Drought Induction of Arabidopsis 9-cis-Epoxycarotenoid Dioxygenase Occurs in Vascular Parenchyma Cells 1[W][OA] Akira Endo 2 , Yoshiaki Sawada, Hirokazu Takahashi, Masanori Okamoto 3 , Keiichi Ikegami, Hanae Koiwai 4 , Mitsunori Seo 5 , Tomonobu Toyomasu, Wataru Mitsuhashi, Kazuo Shinozaki, Mikio Nakazono, Yuji Kamiya, Tomokazu Koshiba, and Eiji Nambara 2 * Department of Biological Sciences, Tokyo Metropolitan University, Hachiouji, Tokyo 192–0397, Japan (A.E., M.O., K.I., H.K., M.S., T.K.); Growth Regulation Research Group, RIKEN Plant Science Center, Tsurumi, Yokohama, Kanagawa 230–0045, Japan (A.E., M.O., Y.K., E.N.); Course of the Science of Bioresource, United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020–8550, Japan (Y.S., T.T., W.M.); Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997–8555, Japan (T.T., W.M.); Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (H.T., M.N.); and Gene Discovery Research Group, RIKEN Plant Science Center, Tsurumi, Yokohama, Kanagawa 230–0045, Japan (K.S.) The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought- induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses. The phytohormone abscisic acid (ABA) plays a central role in responses to abiotic and biotic stresses, such as drought, salinity, low temperature, and path- ogen 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 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 in- volved in the ABA biosynthetic pathway (Seo and Koshiba, 2002; Schwartz et al., 2003; Xiong and Zhu, 2003). Based on these studies, it has become clear that ABA is synthesized from zeaxanthin, a C 40 carotenoid. The conversion of zeaxanthin to xanthoxin, which is the C 15 intermediate, is catalyzed in plastids by pos- sibly 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 1 This work was supported by a Grant-in-Aid for Scientific Research B (grant no. 16370026) to T.K. 2 Present address: Department of Cell and Systems Biology, University of Toronto, 25 Willcocks St., Toronto, Ontario, Canada M5S 3B2. 3 Present address: Plant Functional Genomics Research Group, RIKEN Plant Science Center, Suehiro-cho 1–7–22, Tsurumi, Yokohama, Kanagawa 230–0045, Japan. 4 Present address: National Institute of Landstock and Grassland Science, Senbonmatsu, Nasushiobara, Tochigi 329–2793, Japan. 5 Present address: Dormancy and Adaptation Research Unit, RIKEN Plant Science Center, 1–7–22, Suehiro-cho, Tsurumi, Yokohama 230–0045, Japan. * Corresponding author; e-mail eiji.nambara@utoronto.ca. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eiji Nambara (eiji.nambara@utoronto.ca). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscrip- tion. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116632 1984 Plant Physiology, August 2008, Vol. 147, pp. 1984–1993, www.plantphysiol.org Ó 2008 American Society of Plant Biologists Downloaded from https://academic.oup.com/plphys/article/147/4/1984/6107658 by guest on 01 August 2023