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J. Biol. Chem., Vol. 279, Issue 14, 14049-14054, April 2, 2004

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Distinct Isoprenoid Origins of cis- and trans-Zeatin Biosyntheses in Arabidopsis^*

Hiroyuki Kasahara, Kentaro Takei¶, Nanae Ueda¶, Shojiro Hishiyama||, Tomoyuki Yamaya¶, Yuji Kamiya, Shinjiro Yamaguchi, and Hitoshi Sakakibara¶**

From the Laboratory for Cellular Growth and Development and the ^¶Laboratory for Communication Mechanisms, Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), Yokohama 230-0045, Japan and the ^||Forestry and Forest Products Research Institute, Ibaraki 305-8687, Japan

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with ¹³C in Arabidopsis seedlings. Efficient ¹³C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a ¹³C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.

Received for publication, December 26, 2003

^* This study was supported in part by Grants-in-aid for scientific research (12142202 (to H. S.) and 15770035 (to K. T.)) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^** To whom correspondence should be addressed: Plant Science Center, RIKEN, Suehiro 1-7-22, Tsurumi, Yokohama 230-0045, Japan. Tel.: 81-45-503-9576; Fax: 81-45-503-9609; E-mail: sakaki{at}postman.riken.go.jp.

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