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- MATERIALS AND METHODS
Mastocarpus stellatus and Chondrus crispus are morphologically similar red seaweeds that co-occur on rocky intertidal seashores in the Northern Atlantic. Mastocarpus stellatus grows higher on the shore and is more tolerant of environmental stress, caused by factors such as freezing and desiccation, than C. crispus. Here we report a correlation between reactive oxygen metabolism and stress tolerance, which suggests that reactive oxygen metabolism may play a role in stress tolerance of intertidal red seaweeds. Mastocarpus stellatus scavenged added H2O2 slightly faster, and was more resistant to oxidative stress induced by addition of H2O2 and Rose Bengal, than C. crispus. These data were consistent with higher levels of ascorbate and β-carotene and higher activities of catalase and glutathione reductase, in M. stellatus. Tocopherol content and activities of superoxide dismutase and ascorbate peroxidase were similar in both species. Activities of reactive oxygen scavenging enzymes generally increased with tidal height in M. stellatus; this was, however, not a consistent trend in C. crispus.
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- MATERIALS AND METHODS
A common denominator of many types of stress afflicting photosynthetic organisms, including freezing, high light, temperature, ozone and desiccation stress, is the increased formation of reactive oxygen ( McKersie & Leshem 1994). Reactive oxygen includes a number of compounds; these differ in reactivity, but all are more reactive than ground state oxygen. Examples of reactive oxygen are singlet oxygen (1O2), superoxide ions (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (OH·). Formation of these compounds is a normal part of the metabolism of plants and animals but excessive production can cause damage to DNA, proteins and lipids ( Halliwell & Gutteridge 1989). Reactive oxygen metabolism in plants has been reviewed by Alscher, Donahue & Cramer (1997), McKersie & Leshem (1994) and Asada & Takahashi (1987).
The defence system against reactive oxygen in plants includes anti-oxidants such as ascorbate, glutathione, β-carotene and α-tocopherol and reactive oxygen scavenging enzymes, such as catalase (EC 1·11·1·6), superoxide dismutase (SOD, EC 1·15·1·1), glutathione reductase (GR, EC 1·6·4·2) and ascorbate peroxidase (APX, EC 1·11·1·11). These mechanisms work in concert, or alone, to reduce the levels of reactive oxygen in the cells. Generally, stress-tolerant plants have a more effective defence system against reactive oxygen than stress-susceptible species; this may be constitutive or induced by exposure to stress. Higher content of anti-oxidants, and increased activities of reactive oxygen scavenging enzymes, have been documented after stress, or correlated with stress tolerance, in, for example, the dinoflagellate Peridinium gatunense ( Butow, Wynne & Tel-Or 1994), brown algae of the genus Fucus ( Collén & Davison 1999a, b), spinach (Spinacia oleracea) ( Schöner & Krause 1990), alfalfa (Medicago sativa) ( McKersie et al. 1993 ), maize (Zea mays) ( Pastori & Trippi 1993), tobacco (Nicotiana tabacum) ( Sen Gupta et al. 1993 ), cotton cells (Gossypium hirsutum) ( Gossett et al. 1996 ), and pea leaves (Pisum sativum) ( Donahue et al. 1997 ). In alpine environments, where light, ultraviolet and freezing stress increase with altitude, so do levels of anti-oxidants in alpine herbs and trees ( Bermadinger-Stabentheiner 1996; Wildi & Lütz 1996). Overall, these findings suggest that more effective reactive oxygen metabolism may increase stress tolerance.
Intertidal seaweeds experience drastic environmental changes on a daily basis. During low tide they may be exposed to desiccation, hyper- or hypo-osmotic shock, high or freezing temperatures or high light, depending on season and latitude. The frequency and duration of these stresses increases with tidal elevation. The zonation patterns typical of rocky intertidal seashores, with different species occupying different levels, sometimes only centimetres apart, can partially be explained by differences in stress tolerance ( Davison & Pearson 1996). Generally, stress tolerance against, for example, high light, freezing and desiccation increases with tidal elevation from stress-susceptible low-shore species to stress-tolerant high-shore forms. The mechanisms of environmental tolerance in intertidal seaweeds are not thoroughly known, but it has been suggested that reactive oxygen metabolism may play a key role ( Davison & Pearson 1996; Collén & Davison 1999a, b).
We previously found a correlation between stress tolerance and reactive oxygen metabolism in three species of the brown algal genus Fucus ( Collén & Davison 1999a, b). The least stress-tolerant species, F. distichus, which occupies tidal pools and thereby inhabits a less stressful environment, generally had the lowest activities of reactive oxygen scavenging enzymes and the lowest content of anti-oxidants and showed a drastic increase of reactive oxygen production after exposure to desiccation or freezing stress. The most stress-tolerant species, F. spiralis, which lives highest on the shore, had higher activities of SOD and APX than F. evanescens, which lives in the lower to middle intertidal. Here we describe a series of experiments designed to determine whether the stress tolerance of red algae could also be explained by reactive oxygen metabolism.
Mastocarpus stellatus Stackhouse and Chondrus crispus (Stackhouse) Guiry are two morphologically similar intertidal red macroalgae, belonging to the order Gigartinales, that occur on the western and eastern coasts of the northern to middle Atlantic ( Guiry & West 1983). At the site of collection and at many other places along the Maine coast, C. crispus occurs in the low intertidal, with M. stellatus occurring higher on the shore ( Dudgeon, Davison & Vadas 1989). Between the pure stands of two species is a mixed zone where both species occur at the same tidal elevation. Mastocarpus stellatus is more desiccation- and freezing- tolerant than C. crispus ( Davison, Dudgeon & Ruan 1989; Dudgeon, Davison & Vadas 1989, 1990, Dudgeon et al. 1995 ). However, C. crispus has the ability to acclimatize to freezing stress; photosynthesis of C. crispus subjected to moderate repeated freezing was more tolerant to freezing than control plants ( Dudgeon et al. 1990 ). This acclimatization to freezing stress was not found in M. stellatus. We wanted to determine whether part of the differences in stress tolerance between the two species could be explained by differences in reactive oxygen metabolism.
Here we test three hypotheses.
(1) M. stellatus is better able to tolerate reactive oxygen than C. crispus, which could partly explain the higher stress tolerance of M. stellatus.
(2) Mastocarpus stellatus scavenges reactive oxygen more efficiently than C. crispus, with higher contents of anti-oxidants and/or higher activities of reactive oxygen scavenging enzymes.
(3) Chondrus crispus, but not M. stellatus, exhibits changes in reactive oxygen metabolism with tidal height, caused by its ability to acclimatize.