The Impact of Water Deficiency on Leaf Cuticle Lipids of Arabidopsis

Arabidopsis (Arabidopsis thaliana) plants subjected to water deficit, sodium chloride (NaCl), or abscisic acid treatments were shown to exhibit a significant increase in the amount of leaf cuticular lipids. These stress treatments led to increases in cuticular wax amount per unit area of 32% to 80%, due primarily to 29% to 98% increases in wax alkanes. Of these treatments, only water deficit increased the total cutin monomer amount (by 65%), whereas both water deficit and NaCl altered the proportional amounts of cutin monomers. Abscisic acid had little effect on cutin composition. Water deficit, but not NaCl, increased leaf cuticle thickness (by 49%). Electron micrographs revealed that both water-deprived and NaCl-treated plants had elevated osmium accumulation in their cuticles. The abundance of cuticle-associated gene transcripts in leaves was altered by all treatments, including those performed in both pot-grown and in vitro conditions. Notably, the abundance of the ECERIFERUM1 gene transcript, predicted to function in alkane synthesis, was highly induced by all treatments, results consistent with the elevated alkane amounts observed in all treatments. Further, this induction of cuticle lipids was associated with reduced cuticle permeability and may be important for plant acclimation to subsequent water-limited conditions. Taken together, these results show that Arabidopsis provides an excellent model system to study the role of the cuticle in plant response to drought and related stresses, and its associated genetic and cellular regulation.The plant cuticle is a lipidic layer of cutin intermeshed and coated with waxes that covers essentially all aerial organs and functions to restrict transpiration. By this mechanism, the cuticle is thought to play a critical role in plant drought tolerance through its ability to postpone the onset of cellular dehydration stress during drought (Jenks, 2002; Goodwin and Jenks, 2005; Kosma and Jenks, 2007; Samuels et al., 2008). During severe water deficit, when stomata close, it has been proposed that nanoscale diffusion pathways traversing the cuticle become the primary conduit of plant water loss. These pathways are defined by long-chain aliphatic waxes assembled as water-inaccessible crystalline wax domains within the cutin matrix of the cuticle membrane, and these crystalline domains are thought to be a major determinant of cuticle permeability (Riederer and Schreiber, 1995; Kerstiens, 1996; Burghardt and Riederer, 2006; Schreiber, 2006; Kosma and Jenks, 2007). Typical plant waxes are comprised of a homologous series of primary alcohols, aldehydes, alkanes, fatty acids, esters, and sometimes cyclic compounds like triterpenoids and sterols (Jetter et al., 2006). Cuticular wax alkanes, alcohols, and aldehydes have been shown to confer greater resistance to water diffusion, in an artificial membrane, than either very-long-chain fatty acids or the triterpenoids oleanolic and ursolic acid (Grncarevic and Radler, 1967). Arabidopsis (Arabidopsis thaliana) cutin is comprised of C₁₆ and C₁₈ fatty acids and their oxygenated derivatives. Oxygenations occur as midchain (C₈, C₉, or C₁₀) hydroxyl or ω-hydroxyl groups and can be linked through ester bonds. Cutin monomers may be linked directly to each other or through esterification to glycerol (Kolattukudy, 2001a, 2001b; Graça et al., 2002; Pollard et al., 2008). To date however, the contribution of specific cutin monomers to cuticle permeability is completely unexplored. Nonetheless, mutants defective in one or more components of the cutin polyester such as att1, bdg, hth, lacs2, and wax2, have significant alteration of their cuticle permeability (Lolle et al., 1998; Schnurr et al., 2004; Goodwin and Jenks, 2005; Kurdyukov et al., 2006; Bessire et al., 2007), indicating that properties of the cutin polyester or specific monomers likewise play an important role in establishing the water-barrier properties of the cuticle membrane. Although many hypotheses have been set forth to explain the function of cuticle lipid composition and structure in plant drought tolerance, rigorous experimentation that confirm these hypotheses have yet to be reported.Recent studies establish that many plants respond to water deficit stress through increased cuticular wax deposition (Shepherd and Wynne Griffiths, 2006; Kosma and Jenks, 2007). For example, plants like tree tobacco (Nicotiana glauca), sesame (Sesamum indicum), soybean (Glycine max), and rose (Rosa × hybrida) possessed more leaf wax per unit area after short periods of water deficit (Jenks et al., 2001; Cameron et al., 2006; Kim et al., 2007a, 2007b). In some studies, stress-induced increases in wax amount were associated with major reductions in leaf water-loss rates (Williams et al., 1999, 2000; Cameron et al., 2006). It is still unclear however, which stress-induced changes in the cuticle are most critical for reducing foliar water loss and drought acclimation. Water deficiency also stimulates production of the phytohormone abscisic acid (ABA), which in turn leads to rapid responses like stomatal closure (Bartels and Sunkar, 2005) and the induction of abiotic stress-responsive genes (Shinozaki and Yamaguchi-Shinozaki, 2007). To date it is unclear whether ABA is involved in abiotic stress-induced cuticle lipid accumulation. Data mining of gene expression databases has recently implicated several cuticle-associated genes as ABA responsive (Kosma and Jenks, 2007), however, only a small number of these have been experimentally verified (Hooker et al., 2002; Duan and Schuler, 2005). Though Arabidopsis typically produces very low amounts of leaf wax and cutin, and is a short-lived ephemeral of assumed limited stress adaptation, we report here that cuticle production and associated gene expression in Arabidopsis is nevertheless highly responsive to water deficit and related treatments including ABA. This report demonstrates the potential of Arabidopsis as an effective model system for use in elucidating determinants of cuticle function in plant drought tolerance.