Amounts of individual compounds are given as percentages of the total 842 coverage of alicyclic wax compounds (mean values ± standard deviation; n = 5). 843 844 Supplementary Table IV. Relative composition of the alicyclic compounds in the cuticular 845 waxes from adaxial leaf sides of Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera 846 deliciosa and Schefflera elegantissima. Amounts of individual compounds are given as 847 percentages of the total coverage of alicyclic wax compounds (mean values ± standard deviation; 848 n = 5). 849 850 Supplementary Table V. Chain length composition of VLCFA derivative classes in the 851 cuticular wax from adaxial leaf sides of Citrus aurantium, Euonymus japonica, Clusia flava and 852 Garcinia spicata. Percentages within individual compound classes are given as mean values ± 853 standard deviation (n = 5). Trace (“tr”) signifies values below 0.05 μg/cm2. Data for other minor 854 chain lengths are not shown. 855 856

Supplementary Table VI. Chain length composition of VLCFA derivative classes in the 857 cuticular wax from adaxial leaf sides of Tetrastigma voinierianum, Oreopanax guatemalensis, 858 Monstera deliciosa and Schefflera elegantissima. Percentages within individual compound 859 classes are given as mean values ± standard deviation (n = 5). Trace (“tr”) signifies values below 860 0.05 μg/cm2. Data for other minor chain lengths are not shown. 861 862

Supplementary Table VII. Multivariate correlation matrix of parameters describing the 863 structure, composition and transpiration barrier properties for the investigated species. 864 Parameters are grouped into resistances, structural characteristics and wax composition, either in 865 absolute units (left half) or normalized to % (right half). Pearson correlation coefficients and 866 significance levels are given for all possible pairwise combinations of parameters. Those 867 parameters considered independent are marked "x" in the row "Bonferroni families". Significant 868 correlations, after Bonferroni-adjustment for 18 independent parameter families to P < 0.0028, 869 are highlighted light-yellow. Correlations with r2 > 0.75 are highlighted light-red. 870

871



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ACKNOWLEDGEMENTS 872

873

The authors thank Cornelia Vermeer for skillfully conducting the experiments. We also thank the 874

Botanical Garden of the University of Würzburg (Germany) for maintaining the plants, and the 875

Bio-imaging Facility at the University of British Columbia for providing microscopy and 876

technical support. 877

878

879

880

LIST OF AUTHOR CONTRIBUTIONS 881

882

Both authors designed the experiments, RJ oversaw their technical execution and analyzed the 883

data, and both authors contributed equally to the manuscript. 884

885 886 887



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888 889 890 891 892 Table I. Amounts of cuticular waxes sampled from adaxial leaf surfaces of eight plant species. In two independent experiments, waxes were removed 893 either in four consecutive treatments (three times with gum arabic and then by a final extraction) or in one step (total extraction). Wax amounts 894 (μg/cm2) were quantified by GC-FID, and results are given as mean values ± standard deviation (n = 5) for each treatment and for the sum of gum 895 arabic and extraction steps. 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915

916

Species Gum arabic Final

extraction Gum arabic

treatments plus final extraction

Total extraction

1st treatment 2nd treatment 3rd treatment Citrus aurantium 4.2 ± 1.8 0.6 ± 0.4 0.3 ± 0.2 1.9 ± 0.6 7.0 ± 2.2 7.0 ± 0.7 Euonymus japonica 3.3 ± 1.3 0.4 ± 0.2 0.2 ± 0.2 26.6 ± 5.2 30.5 ± 4.5 29.2 ± 4.6 Clusia flava 5.8 ± 1.0 0.2 ± 0.1 0.1 ± tr 8.9 ± 1.0 15.0 ± 1.5 12.5 ± 1.2 Garcinia spicata 14.9 ± 3.3 0.6 ± 0.1 0.2 ± 0.1 3.4 ± 1.7 19.1 ± 3.5 19.8 ± 2.6 Tetrastigma voinierianum 5.8 ± 3.0 0.2 ± tr 0.1 ± tr 3.3 ± 0.5 9.4 ± 2.5 11.7 ± 1.3 Oreopanax guatemalensis 1.8 ± 0.5 0.4 ± 0.2 0.4 ± 0.2 6.0 ± 2.3 8.6 ± 1.8 7.6 ± 1.3 Monstera deliciosa 2.6 ± 0.7 1.2 ± 0.6 0.6 ± 0.1 0.7 ± 0.04 5.1 ± 1.0 4.9 ± 0.7 Schefflera elegantissima 10.7 ± 2.9 1.4 ± 0.2 0.8 ± 0.4 3.7 ± 0.7 16.6 ± 2.6 18.4 ± 5.1

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Table II. Permeances of isolated cuticular membranes before and after wax removal. Initially, the water transpiration permeances of intact cuticular 917 membranes (CMs) containing both epi-and intracuticular waxes were measured, then the epicuticular wax was removed (by peeling with gum arabic) 918 and permeances determined again, then finally intracuticular waxes too were removed (by extraction with chloroform) and permeances of the 919 remaining cutin matrix (MX) measured. 920

