91AsPac J. Mol. Biol. Biotechnol., Vol. 15 (2), 2007 Browning in Metroxylon sagu: Mechanism and its preventionAsia Pacific Journal of Molecular Biology and Biotechnology, 2007Vol. 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 Onsa1, Nazamid Bin Saari2*, Jinap Selamat2, Jamilah Bakar2, Abdulkarim Sabo Mohammed2 and Shamsul Bahri3

1Food Processing Research Center, Shambat, Khartoum, Sudan2Faculty of Food Science and Technology, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor, Malaysia 3Biotechnology 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

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properties of the browning enzymes was that enzymes from different sources have different physiological functions (Vamos-Vigyazo, 1981). The objective of this study was to identify the location of the main browning enzymes; PPO and POD at the ultrastructural level in Metroxylon sagu pith tissue. The study employed enzyme histochemical technique and visualized under light microscope and TEM. Such information would be of great importance in developing a suitable strategy to overcome the browning problem.

MAtErIALS And MEtHOdS

Plant Material. Metroxylon sagu was collected from Batu Pahat, Johor State, Malaysia. Two trees were selected at different developmental stages; a young tree aged between 3 - 4 months old (a trunk height of 0.20 m and a diameter of 0.05 m) and a tree aged between 2 - 4 years (a trunk height of 3.0 m and a diameter of 0.50 m) to represent a mature tree. The pith was collected from the middle section of each tree.

Chemicals Used. Basic fuchsine, Agar 100 resin was obtained from Agar Scientific Ltd, Stanstel, UK. Osmium tetraoxide, catechol, kojic acid and tetramethylbenzidene (TMBZ) were obtained from Sigma Chemicals, St. Louis, California USA. Sucrose, ethanol, propylene oxide, gluteraldehyde (25% microscopy grade), formaldehyde, cacodylate buffer pH 6.8, urinyl acetate and H2O2 were purchased from BDH Chemicals Ltd, Poole, England.

Sample Preparation. Sample preparation started in the field immediately after the tree was cut using an electric cutter. Using a razor blade, sago pith blocks of 1-2 cm3 were selected from 5 locations in the log cross sections. Each block was cut into sections of 3 - 4 mm3 and immediately transferred into the Karnovsky’s fixative solution at 4°C as recommended by Glauert and Lewis (1998) for fixing plant tissues. The osmolarity of the fixative solution was adjusted using 0.4 M sucrose (Glauert and Lewis, 1998). In the lab, the tissues were subjected to gentle vacuum for 3 - 4 min in order to draw the fixative solution into the cells and vessels to ensure good penetration of the fixative solution and then left overnight at 4°C (Carde, 1986). The samples were then washed 3 times with 0.1 M sodium cacodylate buffer pH 6.8 for 30 min each. The slices were post fixed in 2% buffered osmium tetraoxide for 1 hr at 4°C and washed subsequently 3 times with 0.1 M sodium cacodylate buffer pH 6.8. They were then dehydrated in a graded ethanol series (35%, 50%, 70% and 95%) for 30 min per step and then in absolute ethanol for 1 hr with 2 changes. Propylene oxide was used as an intermediate step for embedding (Beles et al., 1989).

Embedding and Polymerization. Infiltration was carried out according to the standard procedure described by Lewis and Knight (Lewis, 1992), with some modifications, which includes extended resin infiltration time. The tissues were incubated in a series of resin and absolute acetone at ratios of 1:3 for 2 hrs, 1:2 for 3 hrs, 1:1 and 2:1 for 3 hrs each and finally 100% resin (Agar 100) for overnight. The tissues were subjected to constant agitation at each infiltration step. The specimens were then placed in beam capsules and filled with the 100% resin and polymerized in oven (Memmert 845 Scwach, Germany) for 48 h at 50°C.

Sectioning. Semi-thin sections of 1-2 mm thick were cut with an ultramicrotome (Reichert Ultracut Leica) and mounted on glass slides for viewing under light microscope. For TEM viewing, ultra-thin sections of 30-50 nm were prepared using ultramicrotome with a glass knife. Each thin section was mounted on a copper-coated grid ready for staining and viewing.

