1
1
RUNNING HEAD: 1
Localizing the cuticular transpiration barrier 2
3
4
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
13
14
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
19
20
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
29
30
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.
2
2
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
35
36
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
43
*Corresponding author: Reinhard Jetter 44
Fax: (604) 822-6089 45
Email: [email protected] 46 47
48
49
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
59
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
3
3
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
80
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
4
4
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
5
5
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
6
6
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
7
7
167
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
8
8
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
175
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
9
9
199
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
10
10
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
11
11
209
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
215
216
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
12
12
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
254
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
269
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
13
13
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
14
14
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
15
15
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
310
311
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
16
16
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
17
17
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
18
18
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
19
19
398
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
20
20
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
21
21
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
22
22
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
37
37
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
38
38
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
ww
w.plantphysiol.org
on July 8, 2020 - Published by
Dow
nloaded from
Copyright ©
2015 Am
erican Society of P
lant Biologists. A
ll rights reserved.
39
39
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
w
ww
.plantphysiol.orgon July 8, 2020 - P
ublished by D
ownloaded from
C
opyright © 2015 A
merican S
ociety of Plant B
iologists. All rights reserved.
40
40
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
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
41
41
properties of the cuticular membranes were negatively correlated with the relative amounts of 980 alicyclic compounds in the intracuticular wax mixtures. 981 982
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Parsed CitationsArand K, Stock D, Burghardt M, Riederer M (2010) pH-dependent permeation of amino acids through isolated ivy cuticles isaffected by cuticular water sorption and hydration shell size of the solute. J Exp Bot 61: 3865-3873
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Balmann C, De Oliveira S, Gutenberger A, Waßmann F, Schreiber L (2011) Radioactive assay allowing the quantitativemeasurement of cuticular permeability of intact Arabidopsis thaliana leaves. Planta 234: 9-20
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Barthlott, W. and Neinhuis, C. Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta 202, 1-8. 1997.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Becker M, Kerstiens G, Schönherr J (1986) Water permeability of plant cuticles: permeance, diffusion and partition coefficients.Trees 1: 54-60
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Beyer M, Lau S, Knoche M (2005) Studies on water transport through the sweet cherry fruit surface: 9. Comparing permeability inwater uptake and transpiration. Planta 220: 474-485
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Burghardt M, Riederer M (2006) Cuticular transpiration. In M Riederer, C Müller, eds, Biology of the Plant Cuticle, Ed 1 Vol 23.Blackwell, Oxford, pp 292-311
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Buschhaus C, Herz H, Jetter R (2007a) Chemical composition of the epicuticular and intracuticular wax layers on adaxial sides ofRosa canina leaves. Ann Bot 100: 1557-1564
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Buschhaus C, Herz H, Jetter R (2007b) Chemical composition of the epicuticular and intracuticular wax layers on the adaxial sideof Ligustrum vulgare L. leaves. New Phytol 176: 311-316
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Buschhaus C, Jetter R (2011) Composition differences between epicuticular and intracuticular wax substructures: how do plantsseal their epidermal surfaces? J Exp Bot 62: 841-853
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Buschhaus, C. and Jetter, R. Composition and physiological function of the wax layers coating Arabidopsis leaves: ?-amyrinnegatively affects the intracuticular water barrier. Plant Physiology 160. 2012.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Carver TLW, Gurr SJ (2006) Filamentous fungi on plant surfaces. In M Riederer, C Müller, eds, Biology of the Plant Cuticle, Ed 1Vol 23. Blackwell, Oxford, pp 368-397
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Eigenbrode SD, Espelie KE (1995) Effects of plant epicuticular lipids on insect herbivores. Annu Rev Entomol 40: 171-194Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Geyer U, Schönherr J (1990) The effect of the environment on the permeability and composition of Citrus leaf cuticles. I. Waterpermeability of isolated cuticular membranes. Planta 180: 147-153
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Guhling O, Kinzler C, Dreyer M, Bringmann G, Jetter R (2005) Surface composition of myrmecophytic plants: cuticular wax andglandular trichomes on leaves of Macaranga tanarius. J Chem Ecol 31: 2325-2343 www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Haas K, Schönherr J (1979) Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrusleaves. Planta 146: 399-403
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hansjakob A, Bischof S, Bringmann G, Riederer M, Hildebrandt U (2010) Very-long-chain aldehydes promote in vitroprepenetration processes of Blumeria graminis in a dose- and chain length-dependent manner. New Phytol 188: 1039-1054
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hansjakob A, Riederer M, Hildebrandt U (2011) Wax matters: absence of very-long-chain aldehydes from the leaf cuticular wax ofthe glossy11 mutant of maize compromises the prepenetration processes of Blumeria graminis. Plant Pathol 60: 1151-1161
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Heredia A (2003) Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer. Biochim Biophys Acta 1620: 1-7Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jeffree CE (2006) The fine structure of the plant cuticle. In M Riederer, C Müller, eds, Biology of the Plant Cuticle, Ed 1 Vol 23.Blackwell, Oxford, pp 11-144
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jetter R, Kunst L, Samuels AL (2007) Composition of plant cuticular waxes. In M Riederer, C Müller, eds, Biology of the PlantCuticle, Ed 1 Vol 23. Blackwell, Oxford, pp 145-181
