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Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides1

Naohide Watanabe2, Fang-Sik Che2, *, Megumi Iwano, Seiji Takayama, Takeshi Nakano, Shigeo Yoshida, and Akira Isogai

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama Ikoma, Nara 630-0101, Japan (N.W., F.-S.C., M.I., S.T., A.I.); and The Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama, 351-0198, Japan (T.N., S.Y.)

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.

1   This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan (no. 09760304).
2   These authors contributed equally to this paper.
*   Corresponding author; e-mail fsche{at}bs.aist-nara.ac.jp; fax 81-743-72-5459.

Plant Physiol. (1998) 118: 751-758
Copyright Clearance Center:   0032-0889/98/118//08
© 1998 American Society of Plant Physiologists




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