Arabidopsis ECERIFERUM9 Involvement in Cuticle Formation and Maintenance of Plant Water Status 1[W][OA] Shiyou Lü 2 *, Huayan Zhao 2 , David L. Des Marais, Eugene P. Parsons, Xiaoxue Wen, Xiaojing Xu, Dhinoth K. Bangarusamy, Guangchao Wang, Owen Rowland, Thomas Juenger, Ray A. Bressan, and Matthew A. Jenks Division of Chemical and Life Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955–6900, Kingdom of Saudi Arabia (S.L., H.Z., D.K.B., G.W., R.A.B.); Section of Integrative Biology, University of Texas, Austin, Texas 78712 (D.L.D.M., T.J.); Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907–2054 (E.P.P., X.X., R.A.B.); Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada (X.W., O.R.); and United States Arid Land Agricultural Research Center, United States Department of Agriculture-Agricultural Research Service, Maricopa, Arizona 85138 (M.A.J.) 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; Lü et al., 2009). The cuticle is composed primarily of two lipid classes, the non- polymerized cuticular waxes and the cutin polyester. Most waxes are very-long-chain (C 20 –C 34 ) saturated lipids that occur as epicuticular and intracuticular lipids, whereas the more hydrophilic cutin polyester consists of C 16 and C 18 fatty acid derivatives (e.g. hydroxy fatty acids and dicarboxylic acids) linked primarily by ester bonds. 1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery grant to O.R.), the National Science Foundation (grant no. DEB–0618347 to T.J.), and the U.S. De- partment of Agriculture (National Institute of Food and Agriculture Biomass Research and Development Initiative grant to M.A.J.). 2 These authors contributed equally to the article. * Corresponding author; e-mail shiyou.lu@kaust.edu.sa. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Shiyou Lü (shiyou.lu@kaust.edu.sa). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.112.198697 930 Plant Physiology Ò , July 2012, Vol. 159, pp. 930–944, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. All Rights Reserved.