91 AsPac J. Mol. Biol. Biotechnol., Vol. 15 (2), 2007 Browning in Metroxylon sagu: Mechanism and its prevention Asia Pacific Journal of Molecular Biology and Biotechnology, 2007 Vol. 15 (2) : 91-98 *Author for Correspondence. Mailing address: Dept. of Food Science, Faculty of Food Sci- ence and Technology, Universiti Putra Malaysia, 43400 UPM, Ser- dang, Selangor, Malaysia. Tel:+603 89468371; Fax:+603-89423552; Email: nazamid@putra.upm.edu.my Histochemical Localization of Polyphenol Oxidase and Peroxidase from Metroxylon sagu Galila Hassan Onsa 1 , Nazamid Bin Saari 2 *, Jinap Selamat 2 , Jamilah Bakar 2 , Abdulkarim Sabo Mohammed 2 and Shamsul Bahri 3 1 Food Processing Research Center, Shambat, Khartoum, Sudan 2 Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 3 Biotechnology and Strategic Research Unit, Malaysia Rubber Board, 47000 Sg. Buloh, Selangor Malaysia Received 14 May 2006 / Accepted 30 January 2007 Abstract. Polyphenol oxidase (PPO) and peroxidase (POD) activities were visualized histochemically at the cellular level of a young and a mature tree of Metroxylon sagu employing histochemical technique. In the mature tree, intense PPO activity was observed in the cell wall of the parenchyma and xylem cells when visualized under light microscope. Pith collected from the young tree showed PPO activity in the amyloplast and mitochondria inner membrane and to some extent in the golgi complex and endoplasmic reticulum. Whereas, a positive POD reaction product was visualized in the cell wall, peroxisomes and to some extent in the cytoplasm and the vacuole. The localization of PPO activity in the amyloplast and being adsorbed by the starch granules is in line with the general view that enzyme is involved in the browning of sago starch. Keywords. Metroxylon sagu, polyphenol oxidases, peroxidase, cellular localization INTRODUCTION Enzymatic browning reactions mediated by polyphenol oxidases and peroxidases in Metroxylon sagu have been associated with the low marketability of sago starch (Yatsugi, 1986; Ahmad, 1991; Okamoto et al. 1988 and Onsa et al. 2000). The browning reaction occurs when the cell is ruptured and the indigenous phenolic compounds are oxidized in the presence of molecular oxygen (Mayer, 1986; Mayer and Harel, 1981). Histochemical study offers the advantage of localization of enzymes in intact cells based on specific activity staining and viewing under electron microscope. Transmission electron micrographs of other starch storage plant tissues and in particular sago pith tissues are difficult to find among current literature. This lack of ultrastructural information has been due in part to the difficulties in preparing the tissues for transmission electron microscopy (TEM). High levels of starch, lignin, lipid and phenolic compounds create problems in achieving adequate fixation and infiltration of resin into the cells to achieve successful ultramicrotomy for TEM (Buschmann et al. 2002). The formaldehyde used in the fixative mixture penetrates rapidly into the tissue but reacts slowly with proteins and is not recommended on its own for fixing plant cell. Glauert and Lewis (1998) recommend the use of Karnovsky’s fixative solution, which is a mixture of formaldehyde and gluteraldehyde. The nature and occurrence of the oxidative enzymes, polyphenol oxidase (PPO) and peroxidase (POD) have been studied comprehensively in fruits and vegetables (Mayer and Harel, 1978; Zawisstowski et al. 1991; Robinson, 1991). There are very few publications on the localization of these browning enzymes at the ultrastructural level using histochemical techniques and there is no previous work reported for Metroxylon sagu. In specific differentiating plant tissues of Parthenium argentatum stem, an intense oxidative enzymes activity is localized in the parenchyma, xylem and phloem cells (Jayabalan et al. 1995). PPO in some higher plants is considered as a plastid enzyme localized in a diverse series of plastids such as the chloroplast and in the thylakoid membrane (Mayer and Harel, 1978). In potato tuber, a heavy PPO reaction was observed in the thylakoids, vesicles and in the stroma when the specimen was stained with DOPA (dihydroxyphenylalanine) (Czaninski and Catesson, 1974). The reasons suggested for the variation in histochemical