Naphthoquinones as allelochemical triggers of programmed cell death

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Abstract

Juglone and plumbagin are plant bioactive derivatives of 1,4-naphthoquinone occurring in plants, whereas lots of these plants belong to invasive species. Clarifying of action of juglone and plumbagin applied on plant cell model represented by tobacco BY-2 cells was the basic aim of this work. It was shown that naphthoquinones are able to induce various structural, functional and enzymatic changes leading to processes of apoptic-like cell death. Using dihydroethidium as fluorescent probe the mechanism of naphthoquinones action was explained. They are able to generate reactive oxygen species, which play important role in processes of programmed cell death. Disruption of mitochondrial respiratory chain was detected too. This study shown that mechanism of naphthoquinones action to plant cells is very complex and predestine them to be very effective compounds in plant competition fight.

Introduction

The term alellopathy denotes the production of specific biomolecules (alellochemicals) by one plant that can induce suffering in, or give benefit to, another plant. However, it is most commonly used in the former sense—an interaction in which one plant causes suffering to another plant. There are several ways in which an alellopathic plant can release its protective chemicals: (a) volatilization—alellochemicals can release specific compounds in the form of gas through stomata in their leaves, (b) exudation—beneficial chemicals are released into the soil through roots, (c) leaching—protective chemicals are stored in the leaves after their dropping they decompose and release chemicals.

The allelopathic events are usually caused by secondary metabolites. There are hundreds of them in the plant kingdom (Einhellig, 1995, Barkosky and Einhellig, 2003). The compounds exhibit a wide range of mechanisms of action, from affects on DNA (alkaloids), phytohormone activity, ion uptake, and water balance (phenolics) as well as photosynthetic and mitochondrial function (quinones). Interpretations of mechanisms of action are complicated by the fact that individual compounds can have multiple phytotoxic effects (Einhellig, 1995).

One group of such allelophatic compounds are naphthoquinones, oxygen-derivatives of naphthalene. They are widespread in nature as secondary metabolites of micro-organisms and fungi as well as plants. They have been detected in many vascular plant families (cf. Droseraceae, Juglandaceae, Nepenthaceae and Plumbaginaceae (Binder et al., 1989, Crouch et al., 1990, Lin et al., 2003). The most widespread naphthoquinones originated from shikimic acid are juglone (5-hydroxy-1,4-naphthoquinone, amber-colored compound isolated from numerous Juglans spp.) and plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, orange-colored compound isolated from Plumbago, Drosera, Dionaea and Nepenthes). The interest of many investigators in these compounds is due to their broad-range of activities as antibacterial, fungicidal, antiparasitic and insecticidal effects (Babula et al., 2009, Lee and Lee, 2008). They have inhibitory effect on insect larval development and sedative or toxic effect on fish and animals (Higa et al., 1998) besides that they have also cytostatic and anticarcinogenic properties. These properties emerge due to their ability to act as potent inhibitors of electron transport (Vennerstrom and Eaton, 1988), as uncouplers of oxidative phosphorylation (Ferraz et al., 2001), as intercalating agents in the DNA double helix, as bioreductive alkylating agents of biomolecules, and as producers of reactive oxygen radicals by redox cycling under aerobic conditions (Monks et al., 1992).

Plumbagin and especially juglone are widely studied for their allelopathic activities. Massey (1925) hypothesized that inhibitory effect of black walnut (Juglans nigra L.) on growth of some associated species is caused by a compound exuded by roots (Massey, 1925) and this compound was determined by Davis (1928) as juglone (Davis, 1928). Juglone is stored in plant tissues in vacuoles as hydrojuglone-β-d-glucopyranoside, which can be decomposed by enzyme hydrojuglone-β-d-glucopyranosid-β-glucosidase to juglone (Duroux et al., 1998). Juglone, as well as plumbagin, is able to be released to soil and surrounding trough necrotic plant tissues decomposition, may persist in the soil for weeks and months and can be attributed to alellopathy. Growth and germination inhibition at micromolar concentration has been reported for numerous plant species (Marion Meyer et al., 2007, Terzi, 2008, Topal et al., 2007). Mechanisms of plant growth depression by juglone bases in reducing of H+-ATP-ase activity, which was shown at corn and soybean root microsomal fraction treated with juglone in concentration from 0 to 1000 μM. Other effects are inhibition of p-hydroxyphenylpyruvate dioxygenase, the crucial enzyme of plastoquinone synthesis, decrement photosynthesis in leaf tissues and transpiration and stomatal conductance inhibition (Hejl and Koster, 2004). Important fact is ability of naphthoquinones to generate active oxygen species that may play a critical role in the protection of plants against pathogens. This work was aimed on investigating the way of cell death observed in cultures of tobacco BY-2 cells exposed to various doses of juglone as well as plumbagin.

Section snippets

Plant material

Nicotina tabacum L. cv. Bright Yellow-2 suspension-cultured cells (BY-2) were grown in liquid medium according Murashige and Skoog (1962), modified by Nagata et al. (1992) with constant shaking (Kühner Shaker LT-W, Adolf Kühner AG, Switzerland, 130 rpm) at 27 °C in the dark in 250 ml Erlenmeyer flasks.

Cells in exponential growth phase were exposed to naphthoquinones plumbagin and juglone for various times under culture conditions. Naphthoquinones were added to the cell suspension at final

Effect of naphthoquinones on growth and viability of tobacco BY-2 cells

As it is shown in Fig. 1, the exposure of tobacco BY-2 cell to naphthoquinones plumbagin and juglone for 0–240 h resulted in a dose-dependent growth deceleration and cell viability reduction compared with control cells.

The presence of both naphthoquinones decreased cell number in culture at concentration dependent manner. The plumbagin had a stronger influence in all concentration than juglone except the highest used concentrations (50 and 500 μg l−1). The maximum of the cell number was recorded 48

Discussion

Two studied naphthoquinones in this work, juglone and plumbagin, play important role in ecological interactions as protective compounds. They can protect plants against insectual pests (herbivores) as well as fungal pathogens. However, what functions do naphthoquinones have in the relations between plants, respectively? Necrotic plant tissues can release naphthoquinones to the surroundings and than the naphthoquinones can affect other plants. Previous essays demonstrated that naphthoquinones,

Conclusion

Naphthoquinones, the natural occurring phenolic compounds derived from naphthalene, were tested for cytotoxic properties using plant cell suspension culture of tobacco BY-2 as. Our results demonstrate that cytotoxic effect of naphthoquinones plumbagin and juglone is caused by their ability to generate reactive oxygen species (ROS) as well as DNA damage and disturbance of mitosis. All these plumbagin and juglone features lead to programmed cell death that was observable as chromatine

Acknowledgment

This work was supported by grant 1M06030.

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