plantsReviewCuticular Waxes of Arabidopsis thaliana Shoots:Cell-Type-Specific Composition and BiosynthesisDaniela Hegebarth 1 and Reinhard Jetter 1,2,*1 Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4,Canada; daniela.hegebarth@botany.ubc.ca2 Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada* Correspondence: reinhard.jetter@botany.ubc.ca; Tel.: +1-604-822-2477Received: 1 June 2017; Accepted: 2 July 2017; Published: 7 July 2017Abstract: It is generally assumed that all plant epidermis cells are covered with cuticles, andthe distinct surface geometries of pavement cells, guard cells, and trichomes imply functionaldifferences and possibly different wax compositions. However, experiments probing cell-type-specific waxcompositions and biosynthesis have been lacking until recently. This review summarizes new evidenceshowing that Arabidopsis trichomes have fewer wax compound classes than pavement cells, and higheramounts of especially long-chain hydrocarbons. The biosynthesis machinery generating this characteristicsurface coating is discussed. Interestingly, wax compounds with similar, long hydrocarbon chains had beenidentified previously in some unrelated species, not all of them bearing trichomes.Keywords: trichomes; cuticular wax; chain length; Arabidopsis thaliana; KCS; elongation; fatty acidelongase complex; ketoacyl-CoA synthase1. IntroductionAll above-ground plant parts, in the primary state of development, are lined by a layer ofepidermis cells that serve crucial functions for protecting the various organs and, thus, for plantsurvival. The epidermis consists of three different cell types, the pavement cells, guard cells,and trichomes, in characteristic numbers, shapes and geometric arrangements depending on thespecies, organ, and developmental state.The different epidermis cell types serve very different functions (Figure 1; Table 1): pavement cellsform the major protective surface barrier [1], and mature Arabidopsis leaves contain about 29,000 cellseach with a surface area of ca. 4000 µm2 [2] (Table 1). Guard cells, on the other hand, are important forregulating gas exchange and for protecting the surface around stomata [3]. They are less abundantand smaller than pavement cells (Figure 1b), with ca. 10,000 guard cells on average per Arabidopsisleaf and average sizes of about 280 µm2 [2,4] (Figure 1b, Table 1). Finally, trichomes emerge verticallyout of the surface (Figure 1a), serving a variety of roles including UV protection, heat insulation,transpiration control, and insect deterrence [5]. Arabidopsis trichomes consist of a stalk with two orthree perpendicular arms (Figure 1a,b), and they are far less abundant (about 75 trichomes per leaf)but larger (surface areas about 40,000 µm2) than the other epidermal cell types [2] (Table 1).Plants 2017, 6, 27; doi:10.3390/plants6030027 www.mdpi.com/journal/plantsPlants 2017, 6, 27 2 of 19Plants 2017, 6, 27  2 of 19  (a) (b) Figure 1. Cryo-SEM images of abaxial Arabidopsis leaf surfaces. (a) Comparison of abundance and size of pavement and guard cells covering the leaf surface, relative to the trichome cells protruding out of the surface. (b) Detailed view of a single trichome, showing its shape and cell size relative to pavement and guard cells (reprinted from [6,7] with permission). Table 1. Trichome, guard cell, and pavement cell surface areas and cell numbers on adaxial Arabidopsis leaves (adapted from [2]). Projected Surface Area of Blade  (mm2) Number of Surface Area of Trichomes (Blade−1) Guard Cell Pairs (Blade−1) Pavement Cells (Blade−1) Pavement Cells (μm2 cell−1) Trichome Cells(μm2 cell−1) 138 72 10366 29602 4646 40000 It is generally assumed that all three epidermal cell types are covered with an uninterrupted cuticle, a hydrophobic surface consisting of a cutin matrix [8] and solvent-soluble waxes embedded in, and deposited onto, it. Cutin is a polyester of saturated and unsaturated C16 and C18  ω-hydroxyacids, polyhydroxyacids, or epoxyacids and glycerol [9,10]. Cuticular wax usually comprises a variety of aliphatic compound classes such as fatty acids, primary n-alcohols, secondary alcohols, alkyl esters, aldehydes, and alkanes, but also polyketides and terpenoids (Figure 2). Within the compound classes, usually compounds with varying carbon numbers in the hydrocarbon chains are found, thus defining series of aliphatic homologs. Both the abundances of individual constituents within the wax mixture and the relative amounts of wax and cutin vary greatly between plant species, organs, and developmental stages. For instance, in leaf blades of Triticum aestivum seedlings, primary n-alcohols are the predominant compound class, whereas on flag leaf sheaths β-diketones are predominant [11]. Arabidopsis leaf wax contains alkanes with a broad chain length ranging from C25 to C34, while Arabidopsis stem wax consists mainly of C29 alkane.  Figure 2. Chemical structures of major cuticular wax compound classes. 1 mm 300 μm Trichome Pavement CellGuard Cells Trichome i r . r - i es f i l rabi o sis le f s rf ces. ( ) ris f ce si ef t r ll ri t l f rf , r l ti t t tri ll r tr i t ft f ; ( ) t il i f i l t i , i it ll i r l ti t tll i , i i i ).a le 1. Trichome, guard cell, and pavement cell surface areas and cell numbers on adaxial Arabidopsisleaves (adapted from [2]).P ojected SurfaceArea of Blade (mm2)Number of Surface Area ofTrichomes(Blade−1)Guard Cell Pairs(Blade−1)Pavement Cells(Blade−1)Pavement Cells(µm2 cell−1)Trichome Cells(µm2 cell−1)138 72 10366 29602 4646 40000It is generally assumed that all three epidermal cell types are covered with an uninterruptecuticle, a hydrophobic surface consisting of a cutin matrix [8] and solve t-soluble waxes embedded in,and deposited onto, it. Cutin is a polyester of saturated and unsaturated C16 and C18 ω-hydroxyacids,p lyhydroxyacids, r epoxyacids and glycerol [9,10]. Cuticular wax usually comprises a varietof aliphatic compound classes such as fatty acids, primary n-alcohols, secon ary alcohols, alkylesters, aldehy es, and alkanes, but also polyketides and terpenoids (Figure 2). Within the c mpou dclasses, usually compounds with varying carbon numbers in the hydrocarbon chains are fou d,thus defining series of liphatic homologs. Both the abundances of individual constitue ts withinthe wax mixture and the rel tive amounts of wax and cutin vary gre tly between plant species,organs, and developmental stages. For instance, in leaf blades f Triticum aestivum seedlings, primaryn-alc hols are the predominant compound class, whereas on fl g le f sheaths β-diketones arepredominant [11]. Arabidopsis leaf wax contains alkanes with a broad chain length ranging fromC25 to C34, while Arabidopsis stem wax consists mainly of C29 alkane.The mechanisms underlying wax biosynthesis have been largely elucidated using modelorganisms such as Arabidopsis and tomato. First, C16 and C18 fatty acid thioesters are synthesizedde novo in the plastids of epidermal cells. These precursors are then hydrolyzed to free acids,exported to the endoplasmic reticulum (ER), and activated to acyl-CoAs by long chain acyl-CoAsynthases (LACSs) [12]. At the ER, acyl-CoAs are elongated in several elongation cycles from C16and C18 to very-long-chain fatty acids (VLCFA), which usually have aliphatic chains with 24–34carbons [13] (Figure 3).Plants 2017, 6, 27 3 of 19Plants 2017, 6, 27  2 of 19  (a) (b) Figure 1. Cryo-SEM images of abaxial Arabidopsis leaf surfaces. (a) Comparison of abundance and size of pavement and guard cells covering the leaf surface, relative to the trichome cells protruding out of the surface. (b) Detailed view of a single trichome, showing its shape and cell size relative to pavement and guard cells (reprinted from [6,7] with permission). Table 1. Trichome, guard cell, and pavement cell surface areas and cell numbers on adaxial Arabidopsis leaves (adapted from [2]). Projected Surface Area of Blade  (mm2) Number of Surface Area of Trichomes (Blade−1) Guard Cell Pairs (Blade−1) Pavement Cells (Blade−1) Pavement Cells (μm2 cell−1) Trichome Cells(μm2 cell−1) 138 72 10366 29602 4646 40000 It is generally assumed that all three epidermal cell types are covered with an uninterrupted cuticle, a hydrophobic surface consisting of a cutin matrix [8] and solvent-soluble waxes embedded in, and deposited onto, it. Cutin is a polyester of saturated and unsaturated C16 and C18  ω-hydroxyacids, polyhydroxyacids, or epoxyacids and glycerol [9,10]. Cuticular wax usually comprises a variety of aliphatic compound classes such as fatty acids, primary n-alcohols, secondary alcohols, alkyl esters, aldehydes, and alkanes, but also polyketides and terpenoids (Figure 2). Within the compound classes, usually compounds with varying carbon numbers in the hydrocarbon chains are found, thus defining series of aliphatic homologs. Both the abundances of individual constituents within the wax mixture and the relative amounts of wax and cutin vary greatly between plant species, organs, and developmental stages. For instance, in leaf blades of Triticum aestivum seedlings, primary n-alcohols are the predominant compound class, whereas on flag leaf sheaths β-diketones are predominant [11]. Arabidopsis leaf wax contains alkanes with a broad chain length ranging from C25 to C34, while Arabidopsis stem wax consists mainly of C29 alkane.  Figure 2. Chemical structures of major cuticular wax compound classes. 1 mm 300 μm Trichome Pavement CellGuard Cells Trichome Figure 2. Chemical structures of major cuticular wax compound classes.Plants 2017, 6, 27  3 of 19 The mechanisms underlying wax biosynthesis have been largely elucidated using model organisms such as Arabidopsis and tomato. First, C16 and C18 fatty acid thioesters are synthesized de novo in the plastids of epidermal cells. These precursors are then hydrolyzed to free acids, exported to the endoplasmic reticulum (ER), and activated to acyl-CoAs by long chain acyl-CoA synthases (LACSs) [12]. At the ER, acyl-CoAs are elongated in several elongation cycles from C16 and C18 to very-long-chain fatty acids (VLCFA), which usually have aliphatic chains with 24–34 carbons [13]  (Figure 3).   