Journal of Experimental Botany, Vol. 54, No. 384, pp. 1093-1100,
March 1, 2003
© 2003 Oxford University Press
Tissular localization of coumarins in the green alga Dasycladus vermicularis (Scopoli) Krasser: a photoprotective role?
Received 21 June 2002; Accepted 5 December 2002
Universidad de Málaga, Facultad de Ciencias, Departamento de Ecología, Campus Universitario de Teatinos s/n, 29071 Málaga, Spain
1 To whom correspondence should be addressed. Fax: +34 952 13 20 00. E-mail: jaguilera{at}uma.es
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Abstract |
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Cell distribution of coumarins, a group of UV-absorbing substances, was analysed by epifluorescence optical microscopy in the green macroalga Dasycladus vermicularis. Maximal concentration of 3,6,7-trihydroxycoumarin (THC), which corresponds to almost 100% of the total coumarins in D. vermicularis, was found in the apical part of the thallus, which is more exposed to solar radiation. At a cell level, two blue, highly fluorescent layers, corresponding to a large accumulation of THC, were found in the internal part of the cell wall and around the vacuolar membrane. The percentage of UV radiation absorbed by each THC layer could be measured from the in vitro total thallus concentration of THC and histological measurements of the layers. The THC layer close to the cell wall absorbed 88% of the incident irradiance at 346 nm (corresponding to the maximum of absorbance of THC in the UVA region), while that close to the vacuole membrane absorbed 87.5%. These results agree with the hypothesis of a natural sunscreen role, significantly reducing harmful UV radiation reaching the cell. Owing to the release of this substance into the medium under different stress conditions, its capacity as a UV filter for other macroalgae has been tested. The ecological relevance of the release process of this UV-absorbing substance in specific environments is discussed.
Key words: Coumarins, Dasycladus vermicularis, epifluorescence optical microscopy, fluorescence parameters, photoprotection.
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Introduction |
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Increase of ultraviolet radiation on the earth’s surface caused by ozone depletion has been well documented in recent years, not only for polar but also for temperate regions (Smith et al., 1992; Frederick et al., 1993; Pearce, 1996). Accurate information is needed to assess the biological effects of a thinning ozone layer and the consequently increasing detrimental ultraviolet (UV) radiation on terrestrial and aquatic ecosystems. Damage in DNA, RNA, proteins, photosynthesis, and growth by excess UV are relatively well documented in a range of higher and lower plants, including phytoplankton (Tevini and Teramura, 1989; Strid et al., 1990; Karentz et al., 1991; Smith et al., 1992; Buma et al., 1995; Clendennen et al., 1996; Figueroa et al., 1997; Häder and Figueroa, 1997; Aguilera et al., 1999a, b; Bischof et al., 2000) while investigations on benthic marine macroalgae are scarce, despite the important ecological role played by this group in the marine environment. Macroalgae have to cope with significant changes in the incident solar radiation and other environmental factors. One important physiological strategy of various marine organisms inhabiting shallow waters is the prevention of UV photodamage by the accumulation of UV-absorbing substances in their cells (Dunlap et al, 1986; Karentz et al., 1991; Karsten et al., 1998). In red macroalgae, the most common potential UV sunscreens are the mycosporine-like amino acids (MAAs), an array of chemically closely related, water-soluble compounds (Karsten et al., 1998). Riegger and Robinson (1997) reported for various phytoplankton species from Antarctica that high MAAs concentration correlated with an increased, albeit not complete, resistance to UV photodamage.
On the other hand, the presence of very high concentrations of phenolic compounds in brown macroalgae is well known (Van Alstyne and Paul, 1990; Schoenwaelder and Clayton, 1998). The chemical structure of these compounds allows high absorption properties in the UV region of the light spectrum. The phlorotanins, a family of phenolic compounds in brown algae, can reach 10% of algal dried weight (Ragan and Glombitza, 1986). There is also some evidence of the accumulation of high concentrations of other phenolic compounds, namely coumarins, with high UV-absorption properties in green macroalgae like Caulerpa and Dasycladus (Kubitzki, 1987). Menzel et al. (1983) isolated and identified the main compound in this species, 3,6,7-trihydroxycoumarin (THC) as responsible for the very high absorption in the UVA region of the light spectrum.
