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Plastid-To-Nucleus Retrograde Signals Are Essential for the Expression Of Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA] Kazuhiko Enami, Tomoki Ozawa, Noriko Motohashi, Masayuki Nakamura, Kan Tanaka2, and Mitsumasa Hanaoka* Downloaded from https://academic.oup.com/plphys/article/157/1/518/6108903 by guest on 01 October 2021 Division of Applied Biological Chemistry, Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271–8510, Japan (K.E., N.M., K.T., M.H.); Laboratory of Molecular Genetics, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–0032, Japan (T.O., K.T., M.H.); and Graduate School of Natural Sciences, Nagoya City University, Mizuho-ku, Nagoya 467–8501, Japan (M.N.) Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS)andGranule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. Plastids are plant-specific, semiautonomous organ- different subtypes in response to several developmen- elles that have important metabolic functions, such tal and environmental conditions. Each differentiated as photosynthesis, carbon and nitrogen assimilation, form of plastid plays a specific role in maintaining a and biosynthesis of lipids and amino acids. In higher variety of metabolic and physiological functions as a plants, plastids are known to differentiate between component of plant cells or tissues (for review, see Lopez-Juez and Pyke, 2005). For instance, chloroplasts, 1 the most characterized form of plastid, are found in the This work was supported by a Special Coordination Fund for majority of green tissues and are involved in photo- Promoting Science and Technology to M.H., by a Grant-in-Aid for synthesis. Chromoplasts occur in limited organs, such Creative Scientific Research (grant no. 16GS0304 to K.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of as fruit and floral petals, or root tissues in carrot Japan, by Grants-in-Aid for Scientific Research (B; grant no. 21370015 (Daucus carota), and are engaged in the synthesis and to K.T.) and for Young Scientists (B; grant no. 21770034 to M.H.) from storage of carotenoid pigments. Leucoplasts are color- the Japan Society for the Promotion of Science, and by the Centers less plastids mainly existing in root tissues. Several of Excellence StartUp Program of Chiba University to K.T. reactions for terpene biosynthesis take place in leuco- 2 Present address: Chemical Resources Laboratory, Tokyo Insti- plasts as well as in chloroplasts. In meristematic tis- tute of Technology, Midori-ku, Yokohama 226–8503, Japan. sues, plastids can be identified as an undifferentiated * Corresponding author; e-mail [email protected]. form, termed proplastids, which are specialized for The author responsible for distribution of materials integral to the proliferation instead of any metabolic reactions. Am- findings presented in this article in accordance with the policy yloplasts, which are thought to be a specialized form described in the Instructions for Authors (www.plantphysiol.org) is: Mitsumasa Hanaoka ([email protected]). of leucoplasts, exist in sink tissues such as endosperms [W] The online version of this article contains Web-only data. and tubers and are characterized as plastids for starch [OA] Open Access articles can be viewed online without a sub- synthesis and storage. A series of enzymes related scription. to starch synthesis, such as ADP-Glucose Pyrophos- www.plantphysiol.org/cgi/doi/10.1104/pp.111.178897 phorylase (AGPase), Granule-Bound Starch Synthase Ò 518 Plant Physiology , September 2011, Vol. 157, pp. 518–530, www.plantphysiol.org Ó 2011 American Society of Plant Biologists. All Rights Reserved. Role of Plastid Gene Expression in Amyloplast Biogenesis (GBSS), and Starch Branching Enzyme, are specifically mutant, for instance, abnormal chloroplast develop- enriched in amyloplasts (Martin and Smith, 1995; ment resulted from the decline of glutamyl-tRNA Fernie et al., 2002). The stored starch can be broken amount, which caused the inhibition of plastid trans- down to free saccharides or sugar derivatives and lation as well as chlorophyll biosynthesis (Kanamaru supplied for germination and plant growth by trans- et al., 2001; Hanaoka et al., 2003). Furthermore, glutamyl- location. In addition, amyloplasts in root cap cells and tRNA directly bound to and inhibited the transcription endodermal cells of inflorescence stems play an es- activity of NEP in vitro, suggesting that SIG2 mediates sential role in gravity sensing as a kind of statolith, and the switching of transcriptional machinery from NEP that specific accumulation of starch granules in the to PEP during chloroplast development (Hanaoka amyloplasts is important in gravity sensing (for re- et al., 2005). Thus, recent studies of the transcriptional view, see Morita and Tasaka, 2004). regulation of plastid genes have focused mainly on the It is widely believed that plastids originated from an step of differentiation into chloroplasts. On the other Downloaded from https://academic.oup.com/plphys/article/157/1/518/6108903 by guest on 01 October 2021 endosymbiosis between an ancient cyanobacterial cell hand, little is known about transcriptional regulation and a primordial eukaryotic cell. Accordingly, plastids during differentiation into other nonphotosynthetic contain their own genomic DNA and gene expression plastids. machineries of cyanobacterial origin. However, after As a model system of plastid differentiation in non- the long history of evolution, a large number of genes photosynthetic tissues, we chose to use tobacco (Nicoti- were lost from the plastid genome or transferred into ana tabacum) Bright Yellow-2 (BY-2) cultured cells, which the nuclear genome (Leister, 2003). The plastid ge- is a model plant cultured cell system with nonphoto- nomes of higher plants are normally double-stranded, synthetic, highly prolific properties (Nagata et al., 1992). circular DNAs containing about 120 genes that are This cell line is usually maintained in a medium composed of four ribosomal RNAs, 30 transfer RNAs, containing the auxin 2,4-dichlorophenoxyacetic acid and 80 proteins required for transcription, translation, (2,4-D), in which plastids are maintained as undifferen- and a variety of plastid functions such as photosyn- tiated proplastids. Differentiation into amyloplasts from thesis (Sugita and Sugiura, 1996). However, the vast proplastids is induced by the addition of synthetic majority of plastid proteins are encoded in the nuclear cytokinin, 6-benzylaminopurine (BA; Sakai et al., 1992; genome. Once expressed and translated in the cytosol, Miyazawa et al., 1999). During this step, expression of precursors of plastid proteins are imported into plas- several nucleus-encoded genes related to starch biosyn- tids via translocating complexes, processed and mod- thesis, such as AgpS and GBSS,isknowntobespecif- ified into mature proteins, and function individually ically induced by the addition of BA (Miyazawa et al., or by forming functional complexes with other pro- 1999). It was also reported that the transcription activity teins, whether these are originally plastid encoded or of several plastid-encoded genes in amyloplasts was nuclear encoded. In this sense, coordinated expression lower than that in proplastids and chloroplasts (Sakai of the nuclear and plastid genes is considered impor- et al., 1992; Valkov et al., 2009). However, the importance tant for the maintenance of normal plastid functions and requirement of plastid gene expression in amylo- (Goldschmidt-Clermont, 1998). plasts have not been clearly elucidated yet. Expression of plastid genes is strictly controlled by In this paper, we investigate the roles of plastid gene the nucleus, depending on tissues
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