Figure 3. Wax biosynthesis pathways in Arabidopsis. First, acyl-CoAs are elongated by fatty acid elongase (FAE) complexes (black), then their head groups are modified along either the alkane-forming pathway (blue) or the alcohol-forming pathway (red), and the wax compounds are exported to the cuticle (dashed gray arrows). Each elongation cycle is carried out by a fatty acid elongase (FAE), an enzyme complex catalyzing four sequential reactions effecting the overall extension of the hydrocarbon chain by two carbons (Figures 3 and 4). In each cycle, first a β-ketoacyl-CoA synthase (KCS) fuses the incoming chain with a C2 unit from malonate, and then a β-ketoacyl-CoA reductase (KCR), a β-hydroxyacyl-CoA dehydratase (HCD), and an enoyl-CoA reductase (ECR) reduce the functional group of the intermediate into a methylene (CH2) unit [14–20] (Figure 4). The initial condensing reaction, catalyzed by KCS enzymes, is the rate-limiting step and determines the chain length range of substrates and products of the FAE complex [19], while the other three FAE enzymes, the KCR, HCD, and ECR, are used ubiquitously by all FAE complexes [19,21]. It is likely that different FAEs co-exist within one cell, together generating a broad chain length range of acyls with predominantly even carbon numbers. Based on Arabidopsis ksc mutant analyses, the KCS enzyme KCS6/CER6 was found to be central for cuticular wax biosynthesis, as it elongates C24 to C28 acyl-CoAs. More recently, it was shown that the KCS6/CER6 FAE complex may be associated with CER2-LIKE proteins, then enabling elongation of acyl-CoAs up to C34 (Figure 3), yet the mechanism of action of CER2-LIKE proteins remains unknown [22–25]. After elongation, acyl-CoAs are modified on two branch pathways: on the alkane-forming pathway, the CER3 and CER1 enzymes consecutively reduce and decarbonylate acyls, into aldehydes with predominantly even carbon numbers, and alkanes one carbon shorter, and thus odd C-numbers, respectively [26–28] (Figure 3). The alkanes may be converted into secondary alcohols and ketones by the mid-chain alkane hydroxylase1 (MAH1) [29]. On the alcohol-forming pathway, fatty acyl-CoA Figure 3. Wax biosynthesis pathways in Arabidopsis. First, acyl-CoAs are elongated by fatty acidelongase (FAE) complexes (black), then their head groups are modified along either the alkane-formingpathway (blue) or the alcohol-forming pathway (red), and the wax compounds are exported to thecuticle (dashed gray arrows).Each elongation cycle is carried out by a fatty acid elongase (FAE), an enzyme complexcatalyzing four sequential reactions effecting the overall extension of the hydrocarbon chain by twocarbons (Figures 3 and 4). In each cycle, first a β-ketoacyl-CoA synthase (KCS) fuses the incomingchain with a C2 unit from malonate, and then aβ-ketoacyl-CoA reductase (KCR), aβ-hydroxyacyl-CoAdehydratase (HCD), and an enoyl-CoA reductase (ECR) reduce the functional group of the intermediateinto a methylene (CH2) unit [14–20] (Figure 4). The initial condensing reaction, catalyzed by KCSenzym s, is the rate-limiting step and d termines he chain length ra ge of ubstrates and productsof the FAE complex [19], while the other three FAE nzyme , the KCR, HCD, and ECR, are usedubiquitously by all FAE com lexes [19,21]. It is likely that different FAEs co-exist within one cell,together generating a broad chain length range of acyls with predominantly even carbon numbers.Based on Arabidopsis ksc mutant analyses, the KCS enzyme KCS6/CER6 was found to be central forcuticular wax biosynthesis, as it elongates C24 to C28 acyl-CoAs. More recently, it was shown thatthe KCS6/CER6 FAE complex may be associated with CER2-LIKE proteins, then enabling elongationof acyl-CoAs up to C34 (Figure 3), yet the mechanism of action of CER2-LIKE proteins remainsunknown [22–25].Plants 2017, 6, 27 4 of 19Plants 2017, 6, 27  4 of 19 reductases (FARs) form even-numbered n-alcohols [30], which can be converted into alkyl esters by the wax synthase/diacylglycerol acyltransferase1 (WSD1) enzyme [31] (Figure 3).  Figure 4. Elongation of acyl-CoAs by the fatty acid elongase (FAE) complex. It is well established that cuticular waxes are the crucial component of the cuticle, serving its primary physiological function as a barrier that limits transpirational water loss [32]. Therefore, it is generally assumed that all three epidermal cell types must be lined by continuous cuticles comprising similar wax mixtures to protect the entire plant tissue. However, it is not clear whether the pavement cells, guard cells and trichomes have autonomous wax biosynthesis machineries, and whether they produce different wax mixtures to serve slightly different functions in the different geometric contexts of the three cell types. Indirect evidence had gradually accumulated, suggesting that at least trichomes may have cuticular wax compositions and biosynthesis distinct from those of pavement cells, and very recently also direct evidence on trichome waxes has emerged. The current review will summarize all this evidence, focusing first on the composition of Arabidopsis trichome waxes (Section 2) and the trichome-specific wax biosynthesis mechanisms in Arabidopsis (Section 3), and then providing context on similar wax compositions in other plant species (Section 4), as well as their possible implications on wax structure, properties, and functions (Section 5). 2. Composition of Cuticular Wax Covering Arabidopsis Trichomes  Several lines of evidence have addressed the question whether cuticular wax on Arabidopsis trichomes differs from that on pavement cells, first using indirect approaches and, in recent years, also direct chemical analyses. Initially, indirect evidence came from SEM investigations of Arabidopsis stem surfaces. It had repeatedly been reported that the surface of stem pavement cells is covered with epicuticular wax crystals [33–37], while the surface of stem trichomes was devoid of crystals [6,7,38]. It is well established that epicuticular wax crystals form due to the accumulation of one or a few compounds in the wax mixture, and the absence or presence of crystals, hence, reflects different amounts of the crystal-forming compounds within the overall wax mixture [39,40]. Consequently, the different micro-reliefs of Arabidopsis stem pavement and trichome surfaces suggested that these two cell types have different wax compositions. However, this qualitative comparison did not reveal how exactly both wax mixtures differ and, therefore, additional information was required. Unfortunately, such evidence could not be drawn from analogous SEM studies of other Arabidopsis organ surfaces, because they either lack trichomes or else their pavement and trichome cells do not differ in surface morphology. Other, more quantitative approaches were necessary to gauge the differences between the trichome and pavement wax mixtures. Figure 4. Elongation of acyl-CoAs by the fatty acid elongase (FAE) complex.After elongation, acyl-CoAs are modified on two branch pathways: on the alkane-formingpathway, the CER3 nd CER1 enzymes consecu ively reduc and decarbonylate acyls, i to aldehydeswith predominantly even carbon numbers, and alkanes one carbon shorter, and thus odd C-numbers,respectively [26–28] (Figure 3). The alkanes may be converted into secondary alcohols and ketones bythe mid-chain alkane hydroxylase1 (MAH1) [29]. On the alcohol-forming pathway, fa ty acyl-CoAreductases (FARs) form even-numbered n-alcohols [30], which can be converted into alkyl esters bythe wax synthase/diacylglycerol acyltransferase1 (WSD1) enzyme [31] (Figure 3).It is well established that cuticular waxes are the crucial component of th cu icle, serving itsprimary physiological function as a barrier that limits transpirational water loss [32]. Therefore, it isgenerally assumed that all three epidermal cell types must be lined by continuous cuticles comprisingsimilar wax mixtures to protect the entire plant tissue. However, it is not clear whether the pavementcells, guard cells and tricho es have auto omous wax biosynthesis machineries, and whether theyproduce different wax mixtures to serve slightly different functions in the different geometric contextsof the three cell types. Indirect evidence had gradually accumulated, suggesting that at least trichomesmay have cuticular wax compositions and biosynt esis distinct from those of pavement cells, and veryrecently also direct evidence on trichome waxes has emerged. The current review will summarizeall this evidence, focusing first on the composition of Arabidopsis trichome waxes (Section 2) and thetrichome-spec fic wax biosynthe is echanisms in Arabidopsi (Section 3), and hen providing contexton similar wax compositions in other plant species (Section 4), as well as their possible implications onwax structure, properties, and functions (Section 5).2. Composition of Cuticular Wax Covering Arabidopsis TrichomesSeveral lines of evidence have addressed the question whether cuticular wax on Arabidopsistrichomes differs from that on pavement cells, first using indirect approaches and, in recent years,also direct chemical analyses.Initially, indirect evidence came from SEM investigations of Arabidopsis stem surfaces. It hadrepeatedly been reported that the surface of stem pavement cells is covered with epicuticular waxcrystals [33–37], while the surface of stem trichomes was devoid of crystals [6,7,38]. It is well establishedthat epicuticular wax crystals form due to the accumulation of one or a few compounds in the waxmixture, and the absence or presence of crystals, hence, reflects different amounts of the crystal-formingcompounds within the overall wax mixture [39,40]. Consequently, the different micro-reliefs ofArabidopsis stem pavement and trichome surfaces suggested that these two cell types have differentwax compositions. However, this qualitative comparison did not reveal how