plants Review Cuticular Waxes of Arabidopsis thaliana Shoots: Cell-Type-Specific Composition and Biosynthesis Daniela Hegebarth 1 and Reinhard Jetter 1,2,* 1 Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada; [email protected] 2 Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada * Correspondence: [email protected]; Tel.: +1-604-822-2477 Received: 1 June 2017; Accepted: 2 July 2017; Published: 7 July 2017 Abstract: It is generally assumed that all plant epidermis cells are covered with cuticles, and the distinct surface geometries of pavement cells, guard cells, and trichomes imply functional differences and possibly different wax compositions. However, experiments probing cell-type-specific wax compositions and biosynthesis have been lacking until recently. This review summarizes new evidence showing that Arabidopsis trichomes have fewer wax compound classes than pavement cells, and higher amounts of especially long-chain hydrocarbons. The biosynthesis machinery generating this characteristic surface coating is discussed. Interestingly, wax compounds with similar, long hydrocarbon chains had been identified previously in some unrelated species, not all of them bearing trichomes. Keywords: trichomes; cuticular wax; chain length; Arabidopsis thaliana; KCS; elongation; fatty acid elongase complex; ketoacyl-CoA synthase 1. Introduction All above-ground plant parts, in the primary state of development, are lined by a layer of epidermis cells that serve crucial functions for protecting the various organs and, thus, for plant survival. The epidermis consists of three different cell types, the pavement cells, guard cells, and trichomes, in characteristic numbers, shapes and geometric arrangements depending on the species, organ, and developmental state. The different epidermis cell types serve very different functions (Figure1; Table1): pavement cells form the major protective surface barrier [1], and mature Arabidopsis leaves contain about 29,000 cells each with a surface area of ca. 4000 µm2 [2] (Table1). Guard cells, on the other hand, are important for regulating gas exchange and for protecting the surface around stomata [3]. They are less abundant and smaller than pavement cells (Figure1b), with ca. 10,000 guard cells on average per Arabidopsis leaf and average sizes of about 280 µm2 [2,4] (Figure1b, Table1). Finally, trichomes emerge vertically out of the surface (Figure1a), serving a variety of roles including UV protection, heat insulation, transpiration control, and insect deterrence [5]. Arabidopsis trichomes consist of a stalk with two or three perpendicular arms (Figure1a,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] (Table1). Plants 2017, 6, 27; doi:10.3390/plants6030027 www.mdpi.com/journal/plants Plants 2017, 6, 27 2 of 19 Plants 2017, 6, 27 2 of 19 Trichome Guard Cells Pavement Cell Trichome 1 mm 300 μm (a) (b) Figure 1. Cryo-SEMCryo-SEM images of abaxial abaxial Arabidopsis leafleaf surfaces. surfaces. ( (a)) Comparison Comparison of of abundance abundance and size of pavement and guard cells covering the leaf surface, relative to the trichome cells protruding out of the surface. surface; ( b) Detailed Detailed view of a a single single trichome, trichome, showing showing its shape shape and and cell cell size size relative relative to to pavement pavement and guard cells (reprinted fromfrom [[6,7]6,7] with permission). Table 1.1.Trichome, Trichome, guard guard cell, cell, and and pavement pavement cell surface cell surface areas and areas cell and numbers cell onnumbers adaxial onArabidopsis adaxial Arabidopsisleaves (adapted leaves from (adapted [2]). from [2]). Projected Surface Number of Surface Area of Number of Surface Area of AreaProjected of Blade Surface Trichomes Guard Cell Pavement Pavement Cells Trichome Cells 2 Area(mm of Blade2) (mm ) (BladeTrichomes−1) PairsGuard (Blade Cell Pairs−1) CellsPavement (Blade−1 Cells) (μPavementm2 cell−1) Cells Trichome(μm2 cell Cells−1) (Blade−1) (Blade−1) (Blade−1) (µm2 cell−1) (µm2 cell−1) 138 72 10366 29602 4646 40000 138 72 10366 29602 4646 40000 It is generally assumed that all three epidermal cell types are covered with an uninterrupted cuticle,It isa generallyhydrophobic assumed surface that consisting all three of epidermal a cutin matrix cell types[8] and are solvent-soluble covered with anwaxes uninterrupted embedded in,cuticle, and a hydrophobicdeposited onto, surface it. consistingCutin is ofa apo cutinlyester matrix of [saturated8] and solvent-soluble and unsaturated waxes embeddedC16 and C in,18 ωand-hydroxyacids, deposited onto, polyhydroxyacids, it. Cutin is a polyester or epoxyacids of saturated and and unsaturatedglycerol [9,10]. C16 andCuticular C18 ! -hydroxyacids,wax usually polyhydroxyacids,comprises a variety orof aliphatic epoxyacids compound and glycerol classes [9 such,10]. as Cuticular fatty acids, wax primary usually n-alcohols, comprises secondary a variety alcohols,of aliphatic alkyl compound esters, aldehydes, classes such and alkanes, as fatty but acids, also primary polyketidesn-alcohols, and terpenoids secondary (Figure alcohols, 2). Within alkyl theesters, compound aldehydes, classes, and alkanes,usually compounds but also polyketides with varying and terpenoids carbon numbers (Figure in2). the Within hydrocarbon the compound chains areclasses, found, usually thus defining compounds series with of aliphatic varying homolo carbongs. numbers Both the inabundances the hydrocarbon of individual chains constituents are found, withinthus defining the wax series mixture of aliphaticand the relative homologs. amounts Both of the wax abundances and cutin vary of individual greatly between constituents plant species, within organs,the wax and mixture developmental and the relative stages. For amounts instance, of waxin leaf and blades cutin of varyTriticum greatly aestivum between seedlings, plant primary species, n-organs,alcohols and are developmental the predominant stages. compound For instance, class, in leaf whereas blades ofonTriticum flag leaf aestivum sheathsseedlings, β-diketones primary are predominantn-alcohols are [11]. the Arabidopsis predominant leaf compoundwax contains class, alkanes whereas with a on broad flag chain leaf sheaths length rangingβ-diketones from areC25 predominantto C34, while Arabidopsis [11]. Arabidopsis stem waxleaf consists wax contains mainly of alkanes C29 alkane. with a broad chain length ranging from C25 to C34, while Arabidopsis stem wax consists mainly of C29 alkane. 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] (Figure3). Figure 2. Chemical structures of major cuticular wax compound classes. Plants 2017, 6, 27 2 of 19 Trichome Guard Cells Pavement Cell Trichome 1 mm 300 μm (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 Number of Surface Area of Area of Blade Trichomes Guard Cell Pavement Pavement Cells Trichome Cells (mm2) (Blade−1) Pairs (Blade−1) Cells (Blade−1) (μm2 cell−1) (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 Plants 2017predominant, 6, 27 [11]. Arabidopsis leaf wax contains alkanes with a broad chain length ranging from C25 3 of 19 to C34, while Arabidopsis stem wax consists mainly of C29 alkane. Plants 2017, 6, 27 3 of 19 The mechanisms underlying wax biosynthesis have been largely elucidated using model organisms