Félix L. Figueroa

Photoecophysiology of Marine Macroalgae

Aquatic ecosystems are responsible for about half of the primary biomass production on earth and incorporate about 100 gigatons of atmospheric carbon per year (1). Because less than 0.5% of the water surface area is represented by freshwater systems, marine ecosystems have by far the largest share in productivity. Most carbon fixation is due to phytoplankton, but even though most macroalgae are restricted to coastal areas and the continental shelves they are responsible for a considerable proportion of the biomass productivity in the oceans (2). They play a role as primary producers being at the basis of an intricate food web and are of significant economic importance because several hundred thousand tons of seaweeds are harvested every year for food production and technological exploitation. While phytoplankton can move up and down in the water column (3), most macroalgae are sessile and thus restricted to their growth site (4). There they have to cope with the changing irradiance regime of solar radiation. In addition, being adapted to lower irradiances than those of unfiltered solar radiation, macroalgae face a serious light stress when exposed to higher irradiances (3). Consequently, macroalgae, as well as higher plants, have developed mechanisms to regulate their photosynthetic activity to operate under limited light supply and to protect themselves against excessive radiation (5,6). The mechanism of photoinhibition decreases the photosynthetic activity during periods of bright sunshine. This phenomenon is a reversible interruption of the photosynthetic electron transport chain (7) and facilitates thermal dissipation of excessive irradiation (8). Even higher stress will cause photodamage that is not as readily reversible as photoinhibition. Species of different algal groups show a distinctly different behavior upon irradiation (9-14), and especially the ability to cope with enhanced UV radiation varies widely among species (15-17). Therefore a broad survey is necessary to understand photosynthesis in aquatic ecosystems and its interaction with solar radiation. Typical green macroalgae

An inventory of UV-absorbing mycosporine-like amino acids in macroalgae from polar to warm-temperate regions

A qualitative and quantitative survey of 11 green, 27 brown and 30 red macroalgal species collected from polar (Spitsbergen), cold-temperate (Helgoland) and warm-temperate (southern Spain) regions revealed that all Rhodophyceae from the eulittoral zone contained several UV-absorbing mycosporine-like amino acid compounds (MAAs), which are assumed to function as natural UV-sunscreens. In contrast, deep-water red algae, as well as the Chlorophyceae and Phaeophyceae did not contain MAAs or exhibited only trace concentrations. Within all species investigated 9 distinct compounds were found, of which 7 were identified as mycosporine-glycine, shinorine, porphyra-334, palythine, asterina-330, palythinol and palythene. The remaining two substances are unknown; they had different retention times under the Chromatographie conditions used, but identical absorption spectra with a maximum at 357 nm. Both compounds are restricted to a few polar red algae. In Polysiphonia arctica J. Agardh (Rhodophyta) from Spitsbergen the concentrations of MAAs decreased from depths of 1 to 7 m. Shallow-water isolates contained > 5-fold higher total MAA amounts compared to deep-water samples. In addition, the contents of all MAAs correlated with the biogeographic region indicating that the lower the natural solar irradiance in the respective habitat the less MAAs are synthesised and accumulated. The red algal samples from Spain exhibited up to 2-fold higher MAA contents compared to species from cold-temperate and polar waters supporting the idea of a UV-dose-dependent induction and/or concentration. The data support the idea that MAAs represent a least for red macroalgae a natural defense system against exposure to biologically harmful UV-radiation.

Antioxidant activity of mycosporine-like amino acids isolated from three red macroalgae and one marine lichen