exactly both wax mixturesdiffer and, therefore, additional information was required. Unfortunately, such evidence could notbe drawn from analogous SEM studies of other Arabidopsis organ surfaces, because they either lacktrichomes or else their pavement and trichome cells do not differ in surface morphology. Other, morePlants 2017, 6, 27 5 of 19quantitative approaches were necessary to gauge the differences between the trichome and pavementwax mixtures.First quantitative distinctions between pavement and trichome surface compositions wereenabled by Arabidopsis mutant studies [6]. In these experiments, compositions of cuticular waxeswere compared between the trichome-free gl1 mutant, the wild type, and the trichome-rich mutantcpc tcl1 etc1 etc3. The leaf wax of the trichome-rich mutant contained higher amounts of C32–C37 waxcompounds compared to the wild type and trichome-free mutant (Figure 5a). The shifts in chain lengthdistributions occurred within all major compound classes, irrespective of their functional groups,whereas the absolute amounts of the compound classes were not significantly different between lineswith different trichome numbers. Moreover, the stem waxes of the trichome mutant lines showedsimilar effects to those observed for leaves, albeit with increases mainly in C32 and C33 compounds [6].Overall, these results revealed a correlation between the abundances of trichomes and extremelylong-chain aliphatic compounds. Pavement and trichome waxes were, thus, found to differ in theirchain length profiles.Plants 2017, 6, 27  5 of 19 First quantitative distinctions between pavement and trichome surface compositions were enabled by Arabidopsis mutant studies [6]. In these experiments, compositions of cuticular waxes were compared between the trichome-free gl1 mutant, the wild type, and the trichome-rich mutant cpc tcl1 etc1 etc3. The leaf wax of the trichome-rich mutant contained higher amounts of C32–C37 wax compounds compared to the wild type and trichome-free mutant (Figure 5a). The shifts in chain length distributions occurred within all major compound classes, irrespective of their functional groups, w ereas the absolute amounts of the compound classes wer  not significantly differe t between lines with different trichome numbers. Moreover, the stem waxes of the trichome mutant lines showed similar effects to those observed for leaves, albeit with increases mainly in C32 and C33 compounds [6]. Overall, these results revealed a correlation between the abundances of trichomes and extremely long-chai  aliphatic compounds. Pavement and trichome waxes were, thus, foun  to differ in th ir chain length profiles.   (a) (b) Figure 5. Wax composition of Arabidopsis leaves and isolated trichomes. (a) Coverage of single wax compounds within each compound class on leaves of the trichome-free mutant gl1, the wild type, and the trichome-rich mutant cpc tcl1 etc1 etc3. (b) Relative distributions of single compounds within each compound class on leaves of the trichome-free mutant gl1, on trichomes isolated from wild type leaves, and on trichomes isolated from trichome-rich mutant leaves. Average values are given with standard deviations (n = 5). The x-axis labels indicate the total carbon chain numbers of compounds. Asterisks indicate discovery of significant differences between coverages based on Student’s t-test (* = p < 0.05) (adapted from [6]). The inferences from mutant comparisons were confirmed by further investigations into wax compositional changes on developing Arabidopsis wild type leaves [2]. It had previously been shown that, in most dicot species, trichome development starts very early during leaf ontogeny, before major pavement cell expansion. Therefore, immature (still expanding) leaves have higher trichome:pavement surface ratios than mature leaves, and trichomes constitute a larger portion of the surface area in younger leaves. Comparisons of cuticular wax compositions between young and mature leaves can thus be interpreted as proxies for differences in cuticular wax composition of trichomes and pavement cells. Most interestingly, young wild type leaves had relatively high abundances of C35+ compounds, which then decreased over the course of leaf development. This shift in chain length profiles was accompanied by a steady decrease of apparent wax coverages, calculated as wax amounts relative to the macroscopic leaf surface and, thus, likely reflecting the steady decrease in microscopic aspect ratios due to declining trichome:pavement ratios. Taken together, the time-dependent wax compositional changes suggested that the levels of C35+ compounds were positively correlated with trichome densities, confirming that trichome and pavement waxes had different chain length profiles. To further corroborate the conclusions from the previous mutant and time course studies, both approaches were combined to monitor the wax development of the trichome-less Arabidopsis mutant 24 26 28 30 32 34 28 30 32 34 27 29 31 33 35 37 35 37 26 28 30 32 34 30 32 340.000.050.100.150.200.250.30Coverage (µg/cm2 )Trichome-free leavesWild type leavesTrichome-rich leaves********** **Fatty acids Aldehydes Alkanes prim. br. Alcoholsprim. n-AlcoholsAlkenes27 29 31 33 35 37 33 35 37 26 28 30 32 3401020304050607080Relative composition (% of compound class)Wild type trichomesAlkanes Alkenes prim. n-AlcoholsTrichome-free leavesTrichome-rich mutant trichomesFigure 5. Wax composition of Arabidopsis leaves and isolated trichomes. (a) Coverage of single waxcompounds within each compound class on leaves of the trichome-free mutant gl1, the wild type, andthe trichome-rich mutant cpc tcl1 etc1 etc3; (b) R lative distribu ions of singl compounds within eachcompound class on leaves of the trichome-free utant gl1, on trichomes isolated from wild type leaves,and on trichomes isolated from trichome-rich mutant leaves. Average values are given with standarddeviations (n = 5). The x-axis labels indicate the total carbon chain numbers of compounds. Asterisksindicate discovery of significant differences between coverages based on Student’s t-test (* = p < 0.05)(adapted from [6]).The inferences from muta t compa isons were confirmed by further investigations into waxcompositional changes o developing Arabidopsis wild type leaves [2]. It had previously been shownthat, in most dicot species, trichome development starts very early during leaf ontogeny, before majorpavement cell expansion. Therefore, immature (still expanding) leaves have higher trichome:pavementsurface ratios than mature leaves, and trichom s constitute a larger porti n of he urface area inyounger leaves. Comparisons of cuticular wax compositi ns between young and mature leavescan thus be interpreted as proxies for differences in cuticular wax composition of trichomes andpavement cells. Most interestingly, young wild type leaves had relatively high abundances of C35+compounds, which en decreased over the course of leaf evelopment. This shift in chain lengthprofiles was accompanied by a steady decrease of apparent wax coverages, calculated as wax amountsrelative to the macroscopic leaf surface and, thus, likely reflecting the steady decrease in microscopicaspect ratios due to declining trichome:pavement ratios. Taken together, the time-dependent waxcompositional changes suggested that the levels of C35+ compounds were positively correlated withtrichome densities, confirming that trichome and pavement waxes had different chain length profiles.To further corroborate the conclusions from the previous mutant and time course studies,both approaches were combined to monitor the wax development of the trichome-less ArabidopsisPlants 2017, 6, 27 6 of 19mutant gl1 [2]. In contrast to wild type, gl1 leaf wax coverages did not change during development,and in this mutant C35+ compounds were detected at relatively low, constant levels throughout leafexpansion. These results were in stark contrast to the previously observed apparent drop in both waxcoverage and C35+ compounds during wild type leaf expansion, thus confirming that both effectsdepended on the presence of trichomes. The mutant time-course data further underline the conclusionthat trichome surfaces are distinguished from those of pavement cells through the higher abundanceof compounds with extremely long aliphatic chains.Finally, the cell-type-specific surface compositions were assessed directly by chemical analyses ofwaxes from isolated trichomes [6]. The selectively sampled wax from Arabidopsis leaf trichomes hadhigher amounts of C32+ compounds than pavement cell wax (as judged by the composition of the gl1leaf wax) (Figure 5b), thus confirming the results from the previous mutant comparisons and timecourse studies. Interestingly, the direct analysis of wax from isolated (leaf) trichomes also revealedthat it contained mainly alkanes and primary n-alcohols, only two of the many compound classesfound in pavement wax. The trichome wax further comprised small amounts of alkenes, a compoundclass not reported for Arabidopsis waxes before. It seems likely that the alkenes had not been noticed inearlier studies, due to detection problems caused by relatively small trichome contributions to totalwax extracts from mature leaves.In summary, all the recent indirect and direct evidence led to matching conclusions, showingthat the cuticular wax lining Arabidopsis trichomes differs significantly from that on pavement cells.Trichome wax is distinguished by a relatively simple composition with only few compound classes,by the presence of alkenes, and by shifts to longer chain lengths in all compound classes.With such differences between Arabidopsis cell types now firmly established, we mustconsider whether the compositional gradients between trichomes and adjacent pavement mightlead to lateral diffusion along the surface of these cells. To address possible lateral exchange ofconstituents, the mobility of compounds within wax mixtures must be assessed. Schreiber [41]determined self-diffusion coefficients of VLC