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DEVELOPMENT AND HORMONE ACTION

Preferential Up-Regulation of G2/M Phase-Specific Genes by Overexpression of the Hyperactive Form of NtmybA2 Lacking Its Negative Regulation Domain in Tobacco BY-2 Cells^1,[W]

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464–8601, Japan (K.K., S.S., M.I.); RIKEN Plant Science Center, Yokohama, Kanagawa 230–0045, Japan (I.G., T.D., H.F., K.M.); Central Research Institute, Ishihara Sangyo Kaisha, Nishi-shibukawa, Kusatsu, Shiga 525–0025, Japan (S.A.); and Institut de Biologie Moléculaire des Plantes, Laboratoire Propre du Centre National de la Recherche Scientifique, Unité Propre de Recherche 2357, Conventionné avec l'Université Louis Pasteur, 67084 Strasbourg, France (M.-C.C., T.P., P.G.)

Many G2/M phase-specific genes in plants contain mitosis-specific activator (MSA) elements, which act as G2/M phase-specific enhancers and bind with R1R2R3-Myb transcription factors. Here, we examined the genome-wide effects of NtmybA2 overexpression, one of the R1R2R3-Myb transcription factors in tobacco (Nicotiana tabacum). We used a custom-made 16-K cDNA microarray for comparative transcriptome analysis of transgenic tobacco BY-2 cell lines that overexpress NtmybA2 or its truncated hyperactive form. The microarray was also used to determine the transcript profile during the cell cycle in synchronized cultures of BY-2 cells. Combined microarray data from transgenic lines and synchronized cells revealed that overexpression of the truncated hyperactive form of NtmybA2, but not its full-length form, preferentially up-regulated many G2/M phase-specific genes in BY-2 cells. We determined promoter sequences of several such up-regulated genes and showed that all contain MSA-like motifs in the proximal regions of their promoters. One of the up-regulated genes, NtE2C, encoding for cyclin-specific ubiquitin carrier proteins, contained a single functional MSA-like motif, which specifically controlled the expression of a reporter gene in the G2/M phase in BY-2 cells. Furthermore, a genomic footprint experiment showed that the MSA element in the NtE2C promoter interacted with nuclear proteins in vivo. Therefore, we propose that the transcription of many G2/M phase-specific genes in tobacco is positively regulated by NtmybA2, in most cases through direct binding to the MSA elements.

² Present address: Department of Molecular Ecology, Max-Planck-Institute for Chemical Ecology, D–07745 Jena, Germany.

³ Present address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7–3–1, Bunkyo-ku, Tokyo 113–0033, Japan.

⁴ Present address: Laboratory of Plant Nutrition, Faculty of Agriculture, Kyushu University, 6–10–1, Hakozaki, Higashi-ku, Fukuoka 812–8581, Japan.

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: Masaki Ito (masakito{at}agr.nagoya-u.ac.jp).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.135582

^* Corresponding author; e-mail masakito{at}agr.nagoya-u.ac.jp.

Received January 12, 2009; accepted February 18, 2009; published February 25, 2009.