Ian R. Davison

STRESS TOLERANCE IN INTERTIDAL SEAWEEDS

Intertidal seaweeds are periodically exposed to air where they experience a variety of potentially stressful environmental conditions, including nutrient limitation, high light, high and low temperature, desiccation, and osmotic stress. This paper considers the current understanding of stress tolerance in intertidal seaweeds and discusses ways in which future research could increase our understanding of the role of environmental factors in the ecology and physiology of these algae. We believe research is required in at least three areas. 1) Laboratory physiological studies have established that correlations exist between stress tolerance and the vertical distribution of species. However, little information is available on the importance of stress in determining community structure in nature. Field experiments are essential to relate the impact of single or multiple stresses on the survival, growth, and reproductive output of macroalgae. In paticular, it is necessary to clarify the role of sublethal stress in determining the outcome of competitive interactions. 2) With the exception of obvious lethal effects or damage associated with extreme environmental conditions, such as unusually hot and dry weather, it is difficult to evaluate the occurrence and severity of stress in natural populations of seaweeds. There is a need to develop molecular and biochemical markers specific for individual stresses or groups of stresses to allow the unambiguous and direct determination of stress in situ. 3) Despite the apparent importance of stress in intertidal seaweeds, we are largely ignorant of the mechanistic basis of tolerance. The application of currently available tools of molecular and cell biology to the investigation of stress‐induced transcriptional and translational changes could enormously increase our understanding of both the sites of, and pathways involved in, stress tolerance. In summary, there are numerous unanswered fundamental questions about the stress tolerance of intertidal seaweeds, providing opportunities for research ranging from field ecology to molecular biology and biochemistry.

ADAPTATION OF PHOTOSYNTHESIS IN LAMINARIA SACCHARINA (PHAEOPHYTA) TO CHANGES IN GROWTH TEMPERATURE

The effect of growth temperature on photosynthetic metabolism was studied in the kelp Laminaria saccharina (L.) Lamour. Photosynthesis was subject to phenotypic adaptation, with almost constant photosynthetic rates being achieved at growth temperatures between 0 and 20° C. This response involved: (1) an inverse relationship between growth temperature and photosynthetic capacity, (2) a reduction in the Q10 value for photosynthesis of L. saccharina grown at 0 and 5° C compared with 10, 15 and 20° C grown sporophytes, and (3) an acquired tolerance of photosynthesis to temperatures between 15–25° C (which inhibited photosynthesis in 0 and 5° C grown L. saccharina) in sporophytes grown at 10, 15 and 20° C. The physiological basis of these adaptations is discussed in terms of observed changes in activities and kinetics of the Calvin cycle enzyme ribulose‐1, 5‐bisphosphate carboxylase (oxygenase) and efficiency of light harvesting‐electron transport systems.

Temperature acclimation of respiration and photosynthesis in the brown algaLaminaria saccharina

Sporophytes of the brown algaLaminaria saccharina (L.) Lamour grown at 15°C contained significantly more chlorophylla (chla) than did similar plants grown at 5°C. The increase in chla in 15°C plants was due to increased numbers of photosystem II reaction centes, and possibly to increased photosynthetic unit size, compared with 5°C plants. These changes were associated with increasedα values (photosynthetic efficiencies) in 15°C-grownL. saccharina relative to 5°C-grown plants. The changes inα together with reduced respiration rates allowed 15°C-grownL. saccharina to achieve net photosynthesis and light-saturated photosynthesis at a lower photon fluence rate (PFR) than 5°C plants when both groups were assayed at the same temperature (15°C). The photon fluence rates necessary to reach the compensation point and achieve light-saturated photosynthesis (Ic andIk, respectively) increased with increasing incubation temperature inL. saccharina grown at both 5 and 15°C. However, acclimation responses to growth temperature compensated for the short-term effect of temperature onIc andIk. Consequently, plants grown at 5 and 15°C were able to achieve similar rates of light-limited photosynthesis, and similarIc andIk values at their respective growth temperatures. These responses are undoubtedly important for perennial seaweeds such asL. saccharina, which frequently grow in light-limited habitats and experience pronounced seasonal changes in water temperature.

