Deuterium-labeled d6-ABA (Icon Services) was added to each sample prior to extraction

Deuterium-labeled d6-ABA (Icon Services) was added to each sample prior to extraction. salinity, low temperature, and pathogen attack (Zeevaart and Creelman, 1988;Zhu, 2002;de Torres-Zabala et al., 2007). Plants accumulate ABA when they are subjected to drought stress, and these changes in cellular ABA levels trigger the activation Lifitegrast of numerous stress-responsive genes and the closure of stomata to restrict transpiration (Schroeder et al., 2001;Shinozaki and Yamaguchi-Shinozaki, 2007). The details of de novo ABA biosynthesis in higher plants have been worked out in the last decade (Nambara and Marion-Poll, 2005). Molecular genetic studies of ABA-deficient mutants from various plant species contributed to the identification of genes involved in the ABA biosynthetic pathway (Seo and Koshiba, 2002;Schwartz et al., 2003;Xiong and Zhu, 2003). Based on these studies, it has become Lifitegrast clear that ABA is synthesized from zeaxanthin, a C40carotenoid. The conversion of zeaxanthin to xanthoxin, which is the C15intermediate, is catalyzed in plastids by possibly four distinct enzymes: zeaxanthin epoxidase (Marin et al., 1996;Agrawal et al., 2001;Xiong et al., 2002), neoxanthin synthase (North et al., 2007), an unidentified epoxycarotenoid isomerase, and 9-cis-epoxycarotenoid dioxygenase (NCED;Schwartz et al., 1997;Tan et al., 1997;Qin and Zeevaart, 1999;Iuchi et al., 2000,2001). Xanthoxin is then converted to ABA via abscisic aldehyde in the cytosol (Sindhu and Walton, 1987). The oxidation of xanthoxin to produce abscisic aldehyde is catalyzed by AtABA2, a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana;Cheng et al., 2002;Gonzalez-Guzman et al., 2002). In turn, the conversion of abscisic aldehyde to ABA is catalyzed by Arabidopsis aldehyde oxidase 3 (AAO3;Seo et al., 2000b). A variety of studies have indicated that the carotenoid cleavage reaction catalyzed by NCEDs is a key regulatory step in ABA biosynthesis (Qin and Zeevaart, 1999;Thompson et al., 2000;Iuchi et al., 2001). In several plant species, it has been shown that transgenic plants constitutively expressing theNCEDgene accumulated higher amounts of ABA in their leaves and seeds compared with the wild type (Thompson et al., 2000;Iuchi et al., 2001;Qin and Zeevaart, 2002). Among the nine Arabidopsis genes encoding carotenoid cleavage dioxygenase, five (AtNCED2, -3, -5, -6, and -9) are implicated in ABA biosynthesis (Iuchi et al., 2001;Toh et al., 2008). Several features make theAtNCED3gene particularly interesting with respect to its role in stress responses. First, the transcript levels ofAtNCED3have been shown to increase rapidly in response to dehydration, while those of otherAtNCEDgenes showed almost no response to drought stress (Iuchi et al., 2001;Tan et al., 2003). Furthermore, plants with a knocked-out (or knocked-down)AtNCED3have been shown to exhibit enhanced transpiration in turgid conditions and higher sensitivity to dehydration. In contrast, transgenic plants overexpressingAtNCED3have enhanced stress tolerance (Iuchi et al., 2001). However, despite its apparent importance in stress physiology, the regulatory mechanisms ofAtNCED3gene expression in response to drought remain elusive. ABA and its catabolites are mobile, possibly through the phloem and xylem flow (Zeevaart and Boyer, 1984;Wilkinson and Davies, 1997;Sauter et CLC al., 2002). Grafting experiments have indicated that the shoot genotype is more important than that of the root to supply the active ABA pools in whole plants (Fambrini et al., 1995;Holbrook et al., 2002). In this respect,Christmann et al. (2005)utilized transgenic plants expressing Lifitegrast an ABA-inducible reporter gene construct to monitor the active ABA pools in whole plants. The induction of the reporter gene was observed primarily in vascular tissues and guard cells in shoots when the root was.