Staining Method. The semi-thin sections were stained with basic fuchsine and examined under the light microscope (Olympus BH2) equipped with color camera to view the general structure of sago pith tissues and to locate zones of some cellular organelles for ultra-thin sectioning (Lewis, 1992). The localization of polyphenol oxidase (PPO) activity at the ultra-structural level was investigated histochemically by incubating the ultra-thin section for 2 hr with 50 mM catechol in 0.1 M acetate buffer pH 4.5 at 4 °C and transferred to room temperature for 2 hr. Catalase at 0.1 mg mL-1 was added to prevent any peroxidases activity by destroying the hydrogen peroxide, which might be present (Czaninski and Catesson, 1974 and Lewis, 1992). Post-staining was done using 2% aqueous uranyl acetate for 5 min followed by lead citrate for 1 min (Bergman et al. 1985 and Burlat et al. 2001). The detection of peroxidase activity at the ultra-structure level was carried out following the procedure described by Olah and Muller (1981). The specimen was incubated in fresh solution of 10 mM tetramethylbenzidine (TMBZ) and 1% H2O2 in 0.1 M acetate buffer pH 4.5 at 4°C for 2 hr followed by 2 hr incubation at room temperature. As a control the specimens were incubated in 10 mM kojic acid to inhibit the activity of PPO and POD and then stained using 2% aqueous uranyl acetate for 5 min followed by lead citrate for 1 min. The specimens were washed and dried before examination under TEM (Philips CM 12) operating at 80 kV.

rESuLtS And dIScuSSIOn

Anatomy of Sago Pith Tissues. The morphology of cross sections from Metroxylon sagu trunk at two developmental stages were visualized to further understand the changes that occur during maturation. In young Metroxylon sagu (3-

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4 months) the pith was fleshy and has a diameter of 0.05 m surrounded by two outer layers; the leaf base making a crescent shape and a thick bark (Figure 1A). With increasing maturity at 2-4 years the pith progressively enlarged, filled with thickened starch material to reach a diameter of 0.5 m and surrounded by a thin layer bark of 2-3 cm. Between the pith and the bark a very thin protective layer of cork tissues was found, immediately below the bark (Figure 1B). This cork tissue contains, a waxy substance, which provides protection for the inner tissues (Buschmann et al. 2002). When the semi-thin sections from each developmental stage were stained with basic fuchsine, irregular spaced vascular bundles could not be seen in the tissues of young tree (Figure 2) but could be visualized in the mature sago tree within the pith tissue as shown in Figure 3. Flash and Schulling (1989) reported a similar observation on Metroxylon sagu at commercial maturity stage (9-12 years old). These vascular bundles were numerous and thick and can be detected by the naked eye. The vascular bundles comprise of a lignified developing xylem cells bordering the crescent-

Leaf base Pith Bark

cyv

papi

2µm

Figure 1. Cross-section of Metroxylon sagu from (A) a young log (B) a mature log showing the pith, bark and the leaf base.

Figure 2. Light micrograph showing the parenchyma cells of young Metroxylon sagu. The semi-thin cross-section from young sago log was stained with basic fuchsine. pa, parenchyma cell; v, vacuole; cy, cytoplasm; and pi, pigmentation deposit. (×165)

Figure 3. Light micrograph showing the xylem cells and paren-chyma cells of mature Metroxylon sagu. The semi-thin section of mature sago pith was stained with basic fuchsine. xl, xylem lu-men; xv, xylem vessel; pa, parenchyma cell; and pi, pigmentation deposit. (×165)

shaped vascular vessels when stained with basic fuchsine. The xylem cells composed of lignin and tannin complexes, which add stiffness to the cell walls (Buschmann et al. 2002). Pattern of lignin deposition within the stem and the developing xylem cells was also reported for Forsythis intermidia (Burlat et al. 2001). The cytoplasmic materials can be visualized in the parenchyma cells of young sago pith (3-4 months) when viewed under light microscope (Figure 2). The vacuoles occupy small part of the cytoplasm and dense pigmentations can be visualized within the cytoplasm. These cytoplasmic materials could not be detected in the parenchyma cells at the mature stage, suggesting that the cells were fully occupied by the vacuoles. Small vacuoles are known to exist in developing cells and become more prominent in maturing cells (Rastogi, 1988).