Phenolic compounds are well known as constituents of higher plant cell walls in which they are frequently bound to matrix polymers (Fry, 1986). The accumulation of these compounds in the outer parts of the cells could have a photoprotective role against UV. Other functions of plant phenols as a protection against herbivores, alellopathic and antibacterial substances have been reported (Conover and Sieburth, 1966). The pollen wall of some higher plant species that accumulates high contents of phenolic substances have been shown to screen out more than 80% of the incident UV radiation (Demchik and Day, 1996). Phenols are predominantly located in the secondary cell walls of vascular plants, with a thickening role, and they are also thought to be biologically active constituents of the primary wall (Fry, 1986; Wallace and Fry, 1994). A number of authors indicate the presence of organelles in brown macroalgae where phenols are accumulated, called physodes, which are distributed on the medullar cell periphery and on the intercellular zone (Ragan, 1976; Schoenwaelder and Clayton, 1998). In thalli of the green Mediterranean macroalgae Dasycladus vermicularis, the high concentration of 3,6,7-trihydroxycoumarin (almost 20 mg g–1 FW) (Pérez-Rodríguez et al., 2001) seems not to be accumulated in physodes, but uniformly in the cell wall and around the vacuole membrane as shown in the present study. These findings support the hypothesis that the coumarins may have a photoprotective function in D. vermicularis as a natural UV filter.
The aim of this work is to analyse the tissular localization of THC in D. vermicularis and to calculate its ability as an in vivo sunscreen. The work also indicates the potential role of released coumarins as a photoprotective mechanism for other algae.
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Materials and methods |
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Biological material
Samples of D. vermicularis were collected in June 2000 from the upper part of the infralittoral zone (0.5 m depth) in the Natural Park of Cabo de Gata-Níjar (36° 52' N; 2° 12' W, Almería, Southern Spain). Bunches of thalli attached to the rocky substrate were removed with a spatula and transported in an ice chest in natural seawater to the laboratory. After removing sand and epibiota, the algae were transferred to an aerated aquarium containing 5 l of filtered natural seawater at 14.5±1 °C and irradiated with 150 µmol photon m–2 s–1 provided by two fluorescent lamps (Truelite 40 W, Durotest, USA) under a light regime of 12:12 h light:dark.
Pigment content
Thalli of D. vermicularis of approximately 4 cm long were divided into four parts from the basal to the apical zone and incubated in 2.5 ml of N,N dimethylformamide (DMF) for 24 h at 4 °C in darkness for chlorophyll a and coumarin extraction. Chl a concentration in the supernatant was determined spectrophotometrically according to Inskeep and Bloom (1985). Absorbance of the same supernatant was also measured at 346 nm, corresponding to the maximum absorbance of THC in the UVA region of the spectrum. THC absorbance was converted into mg g–1 DW by fitting the data set to a calibration curve generated from the coumarin standard scopoletin (Sigma-Aldrich) (Pérez-Rodríguez et al., 2001).
Histological analysis
Localization of coumarins and microscopic measurements were performed by means of a Fluovert FV (Leitz, Wetzlar, Germany) inverted microscope connected to a CCD camera (Polaroid DMC). Digitized images were analysed by means of the Image-Pro (Media Cybernetics, USA) software. Cross-sections of fresh samples of D. vermicularis were obtained with a cryostat microtome (Microm, Heidelberg, Germany) using OCT compound (Tissue-Tek, USA) as the inclusion liquid, a formulation of water-soluble glycols and resins solidifying at temperature below –10 °C. Sections 20 and 40 µm thick were obtained from frozen samples. For visualization of the chloroplasts a filter type TL was used for exciting the chlorophyll in the blue, and fluorescing at red wavelengths. A filter type A was used for coumarin observations exciting between 340–380 nm. The autofluorescence of THC was observed in the blue region. Several classes of phenolic compounds, including the coumarin family (Ibrahim and Barron, 1989) are strongly autofluorescent in blue under UV radiation.
In vivo absorbance properties of THC
In order to analyse the in vivo potential ability of THC as a UV screen in the cell, intracellular localization and its concentration were analysed. Estimations of THC concentration in µmol of THC g–1 FW were converted into concentration per average cell volume (mm3) by considering the cell as a cylinder.
Two main blue fluorescing layers (corresponding to THC) were clearly differentiated in the cell. THC concentration in both layers was estimated by differences in their fluorescence intensity by means of the image analysis system and based on Lambert–Beer’s Law.
Abs = 
CL(1)
where Abs is the absorbance (optical density), is the molar extinction coefficient at the maximum of THC absorbance (346 nm): 12 500 (M cm–1), C the percentage of THC concentration (M) for each layer, and L the distance, in this case the thickness of each layer (cm).