Several standard in vitro assays were performed in order to determine the potential antioxidant capabilities of purified aqueous extracts of the mycosporine-like amino acids (MAAs), porphyra-334 plus shinorine (P-334 + SH), isolated from the red alga Porphyra rosengurttii, asterina-330 plus palythine (AS-330 + PNE), from the red alga Gelidium corneum, shinorine (SH), from the red alga Ahnfeltiopsis devoniensis, and mycosporine -glycine (M-Gly), isolated from the marine lichen Lichina pygmaea. The scavenging potential of hydrosoluble radicals (ABTS+ decolorization method), the antioxidant activity in lipid medium (β-carotene/ linoleate bleaching method) and the scavenging capacity of superoxide radicals (pyrogallol autooxidation assay) were evaluated. In terms of scavenging of hydrosoluble radicals, the antioxidant activity of all MAAs studied was dose-dependent and it increased with the alkalinity of the medium (pH 6 to 8.5). M-Gly presented the highest activity in all pH tested; at pH 8.5 its IC50 was 8-fold that of L-ascorbic acid (L-ASC) followed by AS-330 + PNE while P-334 + SH and SH showed scarce activity of scavenging of hydrosoluble free radicals. AS-330 + PNE showed high activity for inhibition of β-carotene oxidation relative to vitamin E and superoxide radical scavenging whilst the activity of P-334 +SH and SH were moderate. According to these results, the potential of MAAs in photoprotection can be considered high due to a double function: (1) UV chemical screening with high efficiency for UVB and UVA regions of the solar spectrum, and (2) their antioxidant capacity.

AMMONIUM AND UV RADIATION STIMULATE THE ACCUMULATION OF MYCOSPORINE-LIKE AMINO ACIDS IN PORPHYRA COLUMBINA (RHODOPHYTA) FROM PATAGONIA, ARGENTINA1

The combined effects of ammonium concentration and UV radiation on the red alga Porphyra columbina (Montagne) from the Patagonian coast (Chubut, Argentina) was determined using short‐term (less than a week) experimentation. Discs of P. columbina were incubated with three ammonium concentrations (0, 50, and 300 μM NH4Cl) in anilluminated chamber (PAR=300 μmol photons·m−2·s−1, UVA=15 W·m−2, UVB=0.7 W·m−2) at 15°C. Algae incubated at 300 μM ammonium showed a significant increase (P<0.05) in the concentration of mycosporine‐like amino acids (MAAs) compared with the initial value or with the other ammonium treatments. The increase of MAAs was, however, a function of the quality of irradiance received, with a higher increase in samples exposed to UVA compared with UVB (29% and 5% increase, respectively). However, UVB radiation was more effective in inducing MAA synthesis per unit energy received by the algae. Samples exposed to PAR only had an intermediate increase in MAA concentration of 16%. Chl a concentration decreased through the incubation with the greatest decrease at high ammonium concentration. Phycobiliprotein (BP) decreased through time with the smallest decrease occurring at high ammonium concentration. Photoinhibition (as a decrease of optimal quantum yield) was significantly greater under nitrogen‐deprived conditions than that under replete ammonium levels. Maximal gross photosynthesis (GPmax), as oxygen evolution, and maximal electron transport rate (ETRmax), as chl fluorescence, increased with the ammonium concentration. Positive relationships between maximal GP or ETR and pigment ratios (BP/chl a and MAAs/chl a) and negative relationships with chl a concentration were found.

Non-photosynthetic enhancement of growth by high CO2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)

Abstract. The effects of increased CO2 levels (10,000 μl l−1) in cultures of the green nitrophilic macroalga Ulva rigida C. Agardh were tested under conditions of N saturation and N limitation, using nitrate as the only N source. Enrichment with CO2 enhanced growth, while net photosynthesis, gross photosynthesis, dark respiration rates and soluble protein content decreased. The internal C pool remained constant at high CO2, while the assimilated C that was released to the external medium was less than half the values obtained under ambient CO2 levels. This higher retention of C provided the source for extra biomass production under N saturation. In N-sufficient thalli, nitrate-uptake rate and the activity of nitrate reductase (EC 1.6.6.1) increased under high CO2 levels. This did not affect the N content or the internal C:N balance, implying that the extra N-assimilation capacity led to the production of new biomass in proportion to C. Growth enhancement by increased level of CO2 was entirely dependent on the enhancement effect of CO2 on N-assimilation rates. The increase in nitrate reductase activity at high CO2 was not related to soluble carbohydrates or internal C. This indicates that the regulation of N assimilation by CO2 in U. rigida might involve a different pathway from that proposed for higher plants. The role of organic C release as an effective regulatory mechanism maintaining the internal C:N balance in response to different CO2 levels is discussed.