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Cellular Ultrastructures of Metroxylon sagu Tissues. Metroxylon sagu pith, a starch storage tissue is extremely difficult to fix well. Routine preparative methods have not been successful for imaging mature sago pith tissue ultrastructure. Thick-walled cells and the cortical parenchyma filled with starch also contribute to the preparation difficulties. To overcome this problem, young sago palm of 3 - 4 months old having less starch and fleshy pith were selected for this study. Ultra-structural observations of sago pith tissues have typically been made on materials prepared by the devised infiltration method, where the specimens were immersed in 5 series of different ratios of resin in acetone for a longer time at 4°C. The procedure allows a variety of cellular structures to be visualized.

The cell wall (w) forms compartment boundaries, a typical plant cell wall as described by Cardel (1986). The vacuoles and other cytoplasmic ultra-structures of young sago pith are presented in Figures 4A and 4B. The vacuoles (v) are the dominating feature of sago pith cell. It is described as a clear, well-defined area bound by a membrane known as tonoplast (Rastogi, 1988). It was observed that most of the cytoplasmic material was located around the nucleus (n) similar to that reported in the cell cultures of Berberis spp that have a big size nucleolus (n), which occupies about 10% of the cytoplasm (Amann et al. 1986). The protoplastids visualized could be identified by their inclusion of small starch grain as an amyloplast (a) containing distinct starch granules (s). The amyloplast was typical of plant starch storage tissues as reported for potato tuber (Czaninski and Catesson, 1974) and cassava root tissues (Buschmann et al. 2002). Small mitochondria (m) can be identified by the presence of cristae. Several peroxisomes (pe) were visualized in the cytoplasm. The peroxisomes as described by Rastogi (1988) are single membrane microbodies, containing a ground substance rich in oxidative enzymes.

Histochemical Localization of Polyphenol Oxidase. Enzyme histochemical localization study was accomplished using catechol for PPO activity staining. The principle of this reaction involves the development of an insoluble, electron dense reaction product from a synthetic substrate (Czaninski and Catesson, 1974). A dense deposition of the PPO reaction product is readily visualized within the xylem cell walls and the parenchyma cell wall (Figure 5A). This deposition was probably due to an enzymatic reaction as indicated by the result of inhibition test using kojic acid as a control (Plate 5B). Similar observations were reported for PPO activity in the cell walls of mature apple fruits tissues when detected immunochemically (Murata et al. 1997) and the stem of a 4 years old Pistacia vera (Al-Barazi and Schwabe, 1984). Intense PPO activity has been reported to be localized in both the cell wall and the cytoplasm of parenchyma cells of one year old Parthenium argentatum stem (Jayabalan et al. 1995). In order to localize PPO activity at the sub-cellular organelles, it is necessary to subject the catechol stained sections to post-staining employing uranyl acetate (Bergmann et al. 1985; Burlat et al. 2001). By employing post-staining treatment, satisfactory visualization of the reaction product in various cellular organelles is obtained. Figure 6A shows a typical micrograph of a thin section of pith from young (3 - 4 months old) Metroxylon sagu stained for PPO activity. Electron dense deposits are visible within the amyloplast (a). The dense deposit in the amyloplast is adsorbed by the starch granule (s) and has turned the starch granule darker (Figure 6A). The activity appears to be associated with the amyloplast when compared with the control where the amyloplast (a) and the starch granule (s) lack any reaction deposit. These findings are generally consistent with the observation made for PPO activity staining in the amyloplast

Figure 4A. Electron micrograph showing the ultrastructure of Metroxylon sagu. Ultrathin-section of young sago pith tissue stained with uranyl acetate. w, cell wall; a, amyloplast; and s, starch grains. (×10575)