compounds within wax, reaching values of approximately10−20 m2 s−1 for example for C24–C28 compounds. Based on these self-diffusion coefficients, we predictthat wax molecules will not migrate fast enough within the mixture to cause significant exchangeof material between trichome and pavement waxes. Moreover, if diffusion were to occur, it wouldonly lessen differences over time rather than enhancing them. Consequently, the observed differencesin wax compositions of both cell types at certain times after cuticle formation must be regarded asminimum effects and, assuming limited, local migration, even more drastic differences may have beeninitially established during trichome development.3. Wax Biosynthesis in Arabidopsis Trichome CellsBased on the findings that the composition of trichome surface wax differs from that on pavementcells, it must be assumed that both cell types have autonomous wax biosynthesis machinery, and thatat least some of the genes/enzymes involved differ between them. Most investigations into waxbiosynthesis so far used whole tissues, including all types of epidermal cells, and thus mixtureswere strongly dominated by pavement cells rather than trichomes. Therefore, findings from thesewhole-tissue experiments can be taken as proxies for wax biosynthesis in pavement cells, but notnecessarily for trichomes. Some recent reports now add new information on wax metabolism intrichomes, revealing both commonalities and differences between both cell types.Firstly, promoter activity studies using GUS staining or GFP fluorescence identifiedcell-type-specific expression patterns of wax biosynthesis genes. The Arabidopsis mid-chain alkanehydroxylase enzyme MAH1, responsible for formation of the secondary alcohol and ketone productsof the alkane-forming pathway, is expressed in stem pavement cells, but not in stem trichomesor guard cells [29]. Conversely, the Arabidopsis fatty acyl-CoA reductase CER4 synthesizing waxprimary n-alcohols is expressed preferentially in leaf trichomes rather than pavement cells [30].GUS analyses showed also time-dependent changes in expression levels of several wax biosynthesisPlants 2017, 6, 27 7 of 19genes. For instance, the elongase-associated protein CER2 was found expressed in trichomes and guardcells of developing leaves, but not in mature leaves [42]. Similarly, the alkane-forming decarbonylaseCER1 showed strong expression in trichomes of young leaves, and its expression level decreased withleaf maturation [43].Secondly, analyses of transcriptome datasets from Arabidopsis leaf trichome and pavement cellsconfirmed that both cell types have autonomous wax biosynthetic machinery. On the one hand,several wax biosynthetic genes are expressed equally in pavement cells and trichomes, including someencoding ketoacyl-CoA synthetase (KCS) components of the fatty acid elongase (FAE) complexes(KCS3, KCS4, KCS6/CER6, KCS9, KCS11-14, KCS19, and KCS20), other FAE enzymes such as theketoacyl-CoA reductase (KCR1), the β-hydroxyacyl-CoA dehydratase (HCD/PAS2) and the enoyl-CoAreductase (ECR/CER10), as well as proteins associated with the FAE (CER2 and CER2-LIKE2),and head-group-modifying enzymes, such as the alkane-forming reductase (CER3) and decarbonylase(CER1), and the alcohol-forming reductase (FAR3/CER4) [6,38]. Together, these genes are knownto encode a full complement of wax biosynthetic enzymes, and both pavement and trichome cells,thus, likely harbor the entire machinery required to form major wax constituents, such as aldehydes,alkanes, and primary n-alcohols.On the other hand, further homologs of the genes listed above were found expressed differentiallybetween pavement cells and trichomes. Particularly, KCS2/DAISY and KCS16 were expressed, albeitweakly, only in developing trichomes, while KCS1, KCS5/CER60, KCS8, and KCS10 had especially highexpression signals in developing trichomes. Similarly, homologs of other enzymes associated withthe FAE (KCR2 and CER2-like1/CER26) and with head group modification (CER1-like1) were highlyexpressed only in developing trichomes. Finally, the sole homologs of genes encoding two other headgroup modification enzymes, the mid-chain hydroxylase (MAH1) and the wax ester synthase (WSD1),were also expressed preferentially in trichomes.The KCS enzymes are known to confer chain length specificity to the FAE and, thus, to dictateoverall chain length profiles of wax mixtures. Therefore, the finding that several Arabidopsis KCS genesare expressed preferentially in trichome cells was noteworthy, since Arabidopsis trichomes had also beenreported to contain relatively high amounts of especially long-chain wax constituents (C35 and C37).Previously, nothing was known about the enzymatic machinery involved in elongating fatty acylprecursors beyond C34, and several of the KCS homologs in Arabidopsis had not been characterized.From these candidates, KCS16 was recently selected based on its trichome-specific expression. Detailedbiochemical and molecular genetic investigations revealed that ksc16 loss-of-function mutants weredepleted of C35+ products in trichome and pavement cell waxes, whereas expression of KCS16in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C36 and C38 fattyacids [7]. Together, these findings showed that KCS16 is the sole enzyme catalyzing the elongation ofC34 to C38 acyl-CoAs in Arabidopsis leaf trichomes and that it is, thus, crucial for the trichome-specificformation of especially long-chain wax compounds. Overall, the characterization of KCS16 illustrateshow the cell-type-specific composition of trichome wax results from differential expression ofa dedicated enzyme which is homologous to ubiquitous wax biosynthesis enzymes, but has a distinctproduct profile.It is worth noting that the expression patterns of certain wax biosynthesis genes did not matchwax compositional differences between trichomes and pavement cells. For instance, microarray andGUS analyses showed that the CER4 gene, encoding the fatty acyl-CoA reductase responsible for theformation of wax primary n-alcohols, was expressed mostly in leaf trichomes rather than pavementcells [6,30]. However, diverse chemical analyses (of Arabidopsis trichomes and intact leaves, includingontogenetic time course experiments, see above) unambiguously showed that primary n-alcoholsare present in cuticular waxes of both cell types. This seeming contradiction might be explainedon the one hand by relatively high detection limits of the involved microarray experiments, and byhighly-sensitive GUS staining of trichomes on the other. Even a low level of expression in pavementcells over relatively long spans of pavement development might result in sufficient enzyme activity toPlants 2017, 6, 27 8 of 19account for the alcohol products found on the pavement cells. Indeed, non-negligible CER4 expressionin Arabidopsis leaves was reported from qRT-PCR experiments [2], thus, highlighting the need to testexpression data beyond microarray analyses.In summary, all the recent studies involving transcriptome and promoter analyses confirmedthat trichomes have a complete set of wax biosynthesis genes enabling autonomous wax biosynthesis.However, beyond the ubiquitous complement of wax biosynthesis genes shared between all epidermiscells, trichomes may contain (at least some) unique enzymes extending their wax metabolic pathways.Not only the expression of the genes encoding these trichome-specific enzymes differs betweentrichomes and pavement cells, but also the expression of other, ubiquitous wax biosynthesis enzymes.4. Extra-Long Compounds in the Wax Mixtures of Diverse Plant SpeciesAll the evidence discussed so far shows that Arabidopsis thaliana trichomes have distinct waxcomposition from neighboring pavement cells, due to also distinct biosynthetic mechanisms. To furtherunderstand the evolution and possible eco-physiological functions of trichome waxes, it would beinteresting to know whether such cell-type-specific wax compositions and biosynthesis also occurin other species. However, only little is known about the composition of cuticular waxes coveringtrichomes of other plant species.There is scattered, indirect evidence that cuticular waxes covering trichomes of species other thanArabidopsis differ from those on respective pavement cells. For example, the leaves of Puccinelliatenuiflora and Oryza sativa are known to have epicuticular wax crystals on their pavement cell surfaces,but not on adjacent trichomes [44], indicating wax compositional differences between both epidermiscell types analogous to Arabidopsis. Many studies addressed the total chemical compositions ofglandular trichomes, but did not investigate the trichome waxes specifically [45–47]. In one exceptionalinvestigation, the trichomes isolated from peach fruit were analyzed and found to have cuticular waxconsisting mainly of alkanes (92%), with chain lengths ranging from C22 to C34 [48]. In contrast, wax onaccompanying pavement cells comprised several compound classes, with only 72% alkanes, and chainlength profiles (C23–C29) lacking the extremely long homologs. The differences between trichome andpavement wax compositions on peach fruit, thus, strongly resemble those reported for Arabidopsisleaf trichomes.Unfortunately, the trichome waxes of no other species have been investigated to date, and itremains unclear whether cell-type-specific wax compositions similar to those of Arabidopsis and peachexist elsewhere. Further studies into the wax compositions of both non-glandular and glandulartrichomes of diverse taxa would be of great interest to better understand the specific function ofcuticles lining these special epidermis cells.Conversely, wax compounds with extra-long hydrocarbon chains similar to those on Arabidopsistrichomes had previously been described in the bulk wax mixtures (extracted from whole organswithout discriminating between epidermal cell types) of diverse other plant species (Table 2). It isinteresting to now compare the occurrence of such C35+ aliphatics in diverse taxa growing