As in most terrestrial plants, the cuticle on Arabidopsis thaliana (L.) Heynh. forms a continuous lipid membrane over the apical epidermal cell walls of essentially all aerial plant organs. Epicuticular waxes form the outermost layer over this membrane and are visible on Arabidopsis inflorescence stem and silique surfaces as a bluish-white colored coating called glaucousness or waxy bloom. Intracuticular waxes are intermeshed within the cuticle membrane and not visible to the naked eye. Close examination of epicuticular waxes on Arabidopsis stems and siliques using scanning electron microscopy (SEM) at around 3000× magnification best reveals their diverse crystalline structures (Figure 1A). The stem and silique epicuticular wax morphology is composed primarily of columnar-shaped crystals (of ∼1.0 µm diameter), although rods, tubes, vertical plates, dendritic-, and umbrella-like structures are also typically present. The non-glaucous rosette and cauline leaf surfaces of Arabidopsis lack wax crystals detectable at the level of SEM (Figure 1B). Interestingly, other organs possess epicuticular wax crystals, including those of the pistil (Bowman, 1993). Descriptions of wax crystalline morphology on other flower parts, seeds, seedlings, roots, and other organs of Arabidopsis have not been reported. Figure 1. Surface morphology of air-dried stem and leaf surfaces of wild-type Arabidopsis thaliana ecotype Wassilewskija produced using scanning electron microscopy. A. Flowering stem. B. Abaxial blade surface between midrib and margin. Bar equals 1 µm. ... Whereas the term epicuticular wax is used to describe wax crystals above of the cuticle, cuticular wax is being used here to describe those long chain lipids extracted by submersion of tissues in solvents like hexane and chloroform since this extraction procedure likely removes both epicuticular and intracuticular waxes. The chemical composition of cuticular waxes on Arabidopsis leaves and stems are typical of those on many dicotyledonous plants, being composed primarily of saturated free fatty acids, aldehydes, alkanes, primary alcohols, secondary alcohols, ketones, and wax esters (Figure 2). Within these component classes, homologues occur as aliphatic chains of between 16 and 33 carbons, except the wax esters, which are composed of even more carbons. The dominant wax class on Arabidopsis leaves and stems is the alkanes, although primary alcohols comprise a significant wax fraction on these surfaces. Stems and siliques possess relatively high amounts of ketones and secondary alcohols, whereas rosette and cauline leaves possess these constituents in trace or undetectable amounts. Arabidopsis lacks the wax hydroxy-s-diketones, s-diketones, and alkan-2-ol esters found on certain monocots (Bianchi and Bianchi, 1990), the estolides only reported in gymnosperms, and other minor constituents that occur idiosyncratically in plants that have been examined (Walton 1990). Inflorescence stems of wild-type Arabidopsis can produce over ten fold more total wax per area than leaves, and stem wax chain length distribution is shorter than leaves, with the C29 alkane homologue dominating stem waxes but the C31 alkane dominating leaf waxes (Jenks et al., 1995). Rosette leaves possess lower relative amounts of primary alcohols than cauline leaves, whereas rosette and cauline leaves, and siliques, have lower relative amounts of the C30 aldehydes than the stems (Todd et al., 1999). Interestingly, pollen grains are also coated with waxes, these being dominated as for stems by the C29 alkanes, secondary alcohols, and ketones, and the C30 aldehydes (Preuss et al., 1993; Fiebig et al., 2000). Wax composition on other Arabidopsis organs have not been reported. Figure 2. Cuticular wax composition of stems and leaves of Arabidopsis thaliana ecotype C24. Values represent cuticular wax load in µg/dm2 of stem and leaf blade area ± s.d. Chemical classes and chain lengths are labeled on the horizontal axis. ... Little is known about metabolic and regulatory processes associated with synthesis of cuticular waxes by Arabidopsis. Similarities between wax constituents on Arabidopsis and many other plant species however suggest that the basic mechanisms for wax production are highly conserved within the plant kingdom. The Arabidopsis close-relative Brassica oleraceae L. has been used to describe many of these reactions (see Kolattukudy, 1996). According to the developing model (Figure 3), early wax precursors are short acyl chains activated by a soluble plastidic Acyl Carrier Protein (ACP). Acyl chains are elongated by a single plastidic Fatty Acid Synthetase (FAS) complex which condenses acetal groups from malonyl-ACP onto a growing chain (Ohlrogge and Jaworski, 1997). Once C16 and C18 acyl-ACPs are synthesized in the