Figure 4B. Electron micrograph showing the ultrastructural feature of young Metroxylon sagu (3-4 months old). The speci-men was stained with uranyl acetate. w, cell wall; a, amyloplast; s, starch granules; pe, peroxisomes; n, nucleus; and m, mitochon-dria. (×10500)

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of apple fruit (Mayer, 1986; Bar-Nun and Mayer, 1983). In Figure 6A, a dark reaction deposit in the cell wall is visualized, indicating the uniform distribution of the PPO enzyme in the cell wall (w). This oxidation is not due to endogenous peroxidase, as its activity requires H2O2 in the reaction medium. Phenol oxidation mediated by peroxidase was eliminated by the addition of catalase to the staining media to distinguish PPO from POD activity. However, the presence of some other oxidizing enzymes, which may oxidize catechol, cannot be excluded, (Czaninski and Catesson, 1974). Contradictory observations were reported for potato slices and sycamore leaf when stained with 4-methylcatechol (Czaninski and Catesson, 1974) and carrot cell suspension culture (Olah et al. 1981) where the cell wall

lacks any reaction product. The reaction product of PPO in the cytoplasm is more difficult to characterize because a definite site for the electron-dense product cannot be demonstrated but rather the ground substance of the cytoplasm appear granular and the overall contrast was enhanced (Figure 6A). However, the granular structure in the cytoplasm indicates the presence of the enzyme in a soluble form. The occurrence of soluble PPO has been reported for a number of plants (Mayer, 1986; Mayer and Harel, 1981) and a cytoplasmic reaction product has also been observed in carrot suspension cell cultures (Olah, et al. 1981). Catechol reaction product was also visualized in the distal cisternae of the golgi complex and in the smooth tubules associated with the golgi complex (Figure 6B). Some

Figure 5A. Light micrograph showing the browning reac-tion in the xylem cells and parenchyma cells of mature Metroxy-lon sagu. The semi-thin cross-section was stained with catechol. xl, xylem lumen; xc, xylem cells; xv, xylem vessels; pa, parenchyma cell. (×330)

Figure 5B. Light micrograph showing inhibited browning reac-tion in the xylem cells and parenchyma cells of mature Metroxylon sagu as a control. The semi-thin cross-section from mature sago palm pre-treated with 10 mM Kojic acid followed by activity staining. xl, xylem lumen; xc, xylem cells; xv, xylem vessels; and pa, parenchyma cell. (×330)

Figure 6A. Electronmicrograph showing the histochemical localization of polyphenol oxidase in young Metroxylon sagu (3-4 months old). Heavy catechol reaction product is seen in the cell wall (w), amyloplast (a) with starch granules (s) enclosed. The mi-tochondria cristae and inner membrane show a slight positive reac-tion product. (×13250)

Figure 6B. Electron micrograph showing the histochemi-cal localization of polyphenol oxidase stained by catechol in Metroxylon sagu. Catechol reaction product is seen in the dis-tal cisternae of the golgi complex (g) and in smooth tubules associated with the endoplasmic reticulum (er). (×20520)

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oxidative enzymes are reported to originate from the golgi complex and secreted through the endoplasmic reticulum to the cytoplasm and cell wall (Rastogi, 1988; Dupree and Sherrier, 1998). Mitochondrial (m) internal membrane shows a diffused positive PPO activity, while the lumen and the matrix lack any reaction product (Figure 7B) compared to the control (Figure 7A). The mitochondrion is known to be rich in various enzymes, and they are engaged in a variety of functions including electron transport (Rastogi, 1988).

A positive PPO reaction product in the mitochondrial cristae and inner membrane was reported for the sycamore leaf when stained with 4-methylcatechol (Czaninski and Catesson, 1974).

