Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis 1[W][OA] Maozhi Ren, Shuqing Qiu, Prakash Venglat, Daoquan Xiang, Li Feng, Gopalan Selvaraj, and Raju Datla* Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N 0W9 Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5# external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis. Target of rapamycin (TOR) encodes a large (280 kD) Ser/Thr protein kinase and plays a central role in regulation of cell growth and metabolism (Cafferkey et al., 1993; Kunz et al., 1993). This protein is structur- ally and functionally conserved in eukaryotic species ranging from yeast (Saccharomyces cerevisiae) to human (De Virgilio and Loewith, 2006a). In yeast, TOR is encoded by two genes (TOR1 and TOR2) that have 80% overall amino acid similarity and are also par- tially redundant in function (Cafferkey et al., 1993; Kunz et al., 1993). Yeast TOR proteins interact with several other regulatory proteins to form two distinct complexes, TOR complex1 (TORC1) and TORC2 (Kim et al., 2002; Loewith et al., 2002). TORC1 consists of TOR1, LST8, KOG1, and Tco89, and regulates cell growth and metabolism in response to nutrients and energy requirements (Loewith et al., 2002; Reinke et al., 2004; Inoki et al., 2005; Martin and Hall, 2005). Rapamycin, produced by Streptomyces hygroscopicus (Heitman et al., 1991), is an inhibitor of TORC1 (Stan et al., 1994; Zheng et al., 1995; Kim et al., 2002; Loewith et al., 2002). The yeast TORC2 complex consists of TOR2, LST8, AVO1-3, Bit6, Slm1p, and Slm2p (Loewith et al., 2002; Fadri et al., 2005), and has been implicated in the regulation of cytoskeleton structure and activity, as well as spatial features of cell growth such as cell polarity. TORC2, however, is not inhibited by rapa- mycin (Loewith et al., 2002; De Virgilio and Loewith, 2006b). In animals, including humans, there is only a single copy of TOR. However, as in yeast, the TOR protein occurs in two protein complexes (Kim et al., 2002; Martin and Hall, 2005). The core structure of TOR complexes consists of a catalytic subunit (TOR) and a regulatory subunit (LST8). This core dimer recruits Raptor (yeast KOG1) and PRAS40 to form TORC1, or Rictor (yeast AVO3) and SIN1 to form TORC2 (Kim et al., 2002; Wullschleger et al., 2006). As in yeast, the TORC1 complex in animals modulates a variety of cellular responses, such as translation initiation, ribo- some biogenesis, and cell growth, and it is rapamycin sensitive (Bjornsti and Houghton, 2004; Wullschleger et al., 2006). S6K1 (p70 ribosomal protein S6 kinase1) and 4E-BP (translation initiation factor 4E binding protein) are substrates of TORC1 and phosphorylation of these proteins stimulates translation (Proud, 2004). 1 This work was supported by the National Research Council of Canada Genomics and Health Initiative, Genome Canada, and Genome Prairie. * Corresponding author; e-mail raju.datla@nrc-cnrc.gc.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: Raju Datla (raju.datla@nrc-cnrc.gc.ca). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.169045 Plant Physiology Ò , March 2011, Vol. 155, pp. 1367–1382, www.plantphysiol.org Ó 2011 NRC Canada 1367