The absorptance of each layer can be calculated from the absorbance value by the expression:
A = 1 – 10–abs(2)
On the other hand, absorptance is:
A = 1 – T – R(3)
where T is the transmittance and R is the reflectance. Reflectance was assumed as 0 for THC extracts. Reflectance of THC measured by the integration sphere of a Li-Cor spectroradiometer model Li-1800 UW (Li-Cor, USA) was below 1% of transmittance. Thus, transmittance was calculated as:
T = 1 – A(4)
Then, the transmittance at a wavelength of 346 nm, corresponding to the maximum of THC absorbance, for each THC layer could be calculated. All the calculations in the alga were performed using no fewer than 10 replicates.
Coumarins as a UV screen for other macroalgae
Pérez-Rodríguez et al. (2001) described a very significant excretion of THC by D. vermicularis under different sources of stress, such as high solar radiation and high temperature. This THC can be accumulated in intertidal pools at low tide and protects other algae from harmful UV radiation. To determine if the UV-absorbing properties of THC could exert a photoprotective role for other macroalgae, a second set of experiments was designed using different THC concentrations as a UV screen. Field experiments were performed in situ in June 2000. Samples of several species of macroalgae were placed in flasks filled with 500 ml of seawater and exposed for 1 h to direct solar radiation. In order to avoid an increase in seawater temperature, flasks were submerged into a refrigerated water tank. Tanks made of UV-transparent Plexiglass filled with 250 ml of different dilutions of THC (6.4, 12.8, 19.3, and 25.7 µg ml–1) were placed over the flasks containing the algae. The highest THC concentration corresponded to 2 absorbance units at 346 nm.
The species of marine macrophytes studied were: Phaeophyta: Sargassum vulgare, Fucus spiralis; Chlorophyta: Ulva olivascens; Rhodophyta: Jania rubens; marine angiosperms: Posidonia oceanica.
To determine the physiological status of the plants under the different treatments, chlorophyll fluorescence was measured using a portable modulated fluorometer (PAM 2000, Walz, Effeltrich, Germany). The effective quantum yield of photosystem II (
F/F'm; Schreiber et al., 1986) was determined for at least 10 individual thalli. F represents the difference between the maximal fluorescence of a light acclimated plant (F'm) and the current steady-state fluorescence (Ft). Values of Ft were determined at very low intensity pulses of red light (650 nm, 0.3 µmol photons m–2 s–1), while F'm was induced with a saturating white light pulse (0.4 s, approximately 9000 µmol photons m–2 s–1) (see Hanelt (1998) and references therein for details in the use of PAM fluorometry in macroalgae).
The attenuation of solar radiation caused by the highest concentration of THC used is represented in Fig. 1a. The solar spectral composition over and below the tank containing the highest concentration of THC was determined with a Li-Cor spectroradiometer model Li-1800 UW (Li-Cor, USA) coupled to a cosine corrected planar sensor (2
).
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The attenuation of solar UV radiation by the different dilutions of THC was followed throughout the day. Total UV (UVA+UVB) radiation was estimated using biological dosimeters based on a UV-sensitive bacterial spore monolayer system (VioSpor, BioSense, Bornheim, Germany). These data are expressed as minimal erythemal dose (MED) per day (Furusawa et al., 1998). The total daily accumulated doses under different THC solutions are represented in Fig. 1b.
Data treatment
Changes in chlorophyll and THC concentration in thalli of D. vermicularis as well as effective quantum yield of fluorescence during exposures to the different treatments were tested using one-way ANOVA (model II; Sokal and Rohlf, 1995). In cases of significant differences, means were compared using the least significant difference of Fisher’s multi-range test (LSD). A P <0.05 was considered to be statistically significant.
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Results |
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Chlorophyll and THC distribution in thalli of D. vermicularis
D. vermicularis presented a gradual increase of chlorophyll a and b from the basal to the apical part of the thallus. In the apical part four times more chlorophyll was observed with respect to the base (Fig. 2a). While the highest chlorophyll a concentration found was around 0.32 mg g–1 FW, THC concentrations were much higher, reaching 20 mg g–1 FW in the apical part of the thalli (Fig. 2b). THC was homogeneously distributed in the basal parts, with an average concentration of c. 12 mg g–1 FW.
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Histological analysis
Due to the large size of the siphonalean cells of D. vermicularis, intracellular parts could be clearly observed by light microscopy (Fig. 3a). The cells branched in a verticilated manner from a calcified central axis, and they branch, forming ramifications up to the third order. A distribution of chloroplasts in a reticular structure could be observed by means of the autofluorescence of Chl a in red light (Fig. 3b).