in varioushabitats, as a backdrop for future investigations into their formation and function, possibly also in thecontext of trichome-specific accumulation.Extra-long hydrocarbon chains were typically encountered in relatively small amountsaccompanying much larger quantities of the C26–C34 ubiquitous compounds in bell-shaped homologdistributions. Since trace amounts of C35+ wax compounds were detected in fairly diverse analyses,similar, small quantities of them may be surmised in other species as well, but some wax analysesmay have failed to detect longer homologs due to instrument settings with limited sensitivity.The occurrence of very low amounts of compounds at the high end of the homolog distributionsuggested that they are formed merely as by-products of the normal wax biosynthesis machineryrather than through dedicated processes specific to their chain lengths. Characterization of enzymesinvolved in wax precursor elongation in respective species may reveal whether, in these cases, singleFAEs indeed form both the ubiquitous chain lengths and the C35+ homologs [2,6,23,39,49–71].Plants 2017, 6, 27 9 of 19Table 2. Survey of plant species reported to contain cuticular wax compounds with extra-long hydrocarbon chains (C35+). Chain length ranges, most abundantchain lengths (C max.), and relative abundances of C35+ compounds within respective compound classes (+: 0–1%; ++: 1–5%; +++: 5–10%; ++++: >10%) are shown.Comprehensiveness of respective chemical analyses is indicated by information which compound classes were included in the analyses. As additional information,organs from which extra-long compounds were extracted are given, the occurrence of trichomes on respective tissues is assessed where possible, and climatic zonesare listed for each species.Plant Species Family Tissue Analyzed CompoundClasses Compound Class C max.Carbon ChainLength RangeAbundanceof C36/C35Abundance ofC38/C37ClimaticZone Trichomes ReferenceEuphorbia characiasEuphorbiaceae Leaves Complete wax profileAlkanes 31 C19–C37 + +Temperate N/A [48]Aldehydes 31 C24–C36 + N/AEuphorbia cyparissias Alkanes 31 C19–C37 ++ +Aldehydes 31 C24–C36 + N/AEuphorbia lathyris Alkanes 31 ++ +Aldehydes 31 C24–C36 + N/AEuphorbia niccaensis Alkanes 31 C19–C37 + +Euphorbia peplus Alkanes 31 C19–C37 + N/AAustrocedrus chilensis Cupressaceae Leaves Alkanes only Alkanes 33 C21–C37 ++++ + Temperate toSubtropical N/A [57]Eschscholzia californiaPapaveraceaeLeavesComplete wax profile Alkanes29 C21–C37 + N/ATemperate N/A [58]Papaver orientale Leaves 29 C21–C35 + N/APapaver somniferum Capsules 29 C21–C37 + N/AMiscanthus sinensisPoaceaeLeavesAlkanes only Alkanes31C25–C35+++ N/ATemperateYes [49][59]Senescent leaves 31 +++ N/AStems 31 ++++ N/A No [49]Inflorescence 31 ++ N/A N/APleioblastus chino Leaves 31 ++++ N/A Yes [50]Sasa nipponica Leaves 31 +++ N/A N/ASenescent leaves 31 +++ N/AZoysia japonica Leaves 31 ++ N/A N/ASenescent leaves 31 ++++ N/APlants 2017, 6, 27 10 of 19Table 2. Cont.Plant Species Family Tissue Analyzed CompoundClasses Compound Class C max.Carbon ChainLength RangeAbundanceof C36/C35Abundance ofC38/C37ClimaticZone Trichomes ReferenceAustrodanthonia pilosaPoaceaeLeaf bladesAlkanes, prim.alcoholsAlkanes31C25–C35++ N/A TemperateN/A[60]Austrodanthonia racemosa 33 + N/AAxonopus fissifolius Shoot 33 ++++ N/A SubtropicalBothriochloa macra Leaves 27 ++ N/A TemperateBromus catharticus Shoot 29 ++ N/AChloris gayana Leaves 31 ++ N/ASubtropicalN/AShoot33 ++++ N/ACynodon dactylon 33 +++ N/A Yes [51]Digitaria didactyla 33 ++++ N/A N/AElymus scaber Leaves 31 ++ N/A Temperate N/AFestuca arundinaceaShoot31 + N/AN/AImperata cylindrica 31 +++ N/A SubtropicalLotus corniculatus "Prostate"Shoot29 + N/ATemperateNo [52]Lotus pedunculatus cv. Maku 29 + N/A Yes [52]Microlaena stipoides Leaves 31 ++ N/AN/APaspalum dilatatumShoot33 ++++ N/ASubtropicalPaspalum notatum 35 ++++ N/APennisetum clandestinum 35 ++++ N/A N/APhalaris aquatica 29 +++ N/A TemperateN/ASetaria anceps 27 ++ N/A SubtropicalSporobolus indicus cv. Major 33 ++++ N/AN/AThemeda australis Leaves 31/33 +++ N/ATemperateTrifolium repens FabaceaeShoot31 + N/A Yes [53]Vulpia myuros Poaceae 31 + N/A N/ABrassica spp. BrassicalesLeaves Alkanes only Alkanes N/A C17–C35 N/A N/A TemperateYes [54][37]Pisum sativum Fabaceae N/ARosa canina Rosaceae Leaves Complete wax profile Sec. alcohols N/A C29–C35 + N/A Temperate N/A [61]Wollemia nobilis Araucariaceae Leaves Alkanes only Alkanes N/A C33–C35 N/A N/A Temperate N/A [62]Bambusa bambusaPoaceae Leaves alkanes only Alkanes31/33C23–C35+++ N/ATropical N/A [63]Bambusa dendrocalamopsis 29 + N/ABambusa dendrocalamus 29 ++Alternanthera dentataAmaranthaceaeLeaves Alkanes only Alkanes29 C22–C35 ++ N/ASubtropicalN/A[65]Alternanthera versicolor 31 C18–C35 + N/AAraucaria cunninghamii Araucariaceae 31 C22–C35 ++ N/ABothriochloa ischaemum Poaceae 31 C14–C35 ++ N/A N/ACaryota mitis Arecaceae 31 C20–C35 + N/AN/ACinnamomum burmannii Lauraceae 31 C22–C35 + N/ACodiaeum variegatum Euphorbiaceae 33 C22–C35 ++ N/AEuphorbia trigona 33 C24–C37 ++ N/AHolmskioldia sanguinea Lamiaceae 35 C22–C37 ++++ N/AHylocereus undatus Cactaceae 33 C18–C37 +++ N/AImperata cylindrica Poaceae 31 C14–C35 +++ N/AKigelia africana Bignoniaceae 31 C24–C35 +++ N/AOpuntia dillenii Cactaceae 29 C23–C36 ++++ N/A N/AOsmanthus fragrans Oleaceae 31 C24–C35 ++ N/A N/APistia stratiotes Araceae 31 C24–C37 + N/A Yes [55]Swietenia mahagoni Meliaceae 31 C24–C35 + N/A N/AZoysia japonica Poaceae 33 C14–C35 +++ N/A N/APlants 2017, 6, 27 11 of 19Table 2. Cont.Plant Species Family Tissue Analyzed CompoundClasses Compound Class C max.Carbon ChainLength RangeAbundanceof C36/C35Abundance ofC38/C37ClimaticZone Trichomes ReferenceAspidosperma spp. ApocynaceaeLeavesAlkanes, alkanolsAlkanes33 C29–C35 +++ N/A TropicalN/A [66]Cryptomeria japonica Cypressaceae fatty acids 33 C33–C35 ++++ N/A TemperateJuniperus osteosperma 33 C29–C35 +++ N/A TemperateManilkara spp. Sapotaceae 33 C31–C35 +++ N/A TropicalArabidopsis thaliana Brassicaceae Leaves 1 Complete wax profile Alkanes 29 C25–C35 + N/A Temperate Yes [21]Miscanthus sinensis Poaceae Leaves Alkanes, fatty acids Alkanes 31 C25–C37 + + Temperate Yes [52] [67]Lupinus angustifolius FabaceaeLeaves Complete wax profile AlkanesN/AC23–C37 N/A N/A TemperateN/A[68]Triticum aestivum Poaceae 31 N/AOlea europaea OleaceaeLeaves 1 Alkanes only Alkanes29 C27–C35 + N/AMediterranianN/A[69]Olive oil 25 C21–C35 + N/A N/AArabidopsis thaliana BrassicaceaeYoung/mature Complete wax profile Alkanes 31 C27–C37 + + Temperate N/A [2,7]leaves Alkenes 35 C35–C37 + +Arabidopsis thaliana BrassicaceaeLeaves 1Complete wax profileAlkanes 31 C27–C37 + +Temperate N/A [6]Alkenes 35 C35–C37 + +Leaf trichomes 1Alkanes 31/33 C27–C37 + +Alkenes 35 C33–C37 + +Ludwigia octovalvis Onagraceae Young leaves Alkanes, fatty acids Alkanes23C15–C35+ N/A Tropical Yes [56] [70]Mature leaves 23 + N/AN/A: no information available; 1 other organs were included in the analyses, but were found to lack C35+ compounds.Plants 2017, 6, 27 12 of 19However, some plant species tend to accumulate relatively high amounts of extra-longhydrocarbon chains, raising the question whether in these cases C35+ products are made by dedicatedelongase complexes. Among these species, two main distinct chain length distribution patternscan be observed: (1) In some species, the overall homolog profiles peaked at relatively long chains,such as C33 or even C35 and, thus, the further accumulation of substantial quantities of C35+ waxcompounds fell within bell-shaped distributions that are as narrow as those of other species but,overall, shifted towards longer chain lengths. Such distributions were observed, for example,in the wax mixtures of Austrocedrus chilensis [58], Pleioblastus chino [60], Paspalum notatum [61],or Cryptomerica japonica [67]. (2) In other species, substantial amounts of C35+ compounds occurredwithin chain length profiles peaking at C29, and thus revolving around rather normal chain lengthsbut with a characteristically broad spread. Such distributions were observed in the wax mixturesof Phalaris aquatica [60], Opuntia dillenii [66], or Bambusa dendrocalamus [64]. Both these chain lengthprofile types involving the accumulation of relatively high C35+ compound amounts are of note forfurther studies into wax biosynthesis. It will be interesting to characterize the KCS enzymes involved,as well as other proteins associated with the FAE complexes containing them, to understand how thechain length shifts and/or the broadening of the homolog distribution are effected. Of note, for someof the species in which C35 and C37 compounds were identified, genomic sequence data are available(Brassica spp., Pisum sativum, Triticum aestivum, Lupinus angustifolius, and Zea mays), which will allowfurther investigation of biosynthesis and function of extra-long compounds beyond Arabidopsis.Beyond the machineries generating especially long acyl precursors, it will be interesting to alsostudy the chain length specificities of the enzymes that catalyze reactions by which the acyl precursorsare modified into final wax products such as alkanes and alcohols. Interesting candidate species forthis purpose may be selected based on prior wax composition reports, and comprehensive cuticularwax analyses including all major compound classes are required for this. However, of those studiesreporting C35+ wax compounds, relatively few have provided complete wax profiles. Most interestingly,they suggest that extra-long wax constituents may be limited to certain compound classes, in mostcases to alkanes (Table 2). The chain length range of other compound classes, such as aldehydes orprimary n-alcohols, tended to be shorter (usually between C24 and C34). Only rarely were longer chainlengths also described for other compound classes, such as the C29–C35 secondary alcohols in Rosacanina leaf wax [61].Unfortunately, many other studies focused on analyzing alkanes, without reporting chain lengthprofiles of other wax compound classes accompanying them. It is, hence, impossible to