Relations between electron transport rates determined by pulse amplitude modulated chlorophyll fluorescence and oxygen evolution in macroalgae under different light conditions.

The relationship between O2-based gross photosynthesis (GP) and in vivo chlorophyll fluorescence of Photosystem II-based electron transport rate (ETR) as well as the relationship between effective quantum yield of fluorescence (ΦPSII) and quantum yield of oxygen evolution (ΦO_2) were examined in the green algae Ulva rotundata and Ulva olivascens and the red alga Porphyra leucosticta collected from the field and incubated for 3 days at 100 μmol m−2 s−1 in nutrient enriched seawater. Maximal GP was twice as high in Ulva species than that measured in P. leucosticta. In all species ETR was saturated at much higher irradiance than GP. The initial slope of ETR versus absorbed irradiance was higher than that of GP versus absorbed irradiance. Only under absorbed irradiances below saturation or at values of GP <2 μmol O2 m−2 s−1 a linear relationship was observed. In the linear phase, calculated O2 evolved /ETR molar ratios were closed to the theoretical value of 0.25 in Ulva species. In P. leucosticta, the estimated GP was associated to the estimated ETR only at high irradiances. ETR was determined under white light, red light emitting by diodes and solar radiation. In Ulva species the maximal ETR was reached under red light and solar radiation whereas in P. leucosticta the maximal ETR was reached under white light and minimal under red light. These results are in agreement with the known action spectra for photosynthesis in these species. In the case of P. leucosticta, GP and ETR were additionally determined under saturating irradiance in algae pre-incubated for one week under white light at different irradiances and at white light (100 μmol m−2 s−1) enriched with far-red light. GP and growth rate increased at a growth irradiance of 500 μmol m−2 s−1 becoming photoinhibited at higher irradiances, while ETR increased when algae were exposed to the highest growth irradiance applied (2000 μmol m−2 s−1). The calculated O2 evolved /ETR molar ratios were close to the theoretical value of 0.25 when algae were pre-incubated under 500–1000 μmol m−2 s−1. The enrichment by FR light provoked a decrease in both GP and ETR and an increase of nonphotochemical quenching although the irradiance of PAR was maintained at a constant level. In addition to C assimilation, other electron sinks, such as nitrogen assimilation, affected the GP–ETR relationship. The slopes of GP versus ETR or ΦPSIIversus ΦO_2 were lower in the algae with the highest N assimilation capacity, estimated as nitrate reductase activity and internal nitrogen contents, i.e., Ulva rotundata and Porphyra leucosticta, than that observed in U. olivascens. The possible mechanisms to explain this discrepancy between GP and ETR are discussed.

Long-Term Effects of Natural Sunlight under Various Ultraviolet Radiation Conditions on Growth and Photosynthesis of Intertidal Ulva rigida (Chlorophyceae) Cultivated In Situ

Abstract Long-term effects of full-spectrum solar radiation, solar radiation without UVB, and solar radiation without total UV (UVA + UVB) radiation were studied in intertidal Ulva rigida C. Agardh (Chlorophyta). The experiment was carried out under natural conditions and at prevailing winter levels of UV radiation. Changes in relative growth rate, photosynthesis and pigment content were studied after 7 and 20 d of cultivation under the three solar radiation conditions. Relative growth rate was enhanced (50 %) in the absence of UVB radiation after one week, but no difference was found after 20 d. Pigment content (chlorophyll a, chlorophyll b and carotenoids) was significantly higher in the presence of UVB, suggesting an efficient protective-pigment mechanism. When UVB was removed, photosynthetic performance measured as oxygen exchange and fluorescence parameters exhibited higher values than under the other treatments after 7 d of incubation. When samples were cultivated under PAR alone, photosynthetic performance was always negatively affected compared to in the presence of UV radiation. On the whole, intertidal U. rigida exhibited a remarkable capacity to cope with fluctuating light conditions.

