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.

SUMMARYPlant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C 24 ) were elevated more than 155%. The C 16 cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C 18 monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C 20 -C 30 , with highest activity for C 30 acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C 16 but not C 18 cutin monomers are reduced in lacs1, and C 16 acids are the next most preferred acid (behind C 30 ) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C 16 monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C 16 ) fatty acids for cutin synthesis.

Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C 24 ) and hexacosanoic acid (C 26 ). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.Climatological drought is a historic problem for agriculture worldwide, as it limits crop production, and is now increasing as a threat due to climate change as well as dwindling ground and surface water resources. Genetic alterations that reduce overall transpirational water loss by crops are expected to conserve soil moisture and confer drought tolerance by delaying the onset of cellular dehydration stress during prolonged water deprivation (Nobel, 1999;Chaves et al., 2003;Kosma and Jenks, 2007). Stomata play a major role in regulating transpirational water loss through guard cell behavior (regulating stomatal aperture) and/or stomatal density over the leaf surface (Schroeder et al., 2001;Chaerle et al., 2005;Nilson and Assmann, 2007;Sirichandra et al., 2009;Kim et al., 2010). Transpiration is also controlled by the lipidic and hydrophobic plant cuticle, which coats the aerial surfaces of plants. The cuticle controls plant water loss associated with nonstomatal epidermal transpiration as well as transpiration through the stomatal pore via its role in forming the stomatal ledges (lips) and the cuticular coating that covers the mesophyll surfaces of the substomatal chamber (Xiao et al., 2004;Kerstiens, 2006;Kosma et al., 2009;L...