assess whetherindeed the wax biosynthesis pathways leading to wax compound classes other than alkanes maydiscriminate against the exceptionally long-chain intermediates. Therefore, comprehensive analyses ofdiverse plant species detailing the quantities of C35+ constituents of all wax fractions are needed inthe future.In some plant species, the C35+ wax alkanes occurred together with alkenes that also hadexceptionally long chains. For example, Arabidopsis leaf wax contained not only C23–C37 alkanes,but also exceptionally long alkenes, which however were restricted to a relatively narrow range fromC33 to C37 (Figure 5) [2,6]. Similar alkenes were also reported for other species, including C23 to C35alkenes in Hordeum vulgare spikes [72], a broad distribution around C29 in Rosa damascena flowers [73],and chain lengths up to C35 or C37 in cucumber fruits and stems [74], barley leaves [72], tomatofruit [75], maize pollen [76], or olive oil [77].Although C35+ wax constituents have been reported for diverse plant species, the literature onthese compounds remains patchy. The C35+ compounds have been identified in several species ofPoaceae, Cactaceae, or Cupressaceae, and of diverse other families as well (Table 2). Thus, based on therelatively few comprehensive analyses that positively identified such compounds, their distributionacross diverse taxa can hardly be assessed. It appears likely that they have been over-looked in manyplant species, due to difficulties with detection by GC-MS, and that they are occurring more widelythan previously thought. It is interesting to note that, even within the limited number of species wherePlants 2017, 6, 27 13 of 19they have been detected to date, many are native to subtropical climates. Clearly, this preliminaryobservation will have to be corroborated by wax analyses of many more plant species from diversehabitats. Such broader surveys may be used to search for correlations between the relative amountsof C35+ compounds and select parameters in the growth conditions of respective species, to test forpossible adaptive advantages conferred by the C35+ wax compounds in certain climates.It is also interesting to note that extra-long wax compounds were mostly identified in leaf waxesso far. However, the large majority of wax studies to date focused on leaves, and the wax mixtureson other organs, thus, cannot be compared adequately. Therefore, it is not clear whether the C35+compounds indeed accumulate preferentially on certain organs, and particularly on leaves. On the onehand, Arabidopsis may serve as a point in case, as extra-long compounds were detected in its leaf (mainlytrichome) waxes but not in the wax mixtures covering most other organs. On the other hand, in otherspecies such as Papaver somniferum [59], Miscanthus sinensis [60], Lotus corniculatus, and Trifoliumrepens [61], extra-long wax compounds were also identified on stem, fruit, and inflorescence surfaces(Table 2).Finally, the recent findings that Arabidopsis trichome waxes contain relatively high amounts ofC33–C37 alkanes raise the question whether the C35+ compounds in other species may also residemainly, if not entirely, on trichome surfaces. To answer this question, previous reports on the occurrenceof extra-long wax constituents may be integrated with further studies mentioning the presence oftrichomes on relevant organs of respective species. Interestingly, many of the plant species knownto have C35 or C37 alkanes lack trichomes, suggesting that extra-long wax constituents may becharacteristic constituents of pavement cells as well. Unfortunately, for some species with C35+ waxcompounds, there is no information on the presence or absence of trichomes available. In the future,comprehensive micro-morphological characterizations of all plant surfaces are needed in parallel withchemical analyses of their cuticular wax mixtures. Ideally, wax mixtures on the trichomes of diversespecies should be analyzed directly using the methods recently established for direct investigationof Arabidopsis trichome surfaces, to search for differences in wax composition between pavementand trichome cells, and to test whether C35+ compounds tend to accumulate in trichome waxes ofdiverse species.5. Possible Functions of Extra-Long-Chain Compounds in Trichome WaxThe findings that trichomes (at least in Arabidopsis) have distinct wax biosynthesis machineries and,therefore, compositions raise interesting questions regarding trichome surface properties and functions.However, there is currently only very little evidence to answer such questions on the possible adaptivebenefits of trichome waxes in general, and of extra-long wax compounds in particular. Hence, it mayat this point only be speculated how the presence of C35+ constituents may affect the physical structureand, therefore, the physiological properties of respective wax mixtures.Based on the generally accepted models for the physical structures of wax mixtures, it seemsvery plausible that the presence of extra-long wax constituents will have significant effects on themelting behaviour and the crystallinity, two parameters defining wax properties. Firstly, the C35+ waxcompounds have melting points higher than those of the ubiquitous, shorter homologs, and admixturesof the extra-long chains will therefore affect the melting characteristics of the wax. The melting rangesof plant wax mixtures are known to vary drastically, depending on composition, with melting starttemperatures from 40 ◦C to 75 ◦C [40,78,79] and, thus, possibly in the range of ambient temperaturesin certain habitats. The presence of longer homologs will increase the percentage of wax moleculesremaining in the solid state at these temperatures and may, thus, serve to keep the cuticle structureintact in especially hot micro-environments.Secondly, the presence of especially long wax compounds will affect the packing of moleculeswithin the complex solid-state wax mixtures. However, it is currently not clear whether theaccumulation of C35+ compounds would enhance or impede the water barrier properties of thewax mixtures. Whether such admixtures would have positive or negative effects might depend onPlants 2017, 6, 27 14 of 19their concentration as well as the overall chain length distribution [80]. On the one hand, smallamounts of C35+ compounds may be expected to merely broaden the chain length distribution andlead to increased mismatches in the side-by-side packing of shorter and longer homologs within thewax structure. The resulting local disorder would effectively reduce the overall crystallinity of thewax mixture, facilitating access for water molecules and thus negatively affecting the water barrierproperties of the wax mixture. On the other hand, a sufficient admixture of C35+ compounds couldalso lead to (partial) phase separation, generating domains within the wax where higher homologs areconcentrated and, consequently, both the overall crystallinity and the barrier properties of the mixturemay be increased.Finally, other properties might be also, or even more, relevant for the waxes covering trichomes.Due to their extreme architecture and position, the trichomes are exposed to stresses that are eithermore severe or altogether different from those of pavement cells. Of particular importance in thiscontext, trichomes must be exposed to mechanical stress, and their surface structures must, therefore,be relatively flexible to remain functional throughout various movements and upon contact [5]. It isnot clear in how far the special chemical composition of the trichome waxes found so far may affecttheir mechanical properties, and in how far they may be suited to withstand this particular stress.6. ConclusionsIt has recently been found that the cuticular wax covering Arabidopsis trichomes differs fromthose on adjacent pavement cells, mainly by containing C35+ alkanes and alkenes derived fromrespective C36 and C38 acyl-CoA derivatives. These extra-long precursors are formed by elongationcatalyzed by FAE complexes involving KCS16, a condensing enzyme preferentially expressed intrichomes. Thus, it is now established that Arabidopsis trichomes have distinct surface compositiondue to autonomous wax biosynthesis machinery involving many of the same genes as pavement cells,but also additional elements that are trichome-specific. It seems very likely that trichomes on otherspecies as well have wax compositions and biosynthesis apparatuses distinct from the neighboringpavement cells. However, direct evidence is required for detailed comparisons between cell types andspecies, to assess possible commonalities and differences and, thus, to understand how certain waxcompounds may contribute to special wax functions on trichome surfaces.It will also be interesting to investigate in how far the waxes covering trichomes and pavementcells differ from those on the third type of epidermis cells, the guard cells. While it has not been possibleto analyze the wax compositions of pavement and guard cells directly so far, there is some indirectevidence that guard cells may have a distinct wax composition. For example, differences in UV-inducedfluorescence were observed between guard and other epidermal cells, possibly caused by wax-boundphenolic compounds or a thicker cuticular wax layer on guard cells. Accordingly, wax removal led todecreased fluorescence intensities from guard cells of Olea europaea, Vicia faba, and Triticum aestivumleaves [81]. In a separate study, the Arabidopsis HIGH CARBON DIOXIDE (HIC) gene encoding a KCSwas found expressed exclusively in guard cells [82], and hic mutants, as well as the cer1 and cer6 waxbiosynthesis mutants had significant increases in stomatal frequencies [82]. While these findings clearlyshow that cuticular wax composition influences stomata development, it remains to be determinedwhether, conversely, guard cell waxes may also be distinct from those on pavement cells. Unfortunately,the differences in stomatal frequencies in respective mutants were not sufficient