External carbonic anhydrase and affinity for inorganic carbon in intertidal macroalgae

Abstract Sixteen intertidal macroalga species from the Gibraltar Strait (Southern Spain) have been examined for both their external carbonic anhydrase activity (CA; 4.2.1.1.) and their affinity for inorganic carbon (C i ). Information on the ability to use HCO 3 − was obtained by measuring the effect of increasing seawater pH on the photosynthesis and by examining the O 2 evolution curves vs. C i concentration. The species exhibited a range of characteristics of C i utilisation: the green macroalgae were saturated at seawater C i concentration with K m ranging from 250.0 to 493.3 mmol m −3 and the photosynthetic O 2 production, expressed as conductance for CO 2 ranging from 432.8 to 16.1×10 −6 m s −1 . Only three out of the eleven tested red and brown macroalgae were saturated at C i concentration of seawater and for these algae HCO 3 − use was clearly indicated by the high initial slope of the O 2 evolution rate vs. C i curve. The most efficient HCO 3 − users were species from the high intertidal habitats and rockpools. Two methods were used to infer the presence of external CA: standard method (direct enzyme assay) and use of acetazolamide (AZ), a non-permeant inhibitor of CA, in assays of photosynthetic rate. Some discrepancies between the two methods were found and we obtained indications that the use of AZ could be a more sensitive method to detect external CA. It so, we suggest that external CA might be present in all the macroalgae examined. There appeared to be no correlation between the ability to use HCO 3 − and the presence of external CA. Therefore, the presence of external CA was not a strong indication for an efficient use of HCO 3 − , although some dependence between the requirement for external CA and habitats was found.

Utilization of HCO3 − at high pH by the brown macroalga Laminaria saccharina

The brown macroalga Laminaria saccharina exhibits a type of HCO3 − utilization that could be almost completely inhibited either by proton buffers or by acetazolamide, an inhibitor of extracellularly operating carbonic anhydrase. This means of HCO3 − utilization featured properties similar to direct HCO3 − uptake in that photosynthetic rates were proportional to the HCO3 − concentration of the seawater over a wide pH range (pH 7.0−9.5). Despite this, it must be characterized as carbonic anhydrase-catalysed external HCO3 − dehydration and not as direct HCO3 − uptake. A mechanism is suggested involving a CO2-concentrating capability located at the cell membrane. This mechanism, which might be common in brown algae, is suggested to have an adaptational advantage in colder regions of the sea (as compared with the direct HCO3 − uptake of green macroalgae). This means of HCO3 − utilization is inhibited even by fairly low concentrations of buffer, with consequences for the interpretation of earlier experimental studies on L. saccharina (and possibly other brown algae). These consequences relate both to ecology (e.g. determination of inorganic C affinity) and physiology (e.g. assessing mechanisms for inorganic C uptake).

Effects of different levels of CO2 on photosynthesis and cell components of the red alga Porphyra leucosticta

Photosynthesis and cell composition of Porphyraleucosticta discs grown at low (< 0.0001% in air), current (control) and high (1% CO2 in air)inorganic carbon (Ci) concentrations were analyzed. Carbohydrate content in discs grown at high Ci increased (15.1 mg g-1 FW) with respect to the control (6.4 mg g FW-1), whereas soluble protein content decreased to one-third (5.6 to2.1 mg g-1 FW). Carbohydrate content was unaffected and soluble protein slightly increased in discs grown at low Ci. As a consequence of these changes, a lower C/N molar ratio (8.6) was found in the discs grown at low compared to high Ci(12.4). Nitrate reductase activity increased at high Ci from 0.3 ± 0.2 to 1.7 ± 0.4 μmolNO2- g-1 FW h-1indicating that reduction and assimilation of nitrate were uncoupled. The response of photosynthesis to increasing irradiance, estimated from O2evolution vs. irradiance curves, was affected by the treatments. Maximum quantum yield (Φ O2°) and effective quantum yield (Φ O2) at 150 μmol photon m-2s-1 decreased by 20% and 50%, respectively, at low Ci. These differences could be due to changes in photosynthetic electron flow between PSII and PSI. Treatments also produced changes in maximal (Fv/Fm) and effective (ΔF/Fm′)quantum yield for photosystem II charge separation.