SEASONALITY AND THERMAL ACCLIMATION OF REACTIVE OXYGEN METABOLISM IN FUCUS VESICULOSUS (PHAEOPHYCEAE)

The intertidal brown macroalga Fucus vesiculosus L. acclimates its defense against reactive oxygen in response to both (1) growth at different temperatures in laboratory culture and (2) seasonal changes in environmental conditions. Fucus vesiculosus was grown in seawater at 0° C, 20° C, and at 0° C with a 3‐h daily emersion at −10° C. Algae grown at low temperature, both with and without freezing, produced less reactive oxygen after severe freezing stress than those grown at 20° C. These differences were correlated with growth temperature‐induced changes in activities of superoxide dismutase (SOD), glutathione reductase, and ascorbate peroxidase. The contents of tocopherols increased with increased cultivation temperature, whereas the activity of catalase and the content of glutathione and ascorbate did not change. Growth at 0° C increased the resistance of photosynthesis to freezing and reduced photoinhibition in high light at 5° C; the latter effect was further increased in algae subject to daily freezing. These data suggest that elevated activity of reactive oxygen scavenging enzymes, especially SOD, increases the resistance to photoinhibition, at least at low temperature, as well as being important for freezing tolerance. Seasonal changes in reactive oxygen metabolism showed a similar pattern to those elicited by temperature in laboratory culture. Summer samples had lower activities of most reactive oxygen scavenging enzymes than algae collected in autumn and winter when water temperatures were lower. In contrast to the laboratory experiments, ascorbate content did change and was lower during the winter than summer, whereas the content of glutathione was not influenced by season. Overall, the data not only indicate that temperature plays an important role in the regulation of stress tolerance and reactive oxygen metabolism but also suggest that other factors are also involved.

REACTIVE OXYGEN PRODUCTION AND DAMAGE IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)

The research described in this paper was designed to test the hypothesis that the differential stress tolerance associated with the vertical zonation of intertidal seaweeds is attributable to reactive oxygen metabolism. To do so, we measured the production of, and damage caused by, reactive oxygen in three species of intertidal brown seaweeds—Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.— that differ in their ability to withstand freezing, desiccation, and high light stress. Fucus spiralis is the most stress‐tolerant species and F. distichus the least. Reactive oxygen production was determined by measuring the production of H2O2 and the oxidation of dichlorohydrofluorescein diacetate to dichlorofluorescein. Damage caused by freezing, desiccation, and high‐light stress was assessed by measuring variable fluorescence (Fv/Fm) and lipid peroxidation. Production of reactive oxygen increased following freezing, desiccation, or high‐light stress. In general, the data were consistent with the hypothesis that reactive oxygen metabolism is involved in stress tolerance. The production of reactive oxygen was relatively low in unstressed seaweeds, and there was little difference between species. Fucus distichus showed the greatest increase in reactive oxygen production after desiccation and freezing stress. Fucus evanescens produced more reactive oxygen production after desiccation than F. spiralis. Although F. evanescens and F. spiralis produced similar amounts of reactive oxygen after freezing, this treatment resulted in an increase in lipid peroxidation only in F. evanescens (and F. distichus).

REACTIVE OXYGEN METABOLISM IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)

Our previous research suggests that interspecific variation in stress tolerance in intertidal Fucus spp. (Phaeophyceae) is partially mediated by differences in the production of, or ability to detoxify, reactive oxygen. Here we report on the content of antioxidants (ascorbate, glutathione, carotenoids, and tocopherols) and protective enzymes (catalase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase) involved in reactive oxygen metabolism in three species of intertidal brown algae—Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.—that differ in stress tolerance and position in the intertidal zone. Contents of the major antioxidants were similar in the three species and were not correlated with stress tolerance. The least stress tolerant species, F. distichus, had the lowest activity of reactive‐oxygen‐scavenging enzymes, although F. spiralis, the species with the highest stress tolerance, and F. evanescens contained similar activities of antioxidant enzymes on a fresh‐weight basis. However, the activities of superoxide dismutase and ascorbate peroxidase in F. evanescens are lower than those of F. spiralis when expressed on the basis of chlorophyll. These data show that the ratio between reactive oxygen protection and production might be more important than the absolute content of antioxidants and protective enzymes. It also shows the importance of localization of detoxifying mechanisms and avoidance of oxidative stress.

In vivo measurement of active oxygen production in the brown alga Fucus evanescens using 2',7'-dichlorohydrofluorescein diacetate.

Intracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′‐dichlorohydrofluorescein diacetate (DCFH‐DA) to the fluorescent dye 2′,7′‐dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were incubated in seawater containing DCFH‐DA under a range of conditions, and it was also possible to load algae with DCFH‐DA and then follow subsequent DCF production in emersed tissue. DCF formation was linear for at least 2 h in both darkness and light, with the rate of formation increasing with the light level. DCF formation was temperature dependent. It also increased when algae were treated with H2O2 or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O−2 production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentrations of H2O2. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH‐DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds subjected to environmental stresses.