Histochemical Localization of Peroxidase. Although the browning reaction has been attributed to PPO activity, other oxidative enzymes such as peroxidase (POD) have been well documented in other food commodities (Robinson,

Figure 7A. Electron micrograph showing inhibited PPO re-action in the Metroxylon sagu (3 – 4 months) mitochondria and endoplasmic reticulum as a control. The ultrathin-section was incubated in kojic acid followed by staining with catechol and post stained with uranyl acetate. er, endoplasmic reticulum; m, mitochondria; ml, mitochondria lumen; cy, cytoplasm. (×35520)

Figure 7B. Electron micrograph showing the histochemical lo-calization of PPO in Metroxylon sagu (3-4 month old). Catechol reaction product is seen in the endoplasmic reticulum (er), mito-chondria (m) and the inner cisternae showed a diffused positive reaction product. (×37350)

Figure 8. Electron micrograph showing the location of POD in young Metroxylon sagu (3-4 months old). Dense reaction product was observed in the cell wall (w) and to some extend in cytoplasm (cy) but not seen in the inter-cellular space (ics). The ultrathin sec-tion was stained with TMBZ and H2O2. (×17500)

Figure 9. Electron micrograph showing the histochemical local-ization of POD activity in Metroxylon sagu (3-4 months old) pith stained with TMBZ in the presence of H2O2. Heavy peroxidase reaction product is seen in the peroxisomes (pe) and to some ex-tent in the cytoplasm (cy) and the vacuole (v). (×6750)

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1991; Vamos- Vigyazo, 1981). However no studies of POD activity has been carried out at the ultrastructural level. The reason for this shortage in documentation is that PPO and POD oxidize a wide range of similar phenolic compounds and are very difficult to differentiate (Underhill and Critchly, 1995). POD activity was examined using histochemical techniques employing H2O2 and tetramethyl-benzidine (TMBZ) as substrate and viewed under TEM. Dense particles are associated with the cell walls (w) and to a little extent the cytoplasm (cy) and vacuoles (v). While the intercellular space (ics) is free from any POD reaction deposit (Figure 8). However Gasper et al. (1985) claim that the final location of plant peroxidases is likely to be the cell wall, intracellular space and the vacuoles. While in tomato fruit peroxidase is localized in the inner surface of the tonoplast and the cell wall, no reaction product was found in the cytoplasm (Thomas et al. 1981). Robinson (1991) reported that POD activity was visualized in the cell wall, tonoplast and the plasmalema of spinach (Spinacia oleracea). Dense particles of POD reaction are associated with the peroxisome (Figure 9). The peroxisomes are microbodies well known to be rich in oxidative enzymes (Lewis, 1992). According to Rastogi (1988), the enzymes present in the peroxisomes are related to the metabolism of H2O2. The scavenging of H2O2 in cellular compartments such as the microsomes relies on different peroxidases, which need a reducing substrate for their activity (Leonadis et al. 2000). However, in young sycamore leaves and adult carnation leaves, a reaction product with 4-methylcatechol staining has been observed in the peroxisomes of the foliar cells (Czaninski and Catesson, 1974).

cOncLuSIOn

The results revealed that intense PPO and POD activity was observed in the cell walls of parenchyma and xylem cells of mature Metroxylon sagu when visualized under light microscope. At the ultrastructural level, tissues from a young tree exhibited PPO activity in the amyloplast and mitochondria inner membrane and to some extent the golgi complex and endoplasmic reticulum. The localization of PPO activity in the amyloplast and being adsorbed by the starch grain is in line with the general view that the enzyme is involved in the browning of sago starch. The results suggest that, the enzyme was synthesized and differentially translocated from the cellular organelles to the cell wall during maturation of Metroxylon sagu. The procedure used here to differentiate PPO from POD activities was based on their different substrates. A positive POD reaction product when stained with TMBZ and H2O2 was visualized in the cell wall, peroxisomes and to some extent in the cytoplasm and the vacuole. It is possible to assume that the localization of PPO and POD activity in the cytoplasm

of the pith from young Metroxylon sagu indicate their present in a soluble form but at a low intensity. Knowing the location of PPO and POD in the cell wall and cellular organelles is a first step towards developing a strategy for isolating these membrane-bound enzymes.

AcKnOWLEdGMEnt

We would like to extend our gratitude to Ministry of Science and Environment, Malaysia, for their financial assistance under IRPA grant No. 01-02-04-0229, which was awarded to Assoc. Prof. Dr. Nazamid Saari.

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