to interpret them interms of possibly concurring differences in wax composition. Instead, other mutants with dramaticalterations in stomata density will have to be used for comparative analyses of respective wax mixtures,to enable inferences on guard cell surface composition. Several Arabidopsis mutants with increasedstomata density were described previously, including tmm [83–85], sdd1 [86,87], and yda [88,89].Similarly, Arabidopsis lines overexpressing EPIDERMAL PATTERNING FACTOR (EPF) genes alsohave increased density of stomata on the abaxial side of the leaves, while epf mutants are lackingstomata almost completely and have an increased number of pavement cells instead [89,90]. ChemicalPlants 2017, 6, 27 15 of 19analyses of the cuticular waxes on these Arabidopsis lines with vastly differing stomata numbers maywell hold the answer to the question on guard cell wax autonomy and function.Acknowledgments: This work has been supported by the Natural Sciences and Engineering Research Council(Canada) and the Canada Foundation for Innovation.Author Contributions: D.H. collected data for tables and figures, D.H. and R.J. wrote the paper.Conflicts of Interest: The authors declare no conflict of interest.References1. Ramsay, N.A.; Glover, B.J. MYB-bHLH-WD40 protein complex and the evolution of cellular diversity.Trends Plant Sci. 2005, 10, 63–70. [CrossRef] [PubMed]2. Busta, L.; Hegebarth, D.; Kroc, E.; Jetter, R. Changes in cuticular wax coverage and composition on developingArabidopsis leaves are influenced by wax biosynthesis gene expression levels and trichome density. Planta2016. [CrossRef] [PubMed]3. Kearns, E.V.; Assmann, S.M. The guard cell-environment connection. Plant Physiol. 1993, 102, 711–715.[CrossRef] [PubMed]4. Autran, D.; Jonak, C.; Belcram, K.; Beemster, G.T.S.; Kronenberger, J.; Grandjean, O.; Inzé, D.; Traas, J. Cellnumbers and leaf development in Arabidopsis: A functional analysis of the struwwelpeter gene. EMBO J.2002, 21, 6036–6049. [CrossRef] [PubMed]5. Wagner, G.J.; Wang, E.; Shepherd, R.W. New approaches for studying and exploiting an old protuberance,the plant trichome. Ann. Bot. 2004, 93, 3–11. [CrossRef] [PubMed]6. Hegebarth, D.; Buschhaus, C.; Wu, M.; Bird, D.; Jetter, R. The composition of surface wax on trichomes ofArabidopsis thaliana differs from wax on other epidermal cells. Plant J. 2016, 1–13. [CrossRef]7. Hegebarth, D.; Buschhaus, C.; Joubes, J.; Thoroval, D.; Bird, D.; Jetter, J. Arabidopsis ketoacyl-CoA synthase16 forms C36/C38 acyl precursors for leaf trichome and pavement surface wax. Plant Cell Environ. 2017.[CrossRef] [PubMed]8. Kolattukudy, P.E. Biopolyester Membranes of Plants: Cutin and suberin. Science 1980, 208, 990–1000.[CrossRef] [PubMed]9. Graça, J.; Schreiber, L.; Rodrigues, J.; Pereira, H. Glycerol and glyceryl esters ofω-hydroxyacids in cutins.Phytochemistry 2002, 61, 205–215. [CrossRef]10. Xiao, F.; Goodwin, S.M.; Xiao, Y.; Sun, Z.; Baker, D.; Tang, X.; Jenks, M.A.; Zhou, J.-M. Arabidopsis CYP86A2represses Pseudomonas syringae type III genes and is required for cuticle development. EMBO J. 2004, 23,2903–2913. [CrossRef] [PubMed]11. Wang, Y.; Wang, M.; Sun, Y.; Hegebarth, D.; Li, T.; Jetter, R.; Wang, Z. Molecular characterization of TaFAR1involved in primary alcohol biosynthesis of cuticular wax in hexaploid wheat. Plant Cell Physiol. 2015, 56,1944–1961. [CrossRef] [PubMed]12. Pulsifer, I.; Kluge, S.; Rowland, O. Arabidopsis LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1),LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiol. Biochem. 2012, 51, 31–39. [CrossRef][PubMed]13. Joubes, J.; Raffalele, S.; Bourdenx, B.; Garcia, C.; Laroche-Traineau, J.; Morea, P.; Domergue, F.; Lessire, R. TheVLCFA elongase gene family in Arabidopsis thaliana: Phylogenetic analysis, 3D modelling and expressionprofiling. Plant Mol. Biol. 2008, 67, 547–566. [CrossRef] [PubMed]14. Beaudoin, F.; Wu, X.; Li, F.; Haslam, R.P.; Markham, J.E.; Zheng, H.; Napier, J.; Kunst, L. Functionalcharacterization of the Arabidopsis beta-ketoacyl-coenzyme A reductase candidates of the fatty acid elongase.Plant Physiol. 2009, 150, 1174–1191. [CrossRef] [PubMed]15. Domergue, F.; Chevalier, S.; Creach, A.; Cassagne, C.; Lessire, R. Purification of the acyl-CoA elongasecomplex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the3-hydroxyacyl-CoA dehydratase. Lipids 2000, 35, 487–494. [CrossRef] [PubMed]16. Han, G.; Gable, K.; Kohlwein, S.D.; Beaudoin, F.; Napier, J.A.; Dunn, T.M. The Saccharomyces cerevisiaeYBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase. J. Biol. Chem. 2002, 277,35440–35449. [CrossRef] [PubMed]Plants 2017, 6, 27 16 of 1917. Kunst, L.; Samuels, L. Plant cuticles shine: advances in wax biosynthesis and export. Curr. Opin. Plant Biol.2009, 12, 721–727. [CrossRef] [PubMed]18. Li-Beisson, Y.; Shorrosh, B.; Beisson, F.; Andersson, M.X.; Arondel, V.; Bates, P.D.; Baud, S.; Bird, D.;Debono, A.; Durrett, T.P.; et al. Acyl-lipid metabolism. Arabidopsis Book 2013, 11, e0161. [CrossRef] [PubMed]19. Millar, A.A.; Kunst, L. Very-long-chain fatty acid biosynthesis is controlled through the expression andspecificity of the condensing enzyme. Plant J. 1997, 12, 121–131. [CrossRef] [PubMed]20. Paul, S.; Gable, K.; Beaudoin, F.; Cahoon, E.; Jaworski, J.; Napier, J.A.; Dunn, T.M. Members of the ArabidopsisFAE1-like 3-ketoacyl-CoA synthase gene family substitute for the elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006, 281, 9018–9029. [CrossRef] [PubMed]21. Zheng, H.; Rowland, O.; Kunst, L. Disruptions of the Arabidopsis Enoyl-CoA reductase gene revealan essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis.Plant Cell 2005, 17, 1467–1481. [CrossRef] [PubMed]22. Haslam, T.M.; Mañas Fernández, A.; Zhao, L.; Kunst, L. Arabidopsis ECERIFERUM2 is a component of thefatty acid elongation machinery required for fatty acid extension to exceptional lengths. Plant Physiol. 2012.[CrossRef]23. Pascal, S.; Bernard, A.; Sorel, M.; Pervent, M.; Vile, D.; Haslam, R.P.; Napier, J.A.; Lessire, R.; Domergue, F.;Joubès, J. The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very-long-chainfatty acid elongation process. Plant J. 2013, 73, 733–746. [CrossRef] [PubMed]24. Haslam, T.M.; Haslam, R.; Thoraval, D.; Pascal, S.; Delude, C.; Domergue, F.; Fernández, A.M.; Beaudoin, F.;Napier, J.A.; Kunst, L.; Joubès, J. ECERIFERUM2-LIKE proteins have unique biochemical and physiologicalfunctions in very-long-chain fatty acid elongation. Plant Physiol. 2015, 167, 682–692. [CrossRef] [PubMed]25. Haslam, T.M.; Gerelle, W.K.; Graham, S.W.; Kunst, L. The Unique Role of the ECERIFERUM2-LIKE Cladeof the BAHD Acyltransferase Superfamily in Cuticular Wax Metabolism. Plants 2017, 6, 23. [CrossRef][PubMed]26. Bernard, A.; Domergue, F.; Pascal, S.; Jetter, R.; Renne, C.; Faure, J.-D.; Haslam, R.P.; Napier, J.A.; Lessire, R.;Joubès, J. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell 2012,1–14. [CrossRef] [PubMed]27. Cheesbrough, T.M.; Kolattukudy, P.E. Alkane biosynthesis by decarbonylation of aldehydes catalyzed bya particulate preparation from Pisum sativum. Proc. Natl. Acad. Sci. USA 1984, 81, 6613–6617. [CrossRef][PubMed]28. Schneider-Belhaddad, F.; Kolattukudy, P. Solubilization, partial purification, and characterization of a fattyaldehyde decarbonylase from a higher plant, Pisum sativum. Arch. Biochem. Biophys. 2000, 377, 341–349.[CrossRef] [PubMed]29. Greer, S.; Wen, M.; Bird, D.; Wu, X.; Samuels, L.; Kunst, L.; Jetter, R. The cytochrome P450 enzyme CYP96A15is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stemcuticular wax of Arabidopsis. Plant Physiol. 2007, 145, 653–667. [CrossRef] [PubMed]30. Rowland, O.; Zheng, H.; Hepworth, S.R.; Lam, P.; Jetter, R.; Kunst, L. CER4 encodes an alcohol-forming fattyacyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol. 2006, 142,866–877. [CrossRef] [PubMed]31. Li, F.; Wu, X.; Lam, P.; Bird, D.; Zheng, H.; Samuels, L.; Jetter, R.; Kunst, L. Identification of the wax estersynthase/acyl-coenzyme A: Diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesisin Arabidopsis. Plant Physiol. 2008, 148, 97–107. [CrossRef] [PubMed]32. Riederer, M. Thermodynamics of the water permeability of plant cuticles: Characterization of the polarpathway. J. Exp. Bot. 2006, 57, 2937–2942. [CrossRef] [PubMed]33. Goodwin, S.M.; Rashotte, A.M.; Rahman, M.; Feldmann, K.A.; Jenks, M.A. Wax constituents on theinflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions.Phytochemistry 2005, 66, 771–780. [CrossRef] [PubMed]34. Kunst, L.; Jetter, R.; Samuels, A.L. Biosynthesis and transport of plant cuticular waxes. Biol. Plant Cuticle2006, 23, 182–215.35. Koornneef, M.; Hanhart, C.J.; Thiel, F. A Genetic and phenotypic description of Eceriferum (cer) mutants inArabidopsis thaliana. J. Hered. 1989, 80, 118–122. [CrossRef]Plants 2017, 6, 27 17 of 1936. Xue, Y.; Xiao, S.; Kim, J.; Lung, S.-C.; Chen, L.; Tanner, J.A.; Suh, M.C.; Chye, M.-L. Arabidopsismembrane-assoicayted acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation. J. Exp. Bot.2014, 65, 5473–5483. [CrossRef] [PubMed]37. Bird, S.M.; Gray, J.E. Signals from the cuticle affect epidermal cell differentiation. New Phytol. 2003, 157, 9–23.[CrossRef]38. Marks, M.D.; Wenger, J.P.; Gilding, E.; Jilk, R.; Dixon, R.A. Transcriptome analysis of Arabidopsis wild-typeand gl3-sst sim trichomes identifies four additional genes required for trichome development. Mol. Plant2009, 2, 803–822. [CrossRef] [PubMed]39. Jeffree, C.E. The fine structure of the plant cuticle. In Biology of the Plant Cuticle; Riederer, M., Müller, C., Eds.;Blackwell Publishing Ltd: Oxford, UK, 2006; pp. 11–125.40. Jetter, R.; Kunst, L.; Samuels, A.L. Composition of plant cuticular waxes. Annu. Plant Rev. 2007, 23, 145–181.[CrossRef]41. Schreiber, L. Review of sorption and diffusion of lipophilic molecules in cuticular waxes and the effects ofaccelerators on solute mobilities. J. Exp. Bot. 2006, 57, 2515–2523. [CrossRef] [PubMed]42. Xia, Y.; Nikolau, B.J.; Schnable, P.S. Developmental and hormonal regulation of the arabidopsis CER2gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes.Plant Physiol. 