84  2000 American Society for Photobiology 0031-8655/00 $5.00+0.00 Photochemistry and Photobiology, 2000, 71(1): 84–89 Isolation and Initial Characterization of 13 2 -Hydroxychlorophyll a Induced by Cyclohexanedione Derivatives in Tobacco Cell Suspension Cultures Jing-Ming Wang* 1 , Tadao Asami 2 , Noboru Murofushi 1 and Shigeo Yoshida 2 1 Department of Biotechnology, Akita Prefectural University, Nakano Simoshinjo Akitashi Akita, Japan and 2 The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako, Saitama, Japan Received 6 July 1999; accepted 13 October 1999 ABSTRACT This paper reports that a new photobleaching compound, 2-(2-chloro-5-propoxycarbonylphenyl)aminomethylidene- 5,5-dimethyl-cyclohexane-1,3-dione (RWH-21), stimu- lates accumulation of 13 2 -hydroxychlorophyll a in cul- tured tobacco cells. This was shown based on isolation of 13 2 -hydroxychlorophyll a from pigment extracts of cul- tured tobacco cells by diode-array HPLC and subsequent fast atom bombardment mass spectrometry analysis. 13 2 - Hydroxychlorophyll a rapidly accumulates in tobacco cells both in the light and dark in the presence of RWH- 21 (50 M). Analysis of 13 2 -hydroxychlorophyll a for- mation in tobacco cells indicates that 13 2 -hydroxychlo- rophyll a is rapidly accumulated within 20 h incubation time both in the dark and light. Although the amount of 13 2 -hydroxychlorophyll a is continuously increased in the dark, the amount of 13 2 -hydroxychlorophyll a decreased remarkably in the light after 20 h incubation. Analysis of 13 2 -hydroxychlorophyll a formation and lipid peroxi- dation by determination malondialdehyde in tobacco cells suggests that RWH-21-induced 13 2 -hydroxychloro- phyll a has the potential to cause a photodynamic action in cultured tobacco cells. INTRODUCTION The prominent function of chlorophyll (Chl)² pigments in photosynthesis and plant physiology attracts enormous re- search interest. In particular, the biosynthetic pathway and the degradation process of Chl that are thought to be in- volved in plant development and senescence are of substan- tial interest to biochemists and plant physiologists. Com- pared with the biosynthetic pathway of Chl that has been studied in considerable detail, knowledge of the biochemical *To whom correspondence should be addressed at: Department of Biotechnology, Akita Prefectural University, Nakano Simoshinjo Akitashi Akita 010-0146, Japan. Fax: (+81) 18-872-1670; e-mail: jmwang@akita-pu.ac.jp ² Abbreviations: Chl(s), chlorophyll(s); DAD, diode array detector; FAB-MS, fast atom bombardment mass spectrometry; MDA, ma- londialdehyde; RWH-21, 2-(2-chloro-5-propoxycarbonylphenyl)- aminomethylidene-5,5-dimethylcyclohexane-1,3-dione; TBP, tet- rabutylammonium phosphate. mechanism responsible for the degradation of Chl is much more limited. The degradation of Chl has been generally thought to be done through a process that releases Chl from their protein complex followed by dephytylization catalyzed by chloro- phyllase and possibly by pheophytinization with loss of the Mg 2+ ion. Oxidation of the porphyrin ring structure ensues, ultimately leading to the formation of colorless end products (1–3). Another chemical reaction involved in the degradation of Chl is called allomerization (4). Nowadays, knowledge of in vitro allomerization of Chl in alcoholic solution is pro- gressively accumulating (5–10). This process involves oxi- dation of Chl by triplet oxygen. All the products (allomers) have an oxygen atom bonded to a carbon atom at the posi- tion of C-13 2 . Among the allomers of Chl a, 13 2 -hydroxy- chlorophyll a is an early intermediate in the allomerization pathway of Chl in methanol solution (9). Although consid- erable progress has been made in the knowledge of break- down of Chl in senescent leaves (2,3,11–13), there are very few reports about the natural occurrence of 13 2 -hydroxy- chlorophyll a in plant tissues and little is known about the molecular mechanism of the production of Chl allomers in plant tissues. We reported that 2-(2-chloro-5-propoxycarbonylphenyl)a minomethylidene-5,5-dimethylcyclohexane-1,3-dione (RWH-21, the structure is shown in Fig. 1) showed a light- requiring bleaching activity in cultured tobacco cells (14). This observation suggests that RWH-21 may activate a pho- tosensitizer in tobacco cells that is responsible for the bleaching. In the present study, we have shown that RWH- 21 promotes the accumulation of 13 2 -hydroxychlorophyll a in tobacco cells. More intriguing is that accumulation of 13 2 - hydroxychlorophyll a in tobacco cells is independent of ir- radiation, but the action of RWH-21 for lipid peroxidation requires light illumination, implicating a photodynamic pro- cess involved in the bleaching action of RWH-21. MATERIALS AND METHODS Chemicals. The herbicidal compound RWH-21 was prepared by a method described previously (14). The compound for assay was pu- rified by recrystallization. The structure was confirmed by NMR spectroscopy and an elementary analysis for C, H, N and Cl. The compound for assay was dissolved in dimethyl sulfoxide and ster- ilized through a sterile membrane filter (0.2 M Millex-FG: Nihon Millipore Ltd., Tokyo, Japan). All other reagents were of the highest quality commercially available.