Abstract When BY-2 cultured tobacco ( Nicotiana taba- cum L.) cells were transferred to auxin-depleted culture medium containing cytokinin (benzyladenine, 1 mg/l), the starch content per cell started increasing from 18 h of cul- ture and amyloplasts had formed by 48 h. Pulse-treatment of the cells with actinomycin D and cycloheximide for the first 12 h (or longer) of culture significantly decreased the cellular starch content after 48 h, whereas the starch con- tent did not decrease significantly when the cells were re- leased from the inhibition within 6 h. This suggests that nuclear gene expression necessary for amyloplast forma- tion begins 6–12 h after the transfer. Immunoblotting anal- ysis of the accumulation of ADP-glucose starch glycosyl transferase (starch synthase) supported this inference. Key words ADP-glucose starch glycosyl transferase · Amyloplast · BY-2 · Nicotiana tabacum · Transcription/translation inhibitors Abbreviations BA Benzyladenine · 2,4-D 2,4-dichlo- rophenoxyacetic acid · WXP wx + protein Introduction Amyloplasts are non-green plastids specialized for the syn- thesis and accumulation of starch (Kirk and Tilney-Bas- sett 1978). They are observed in various organs and tis- sues, such as cotyledons, tubers, tapetum cells in anthers, pollen grains, and root caps. As amyloplasts are intimately involved in carbon metabolism in plants, analysis of the mechanisms controlling amyloplast formation is a key step toward understanding the regulation of storage potential and/or sink-source relationships in plants. We previously reported a procedure for inducing amy- loplast formation in BY-2 cultured tobacco ( Nicotiana ta- bacum L.) cells and proposed it to be a useful system for analyzing the processes and mechanisms of amyloplast for- mation (Sakai et al. 1992); in this system, depletion of auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) and addi- tion of cytokinin (benzyladenine, BA) induced the dif- ferentiation of leucoplast-like plastids to amyloplasts within 2–3 days. Although BY-2 cells did not proliferate under the amyloplast-inducing conditions, accumulation of starch was not a consequence of the cessation of cell proliferation, being regulated more directly by plant hor- mones (Sakai et al. 1996). Amyloplast formation in BY-2 cells was inhibited by transcription/translation inhibitors for both eukaryotic and prokaryotic systems, indicating that de novo expression of nuclear- and organelle-coded genes is necessary, either directly or indirectly, for amylo- plast development (Sakai et al. 1997). We also reported that continous expression of nuclear genes was required for amyloplast formation, whereas organelle gene expres- sion became less necessary in the late phase of amyloplast development. This observation, together with the fact that genes involved in starch synthesis have not been found in the plastid genome (Sugiura 1992), indicates that amylo- plast formation is predominantly controlled by the cell nu- clear genome. In the present study, we determined the timing, during amyloplast formation in BY-2 cells, of the expression of the cell nuclear gene(s) necessary for amyloplast develop- ment. We examined the kinetics, as well as responses to transcription/translation inhibitors, of starch accumula- tion. We also examined the behavior of an amyloplast-re- lated, nuclear-encoded enzyme, ADP-glucose starch gly- cosyl transferase [starch (amylose) synthase], known as the Plant Cell Reports (1999) 18: 589–594 © Springer-Verlag 1999 Received: 21 August 1998/ Revision received: 3 September 1998/ Accepted: 2 October 1998 A. Sakai · Y. Miyazawa · C. Saito · N. Nagata H. Takano · H.-Y. Hirano · T. Kuroiwa Amyloplast formation in cultured tobacco cells. III Determination of the timing of gene expression necessary for starch accumulation Communicated by R. L. Rodriguez A. Sakai () · Y. Miyazawa · C. Saito · N. Nagata · H. Takano T. Kuroiwa Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113–0033, Japan Fax: +81-3-3814-1408 e-mail: sakai@biol.s.u-tokyo.ac.jp H.-Y. Hirano Laboratory of Plant Breeding and Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Tokyo 113–8657, Japan