921 922 923 924 925 926 927 928 929 930 931 932 933 934

Species Permeance of

intact cuticular membrane Permeance of

cuticular membrane minus epicuticular wax

Permeance of cuticular membrane minus epi- and intracuticular wax

m / s m / s m / s

Citrus aurantium 1.22·10-5 ± 8.81·10-6 2.10·10-5 ± 1.07·10-5 1.72·10-3 ± 1.90·10-4 Euonymus japonica 1.37·10-5 ± 7.70·10-6 2.11·10-5 ± 9.93·10-6 7.93·10-4 ± 1.17·10-4 Clusia flava 7.22·10-6 ± 8.34·10-6 2.17·10-5 ± 1.32·10-5 7.50·10-4 ± 4.23·10-4 Garcinia spicata 5.44·10-6 ± 4.00·10-6 9.32·10-6 ± 8.48·10-6 4.07·10-4 ± 7.74·10-5 Tetrastigma voinierianum 1.36·10-6 ± 4.33·10-7 1.78·10-6 ± 7.59·10-7 7.32·10-4 ± 1.94·10-4 Oreopanax guatemalensis 4.12·10-6 ± 3.14·10-6 5.92·10-6 ± 4.42·10-6 1.07·10-3 ± 1.33·10-3 Monstera deliciosa 1.29·10-6 ± 4.88·10-7 1.49·10-6 ± 6.15·10-7 3.65·10-4 ± 1.02·10-4 Schefflera elegantissima 1.83·10-6 ± 7.89·10-7 2.71·10-6 ± 1.57·10-6 3.92·10-4 ± 1.13·10-4

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935 FIGURE LEGENDS 936 937 Figure 1. Scanning electron micrographs of isolated cuticles. A, B. Side views of cryo-938 fractured cuticles from leaves of Citrus aurantium (A) and Monstera deliciosa (B), illustrating 939 species with relatively thin and thick cuticles, respectively. C, D. Top-down views onto the outer 940 surface of cuticles of Citrus aurantium (C) and Monstera deliciosa (D); the lower portions of 941 the pictures show areas from which the epicuticular wax film was removed with adhesive, while 942 the upper portions show the native cuticle surface. 943 944 Figure 2. Thickness and material coverages of isolated adaxial cuticles from leaves of eight 945 plant species. (A) Cuticular membranes (CMs) were isolated enzymatically, dried, cut 946 transversally and viewed by SEM to assess their cross section. Average values (n ≥ 15, ± 947 standard deviation) are given for the thickness of cuticle areas above the outer periclinal cell 948 walls of normal epidermal cells. (B) Wax coverages (μg/cm2) were calculated from weight 949 differences of isolated cuticular membranes before and after extraction with chloroform. (C) 950 Cutin coverages (μg/cm2) were determined gravimetrically after waxes had been extracted from 951 isolated cuticles. 952 953 Figure 3. Wax composition in isolated adaxial cuticles from leaves of eight plant species. 954 Epicuticular waxes were sampled using gum arabic as an adhesive, intracuticular waxes were 955 extracted after gum arabic treatments. In a separate experiment, total cuticular waxes were 956 extracted without prior adhesive treatment. Relative compositions are given as percentages of 957 compound classes in respective wax mixtures (n = 5; ± standard deviation). 958 959 Figure 4. Transpiration resistances of isolated adaxial cuticles from eight species. Water 960 transport was measured gravimetrically under controlled conditions for intact cuticular 961 membranes (CMs), after removing the epicuticular wax layer, and again after also removing the 962 remaining intracuticular waxes. Based on these data, the transpiration resistance of the total 963 cuticular material and the polymer matrix (MX) could be calculated directly, and the resistances 964 of the epicuticular and the intracuticular waxes were inferred as differences of resistances before 965 and after removing these waxes (n ≥ 15 ± standard deviation). 966 967 Figure 5. Total cuticular resistances plotted against cuticle thicknesses. A correlation could 968 not be found between total cuticular resistances and cuticle thicknesses, two parameters 969 describing the overall structure and physiological properties of the cuticles of eight tested 970 species. 971 972 Figure 6. Polymer matrix resistances plotted against matrix coverages. Two parameters 973 describing the amount and physiological properties of the cutin matrix were selected. For the 974 eight species investigated here, the resistance of the polymer matrix (MX) and its coverage were 975 not correlated (p = 0.11; r2 = 0.21). 976 977 Figure 7. Comparison of relative transpiration resistances and relative composition of 978 intracuticular waxes. The contributions of the intracuticular waxes to the overall barrier 979



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properties of the cuticular membranes were negatively correlated with the relative amounts of 980 alicyclic compounds in the intracuticular wax mixtures. 981 982



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1 running head: 2 localizing the cuticular transpiration ... · 72 percentages of alicyclic...

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