1997, 115, 925–937. [CrossRef] [PubMed]43. Bourdenx, B.; Bernard, A.; Domergue, F.; Pascal, S.; Léger, A.; Roby, D.; Pervent, M.; Vile, D.;Haslam, R.P.; Napier, J.A.; Lessire, R.; Joubès, J. Overexpression of Arabidopsis ECERIFERUM1 promotes waxvery-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Physiol.2011, 156, 29–45. [CrossRef] [PubMed]44. Yang, C.; Ma, S.; Lee, I.; Kim, J.; Liu, S. Saline-induced changes of epicuticular waxy layer on thePuccinellia tenuiflora and Oryza sativa leave surfaces. Biol. Res. 2015, 48, 1–8. [CrossRef] [PubMed]45. Fahn, A. Structural and functional-properties of trichomes of xeromorphic leaves. Ann. Bot. 1986, 57, 631–637.[CrossRef]46. Guhling, O.; Kinzler, C.; Dreyer, M.; Bringmann, G.; Jetter, R. Surface composition of myrmecophilic plants:Cuticular wax and glandular trichomes on leaves of Macaranga tanarius. J. Chem. Ecol. 2005, 31, 2323–2341.[CrossRef] [PubMed]47. Heinrich, G.; Pfeifhofer, H.W.; Stabentheiner, E.; Sawidis, T. Glandular hairs of Sigesbeckia jorullensis Kunth(Asteraceae): Morphology, histochemistry and composition of essential oil. Ann. Bot. 2002, 89, 459–469.[CrossRef] [PubMed]48. Fernández, V.; Khayet, M.; Montero-Prado, P.; Heredia-Guerrero, J.A.; Liakopoulos, G.; Karabourniotis, G.;Del Río, V.; Domínguez, E.; Tacchini, I.; Nerín, C.; et al. New insights into the properties of pubescentsurfaces: Peach fruit as a model. Plant Physiol. 2011, 156, 2098–2108. [CrossRef] [PubMed]49. Hemmers, H.; Gulz, P.G. Waxes of five Euphorbia Species. Phytochemistry 1986, 25, 2103–2107.50. Clark, L.V.; Ryan Stewart, J.; Nishiwaki, A.; Toma, Y.; Kjeldsen, J.B.; Jörgensen, U.; Zhao, H.; Peng, J.; Yoo, J.H.; Heo, K.; et al. Genetic structure of Miscanthus sinensis and Miscanthus sacchariflorus in Japan indicatesa gradient of bidirectional but asymmetric introgression. J. Exp. Bot. 2015, 66, 4213–4225. [CrossRef][PubMed]51. Motomura, H. Distribution of silicified cells in the leaf blades of Pleioblastus chino (Franchet et Savatier)Makino (Bambusoideae). Ann. Bot. 2000, 85, 751–757. [CrossRef]52. Hameed, M.; Ashraf, M.; Naz, N.; Nawaz, T.; Batool, R.; Sajid Aqeel Ahmad, M.; Ahmad, F.; Hussain, M.Anatomical adaptations of Cynodon dactylon (L.) Pers. from the salt range (Pakistan) to salinity stress. II. leafanatomy. Pakistan J. Bot. 2013, 45, 133–142.53. Gruber, M.; Skadhauge, B.; Yu, M.; Muir, A.; Richards, K. and flavonoids within a Lotus germplasm collection.Can. J. Plant Sci. 2008, 88, 121–132. [CrossRef]54. Retallack, B.; Willison, J.H.M. Morphology, anatomy, and distribution of capitate glandular trichomes onselected Trifolium species. Crop Sci. 1988, 28, 677–680. [CrossRef]55. Ågren, J.; Schemske, D.W. Artificial selection on trichome number in Brassica rapa. Theor. Appl. Genet. 1992,83, 673–678. [CrossRef] [PubMed]56. Farnese, F.S.; Oliveira, J.A.; Lima, F.S.; Leao, G.A.; Gusman, G.S.; Silva, L.C. Evaluation of the potentialof Pistia stratiotes L. (water lettuce) for bioindication and phytoremediation of aquatic environmentscontaminated with arsenic. Braz. J. Biol. 2014, 74, 108–112. [CrossRef] [PubMed]Plants 2017, 6, 27 18 of 1957. Titah, H.S.; Abdullah, S.R.S.; Mushrifah, I.; Anuar, N.; Basri, H.; Mukhlisin, M. Effect of applying rhizobacteriaand fertilizer on the growth of Ludwigia octovalvis for arsenic uptake and accumulation in phytoremediation.Ecol. Eng. 2013, 58, 303–313. [CrossRef]58. Dodd, R.S.; Rafii, Z.A.; Power, A.B. Ecotypic adaptation in Austrocedrus chilensis in cuticular hydrocarboncomposition. New Phytol. 1998, 138, 699–708. [CrossRef]59. Jetter, R.; Riederer, M. Cuticular waxes from the leaves and fruit capsules of eight Papaveraceae species.Can. J. Bot. 1996, 74, 419–430. [CrossRef]60. Zhang, Y.; Togamura, Y.; Otsuki, K. Study on the n-alkane patterns in some grasses and factors affecting then-alkane patterns. J. Agric. Sci. 2004, 142, 469–475. [CrossRef]61. Bugalho, M.N.; Dove, H.; Kelman, W.; Wood, J.T.; Mayes, R.W. Plant wax alkanes and alcohols as herbivorediet composition markers. J. Range Manag. 2004, 57, 259–268. [CrossRef]62. Buschhaus, C.; Herz, H.; Jetter, R. Chemical composition of the epicuticular and intracuticular wax layers onadaxial sides of Rosa canina leaves. Ann. Bot. 2007, 100, 1557–1564. [CrossRef] [PubMed]63. Dragota, S.; Riederer, M. Epicuticular wax crystals of Wollemia nobilis: Morphology and chemical composition.Ann. Bot. 2007, 100, 225–231. [CrossRef] [PubMed]64. Li, R.; Luo, G.; Meyers, P.A.; Gu, Y.; Wang, H.; Xie, S. Leaf wax n-alkane chemotaxonomy of bamboo froma tropical rain forest in Southwest China. Plant Syst. Evol. 2012, 298, 731–738. [CrossRef]65. Domínguez, E.; Cuartero, J.; Heredia, A. An overview on plant cuticle biomechanics. Plant Sci. 2011, 181,77–84. [CrossRef] [PubMed]66. Bi, X.; Sheng, G.; Liu, X.; Li, C.; Fu, J. Molecular and carbon and hydrogen isotopic composition of n-alkanesin plant leaf waxes. Org. Geochem. 2005, 36, 1405–1417. [CrossRef]67. Diefendorf, A.F.; Freeman, K.H.; Wing, S.L.; Graham, H. V. Production of n-alkyl lipids in living plants andimplications for the geologic past. Geochim. Cosmochim. Acta 2011, 75, 7472–7485. [CrossRef]68. Gao, L.; Huang, Y. Inverse gradients in leaf wax δD and δ13C values along grass blades of Miscanthus sinensis:Implications for leaf wax reproduction and plant physiology. Oecologia 2013, 172, 347–357. [CrossRef][PubMed]69. Nadiminti, P.P.; Rookes, J.E.; Boyd, B.J.; Cahill, D. M. Confocal laser scanning microscopy elucidation of themicromorphology of the leaf cuticle and analysis of its chemical composition. Protoplasma 2015. [CrossRef][PubMed]70. Mihailova, A.; Abbado, D.; Pedentchouk, N. Differences in n-alkane profiles between olives and olive leavesas potential indicators for the assessment of olive leaf presence in virgin olive oils. Eur. J. Lipid Sci. Technol.2015, 117, 1480–1485. [CrossRef]71. Mitra, S.; Sarkar, N.; Barik, A. Long-chain alkanes and fatty acids from Ludwigia octovalvis weedleaf surface waxes as short-range attractant and ovipositional stimulant to Altica cyanea (Weber)(Coleoptera: Chrysomelidae). Bull. Entomol. Res. 2017, 1–10. [CrossRef] [PubMed]72. Von Wettstein-Knowles, P. Analyses of barley spike mutant waxes identify alkenes, cyclopropanes andinternally branched alkanes with dominating isomers at carbon 9. Plant J. 2007, 49, 250–264. [CrossRef][PubMed]73. Wollrab, V. Olefine und Paraffine aus den Wachsen einiger Pflanzen der Familie Rosaceae. Ueber Naturwachse1986, 33, 1584–1600.74. Wang, W.; Zhang, Y.; Xu, C.; Ren, J.; Liu, X.; Black, K.; Gai, X.; Wang, Q.; Ren, H. Cucumber ECERIFERUM1(CsCER1), which influences the cuticle properties and drought tolerance of cucumber, plays a key role inVLC alkanes biosynthesis. Plant Mol. Biol. 2015, 87, 219–233. [CrossRef] [PubMed]75. Leide, J.; Hildebrandt, U.; Reussing, K.; Riederer, M.; Vogg, G. The developmental pattern of tomato fruitwax accumulation and its impact on cuticular transpiration barrier properties: Effects of a deficiency ina beta-ketoacyl-coenzyme A synthase (LeCER6). Plant Physiol. 2007, 144, 1667–1679. [CrossRef] [PubMed]76. Bianchi, G.; Murelli, C.; Ottaviano, E. Maize pollen lipids. Phytochemistry 1990, 29, 739–744. [CrossRef]77. Bortolomeazzi, R.; Berno, P.; Pizzale, L.; Conte, L.S. Sesquiterpene, alkene, and alkane hydrocarbons in virginolive oils of different varieties and geographical origins. J. Agric. Food Chem. 2001, 49, 3278–3283. [CrossRef][PubMed]78. Piper, S.H.; Chibnall, A.C.; Williams, E. Melting-points and long crystal spacings of the higher primaryalcohols and n-fatty acids. Biochem. J. 1934, 28, 2175–2188. [CrossRef] [PubMed]Plants 2017, 6, 27 19 of 1979. Merk, S. Phase behaviour and crystallinity of plant cuticular waxes studies by Fourier transform infraredspectroscopy. Planta 1998, 204, 44–53. [CrossRef]80. Jetter, R.; Riederer, M. Localization of the transpiration barrier in the epi- and intracuticular waxes of eightplant species: Water transport resistances are associated with fatty acyl rather than alicyclic components.Plant Physiol. 2015. [CrossRef] [PubMed]81. Karabourniotis, G. Epicuticular Phenolics Over Guard Cells: Exploitation for in situ stomatal counting byfluorescence microscopy and combined image analysis. Ann. Bot. 2001, 87, 631–639. [CrossRef]82. Gray, J.E.; Holroyd, G.H.; van der Lee, F.M.; Bahrami, R.; Sijmons, P.C.; Woodward, F.I.; Schuch, W.;Hetherington, A.M. The HIC signalling pathway links CO2 perception to stomatal development. Nature2000, 408, 713–716. [CrossRef] [PubMed]83. Geisler, M.; Nadeau, J.; Sack, F.D. Oriented asymmetric divisions that generate the stomatal spacing patternin arabidopsis are disrupted by the too many mouths mutation. Plant Cell 2000, 12, 2075–2086. [CrossRef][PubMed]84. Nadeau, J.; Sack, F.D. Stomatal development in Arabidopsis. Arabidopsis Book 2002, 1, e0066. [CrossRef][PubMed]85. Yang, M.; Sack, F.D. The too many mouths and four lips mutations affect stomatal production in Arabidopsis.Plant Cell 1995, 7, 2227–2239. [CrossRef] [PubMed]86. Berger, D.; Altmann, T. A subtilisin-like serine protease involved in the regulation of stomatal density anddistribution in Arabidopsis thaliana. Genes Dev. 2000, 14, 1119–1131. [CrossRef] [PubMed]87. Von Groll, U.; Berger, D.; Altmann, T. The subtilisin-like serine protease SDD1 mediates cell-to-cell signalingduring Arabidopsis stomatal development. Plant Cell 2002, 14, 1527–1539. [CrossRef] [PubMed]88. Gray, J.E.; Hetherington, A.M. Plant development: YODA the stomatal switch. Curr. Biol. 2004, 14, 488–490.[CrossRef] [PubMed]89. Hunt, L.; Gray, J.E. The signaling peptide EPF2 controls asymmetric cell divisions during stomataldevelopment. Curr. Biol. 2009, 19, 864–869. [CrossRef] [PubMed]90. Hara, K.; Kajita, R.; Torii, K.U.; Bergmann, D.C.; Kakimoto, T. The secretory peptide gene EPF1 enforces thestomatal one-cell-spacing rule. Genes Dev. 2007, 21, 1720–1725. [CrossRef] [PubMed]© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).