plastids, acyl-ACP thioesterases cleave the ACP and release free C16 and C18 acids (palmitic and stearic acids) into the cytoplasm, where they are activated by Acyl-CoenzymeA (CoA) Synthetase via condensation with CoA. Of those C16 and C18 acyl-CoAs destined for conversion to waxes, most enter a membrane-associated pathway where they are modified by what may be a series of cytoplasmic enzyme complexes called elongases. Unlike short chain synthetases of the plastid, elongases use malonyl-CoA as the two-carbon donor, instead of malonyl-ACP (Agrawal et al., 1984; Bessoule et al., 1989). Studies using chemical inhibitors, used to target single elongation steps, suggested that, unlike short acyl-chain elongation, very long wax acyl-CoAs may be elongated by numerous chain-length-specific acyl-CoA elongase complexes (Mikkelsen, 1978; Agrawal et al., 1984; Wettstein-Knowles, 1985; 1995). Mutations in Arabidopsis and other plant species provide additional evidence for multiple elongases, as individual gene disruptions have been shown to affect single elongation steps primarily, either C24, C26, C28, or C30 acyl-chain elongation (Bianchi et al., 1979; Macey and Barber, 1970a; 1970b; Avato et al., 1982; Wettstein-Knowles, 1982; Jenks et al., 1995; 2000; Rashotte et al., 2001). Whether multiple elongase complexes are present however has not been confirmed. Once the very long acyl-CoA chains are synthesized, they are converted to cuticular waxes after either 1) deactivation by acyl-CoA thioesterases to release free acids (Liu and Post-Beittenmiller, 1995), 2) conversion to aliphatic esters by condensation of the acyl moiety with a primary alcohol by a putative acyl-CoA:fatty alcohol transacylase (Kolattukudy, 1967), or 3) entry into one of two reductive pathways that either convert acyl-CoAs to primary alcohols, or convert acyl-CoAs to aldehydes (Vioque and Kolattukudy, 1997). Clearly, the activity and regulation of very-long-chain acyl-CoA elongation reactions defines a central control point in plant wax biosynthesis since all cuticular waxes are derived from these reactions. Aldehydes generated by acyl-CoA reduction are likely converted, in large part, to alkanes by a putative decarbonylase (Cheesbrough and Kolattukudy, 1984). Much of the alkanes are then converted to secondary alcohols by a putative alkane hydroxylase, and then to ketones by a putative secondary alcohol oxidase (Kolattukudy et al., 1973). Much work is still needed to fully elucidate how the very-long-chain fatty acyl-CoAs are elongated in Arabidopsis, and how their acyl moieties are then shunted through the various networks of the wax biosynthetic pathway. Figure 3. Conversion of major constituents in the stem cuticular wax biosynthetic pathway. [1] 3-ketoacyl-ACP synthetase II (KASII), [2] palmitoyl-ACP thioesterase, [3] stearoyl-ACP thioesterase, [4] acyl-CoA synthetase, [5–12] each a unique chain-length-specific ... It is presently unclear how cuticular waxes of Arabidopsis get to the surface from locations of initial synthesis in epidermal plastids. Likely, Arabidopsis waxes are secreted according to conserved mechanisms as described in comparative studies using plant, yeast, and mammalian cells (Moore et al., 1991; Barinaga, 1993). Wax precursors formed in epidermal plastids appear to be transported into the cytoplasm where various microsomal elongases have been localized (Agrawal et al., 1984; Bessoule et al., 1989). Once the precursors are transferred out of the cytoplasm through the apical plasmalemma via exocytosis (Jenks et al., 1994b), they must then traverse the cell wall and cuticle layers where decarbonylase activity has been localized (Cheesbrough and Kolattukudy, 1984). The pathway most likely involves endoplasmic reticulum, transport vesicles, substrate ligands, vesicle receptors, and many other secretory factors (Barinaga, 1993; Jenks et al., 1994b). For example, Jenks et al., (1994b) showed a dramatic increase in endoplasmic reticulum and vesicle density below wax secretory sites in the Sorghum bicolor (L.) Moench leaf epidermis during wax induction by light. Other plant studies demonstrated that lipid transfer proteins (LTPs) were the most abundant proteins within Brassica cuticular waxes, providing evidence that LTPs may be involved in wax transport to the surface (Pyee et al. 1994). Arabidopsis mutants altered in both cell wall structure and cuticular waxes were recently reported, and a premise was set forth that mutants bearing these phenotypes may possess mutations that alter common processes in secretion of epidermal cell wall and cuticular lipid constituents (Jenks et al. 1996a). Notwithstanding, Arabidopsis cuticular wax secretory mutants, if discovered, would provide a model system for studying gene involvement in basic secretory processes used by plants.