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jetter R, Schäffer S (2001) Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticularwax film during leaf development. Plant Physiol 126: 1725-1737
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jetter R, Schäffer S, Riederer M (2000) Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidencefrom Prunus laurocerasus L. Plant, Cell Environ 23: 619-628
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kerstiens G (1996) Cuticular water permeability and its physiological significance. J Exp Bot 47: 1813-1832Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kerstiens G, Lendzian KJ (1989) Interactions between ozone and plant cuticles II. Water permeability. New Phytologist 112: 21-27Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Knoche M, Peschel S, Hinz M, Bukovac MJ (2000) Studies on water transport through the sweet cherry fruit surface:characterizing conductance of the cuticular membrane using pericarp segments. Planta 212: 127-135
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kosma DK, Jenks MA (2007) Eco-physiological and molecular-genetic determinants of plant cuticle function in drought and saltstress tolerance. In MA Jenks, ed, Advances in Molecular Breeding Towards Salinity and Drought Tolerance, Ed 1 Springer,Heidelberg, pp 91-120
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Krauss P, Markstädter C, Riederer M (1997) Attenuation of UV radiation by plant cuticles from woody species. Plant, Cell Environ20: 1079-1085
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Le Roux JH (1969) Fischer-Tropsch waxes. II. Crystallinity and physical properties. J appl Chem 19: 86-88 www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Leide J, Hildebrandt U, Reussing K, Riederer M, Vogg G (2007) The developmental pattern of tomato fruit wax accumulation andits impact on cuticular transpiration barrier properties: effects of a deficiency in a ?-ketoacyl-CoA synthase (LeCER6). Plant Physiol144: 1667-1679
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lendzian KJ (2006) Survival strategies of plants during secondary growth: barrier properties of phellems and lenticels towardswater, oxygen, and carbon dioxide. J Exp Bot
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lendzian KJ, Nakajima A, Ziegler H (1986) Isolation of cuticular membranes from various conifer needles. Trees 1: 47-53Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Leveau JHJ (2006) Microbial communities in the phyllosphere. In M Riederer, C Müller, eds, Biology of the Plant Cuticle, Ed 1 Vol23. Blackwell, Oxford, pp 334-367
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Medina E, AguiarG, GomezM, MedinaJD (2004) Patterns of leaf epictuciular waxes in species of Clusia: taxonomical implications.Interciencia 29: 579-58s
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Müller C, Riederer M (2005) Plant surface properties in chemical ecology. J Chem Ecol 31: 2621-2651Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nawrath C (2006) Unraveling the complex network of cuticular structure and function. Current Opinion in Plant Biology 9: 281-287Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Popp C, Burghardt M, Friedmann A, Riederer M (2005) Characterization of hydrophilic and lipophilic pathways of Hedera helix L.cuticular membranes: permeation of water and uncharged organic compounds. J Exp Bot 56: 2797-2806
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Reisberg EE, Hildebrandt U, Riederer M, Hentschel U (2012) Phyllosphere bacterial communities of trichome-bearing andtrichomeless Arabidopsis thaliana leaves. Antonie van Leeuwenhoek 101: 551-560
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Riederer M (2006) Thermodynamics of the water permeability of plant cuticles: characterization of the polar pathway. J Exp Bot 27:2937-2942
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Riederer M, Schneider G (1989) Comparative study of the composition of waxes extracted from isolated leaf cuticles and fromwhole leaves of Citrus: Evidence for selective extraction. Physiologia Plantarum 77: 373 - 384.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Riederer M, Schreiber L (2001) Protecting against water loss: analysis of the barrier properties of plant cuticles. J Exp Bot 52:2023-2032
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J (1976) Water permeability of isolated cuticular membranes: the effect of cuticular waxes on diffusion of water. Planta131: 159-164
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J (2006) Characterization of aqueous pores in cuticles and permeation of ionic solutes. J Exp Bot 57: 2471-2491 www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J, Lendzian KJ (1981) A simple and inexpensive method of measuring water permeability of isolated plant cuticularmembranes. Z Pflanzenphysiol 102: 321-327
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J, Riederer M (1986) Plant cuticles sorb lipophilic compounds during enzymatic isolation. Plant Cell and Environment 9:459-46
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J, Riederer M (1988) Desorption of chemicals from plant cuticles: evidence for asymmetry. Arch Environ ContamToxicol 17: 13-19
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J, Schmidt HW (1979) Water permeability of plant cuticles, dependence of permeability coefficients of cuticulartranspiration on vapor pressure saturation deficit. Planta 144: 391-400
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schönherr J, Schreiber L (2004) Size selectivity of aqueous pores in astomatous cuticular membranes isolated from Populuscanescens (Aiton) Sm. leaves. Planta 219: 405-411
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schreiber L (2005) Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis. Ann Bot 95: 1069-1073Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schreiber L, Riederer M (1996) Ecophysiology of cuticular transpiration: comparative investigation of cuticular water permeabilityof plant species from different habitats. Oecologia 107: 426-432
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Small DM (1984) Lateral chain packing in lipids and membranes. J Lipid Res 25: 1490-1500Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Solovchenko A, Merzlyak M (2003) Optical properties and contribution of cuticle to UV protection in plants: experiments with applefruits. Photochem Photobiol Sci 2: 861-866
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tanaka H, Machida Y (2013) The cuticle and cellular interactions. In M Riederer, C Müller, eds, Biology of the Plant Cuticle, Vol 23.Blackwell, Oxford, pp 312-333
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M (2004) Tomato fruit cuticular waxes and their effects ontranspiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid ?-ketoacyl-CoAsynthase. J Exp Bot 55: 1401-1410
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Weichert H, Knoche M (2006) Studies on water transport through the sweet cherry fruit surface. 10. Evidence for polar pathwaysacross the exocarp. J Agr Food Chem 54: 3951-3958
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zeisler V, Schreiber L (2015) Epicuticular wax on cherry laurel (Prunus laurocerasus) leaves does not constitute the cuticulartranspiration barrier. Planta: online publication 0.1007/s00425-015-2397-y
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon July 8, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.