The effect of growth temperature on enzyme activities in the brown alga Laminaria saccharina

The effect of changes in growth temperature on the extractable activities of several key enzymes of carbon and nitrogen metabolism was studied in the brown alga Laminaria saccharina. Standard enzyme activities (measured at 20°C) were found to respond in one of three ways, being (i) unaffected by growth temperature (aspartate: 2-oxyglutarate aminotransferase, malate dehydrogenase, mannitol-1-phosphate dehydrogenase and phosphoenol-pyruvate carboxykinase), (ii) inversely proportional to growth temperature (glyceraldehyde-3-phosphate dehydrogenase (NAD- and NADP-dependent) and ribulose-1,5-bisphosphate carboxylase) or (iii) following changes in growth rate which was maximal at 10°C (glutamine synthetase and nitrate reductase). The photosynthetic capacity of L. saccharina sporophytes responded in a manner similar to the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (NADP-dependent) and ribulose-1,5-bisphosphate carboxylase with standard photosynthetic rates (measured at 15°C) being inversely p...

COEXISTENCE OF SIMILAR SPECIES IN A SPACE-LIMITED INTERTIDAL ZONE

The lower intertidal zone (0.0 to +1.0 m mean low water [MLW]) of rocky shores in New England is a space-limited community occupied by two similar rhodophyte seaweeds, Chondrus crispus and Mastocarpus stellatus, that overlap broadly in their use of three essential resources: space, light, and nutrients. C. crispus coexists primarily with the prostrate-crust generation of M. stellatus lower on the shore (less than +0.35 m MLW) and with the alternate upright-frond generation higher on the shore (greater than +0.35 m MLW). Our objectives were to determine (1) whether these two species compete and (2) if so, what process(es) enable their coexistence. Upright fronds of M. stellatus transplanted to the lowest intertidal zone (less than +0.25 m MLW) where C. crispus predominates grew faster and showed higher survivorship after 2 yr than those transplanted to areas where M. stellatus predominates. However, the failure of upright fronds of M. stellatus to consistently recruit limits their abundance in the lowest z...

PHOSPHORUS AND NITROGEN NUTRITION IN CHONDRUS CRISPUS (RHODOPHYTA): EFFECTS ON TOTAL PHOSPHORUS AND NITROGEN CONTENT, CARRAGEENAN PRODUCTION, AND PHOTOSYNTHETIC PIGMENTS AND METABOLISM1

The existence of a phenomenon in phosphorus (P) nutrition comparable to the “Neish effect” in nitrogen (N) nutrition (an inverse relation between seawater N enrichment and carrageenan content) was investigated in the temperate red alga Chondrus crispus Stackhouse. Plants were preconditioned for 17 d and then cultured under varying enrichments of P (0, 3, 6, 10, 15 μM P·wk−1) and a constant N enrichment (53.5 μM N·wk−1) for 5 wk. Tissue total P, tissue total N, and carrageenan contents were then determined. Identical experiments were performed using C. crispus collected during the fall, winter, spring, and summer seasons. The procedure was repeated using material collected during the following fall season and cultured under constant P (6 μM P·wk−1) and varying N enrichments (0, 3, 6, 10, 25 μM N·wk−1). In the fall (P) experiment, carrageenan content was the highest [53.1 ± 0.3% DW (dry weight)], and tissue total P content was the lowest (1.71 ± 0.27 mg P·g DW−1) in plants that received no P enrichment. Carrageenan content was stable (46.1 ± 1.8% DW) for plants given enrichments of 3 μM P·wk−1 and greater. Thus, a decrease in carrageenan content, concomitant with an increase in tissue total P content, was observed, but only at tissue total P levels below 2 mg P·g DW−1. As these levels were always higher than 2 mg P·g DW−1 in the winter, spring, and summer experiments, carrageenan content remained constant within each season at 46.2 ± 1.3, 43.1 m 0.7, and 44.5 ± 0.6% DW, respectively. Nitrogen enrichment of plants collected in the fall did not affect carrageenan content, which was stable at 49.3 ± 0.9% DW. When these plants were compared with those of the previous fall experiment (6 μM P·wk−1 and 53.5 μM N·wk−1), a slight increase in carrageenan content was noted. Thus, at sufficiently high concentration, N also decreased carrageenan content in C. crispus. Phosphorus nutrition had no significant effect on photosynthesis versus irradiance parameters (Pmax, α, Rd, Ic, and Ik), the contents of the photosynthetic pigments chlorophyll‐a, phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC), and the ratios PE:APC and PC:APC. In contrast, N nutrition affected both Pmaxand the photosynthetic pigment contents. The data indicate that N limitation reduces the number of phycobilisomes but not their size. The greater reduction in phycobiliprotein than chlorophyll‐acontent corroborates the natural bleaching phenomenon regularly observed in C. crispus populations during summer when N levels are generally low in seawater. These results suggest that C. crispus in the temperate waters of the Bay of Fundy may experience N limitation, but P limitation is unlikely.