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In a longitudinal section of the cell, two layers of blue fluorescing THC were clearly differentiated (Fig. 4a). The external one, involving the cell wall, was 5.98±0.91 µm thick, while the second, surrounding the vacuole of the cell, was 14.76±2.35 µm thick. Its fluorescence was lower than the external one. The same was observed in transverse section. Two strong blue fluorescent layers (Fig. 4b) were observed close to the cell wall and surrounding the vacuole.
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Morphometric measurements for calculations of THC content in the different layers as well as calculations of UV-absorbing abilities of the two layers are shown in Table 1. From fluorescence measurements, it could be observed that THC content was included in the two layers. THC concentration was needed to estimate the UV-absorbing ability of each layer. The total cellular THC content (0.44 nmol cell–1) was distributed in the two layers as follows:
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x+y= 0.44 nmol THC(5)
where x is the THC content in outer layer and y is the THC content in inner layer.
According to the image analysis, the external layer showed a 2.5 higher autofluorescence intensity than the internal one. The THC concentration in the outer and inner layers correspond to:
THC content (x)/outer layer volume=2.5xTHCcontent (y)/inner layer volume(6)
x/0.001935 = 2.5y/0.004031(7)
From equations (5) and (7), x and y were obtained. The outer layer THC content (x) was 0.24 nmol and the inner layer THC content (y) was 0.20 nmol. Dividing by the layer volume, the concentration of the both layers were: Outer layer: 0.124 µmol mm–3=0.124 M; Inner layer: 0.049 µmol mm–3=0.049 M.
According to Lambert–Beer’s Law (equation (1) described in the Materials and methods) the absorbance (optical density) of the two coumarin layers at 346 nm was estimated as: Abs (outer layer): 12 500x0.124x 5.98x10–4=0.926; Abs (inner layer): 12 500x0.049x 14.76x10–4=0.904. The absorptance (A) of the two layers was calculated according to equation (2) as: Outer layer A1: 1–10–0.926=0.881; Inner layer A2: 1–10–0.904=0.875. The transmitance (T) of the radiation at 346 nm through the layers was calculated according to equation (4): T1: 12.0%: the outer layer absorbs 88% of the incident radiation at 346 nm.; T2: 12.5%: the inner layer absorbs 87.5% of the radiation at 346 nm transmitted by the outer layer. The percentage of transmittance of the two layers indicated that only 1.5% of the incident radiation at 346 nm was transmitted into the vacuole of the cell.
Coumarins as a UV screen for other macroalgae
Figure 5 shows the effective quantum yield (F/F'm) of photosystem II of different macroalgae after 1 h of exposure to direct and filtered solar radiation.
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In general, algae showed a drastic decrease in
F/F'm ranging from 55% in the brown alga Sargassum up to almost 90% in the brown alga Fucus spiralis after 1 h of exposure to direct solar radiation at midday. Algae covered with increased THC-containing solutions were less inhibited than the controls. The THC solution at a concentration of 12.8 µg ml–1, that resulted in 1 absorbance unit at 346 nm, implied a decrease in less than 50% of F/F'm for algae like Ulva rigida, Fucus spiralis and Jania rubens while the marine angiosperm Posidonia oceanica was more affected (60% of decrease of F/F'm). Algae exposed to the highest THC concentration tested (25.7 µg ml–1) decreased their F/F'm by 25% on average. |
Discussion |
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The survival of algae in the upper part of the rocky shore, where they are exposed to high levels of solar radiation might be related to the presence of different photoprotective mechanisms in the plant. This work has shown that the accumulation and release of UV-absorbing compounds such as coumarins may play an important photoprotective role in the green alga Dasycladus vermicularis. Coumarins had not been documented as phenolic constituents in green algae before Menzel et al. (1983) showed that purified 3,6,7 trihidroxycoumarin represented almost 100% of total coumarins in several members of the siphonous green algae of the family Dasycladaceae.
D. vermicularis has a high THC (20.81±0.67 mg g–1 FW) content which mainly occurs in the apical zone of the thallus. The rest of the plant is semi-buried by sand in the shore, so little or no light reaches the basal part (Pérez-Rodríguez et al., 1998). Thus, almost all THC and chlorophyll were found in the more exposed parts of the thallus.
The distribution of these molecules supports the hypothesis of a photoprotective role for these substances. The large siphonalean cells of D. vermicularis presented two well-defined autofluorescent bands corresponding to an accumulation of THC around the large vacuole and a second external layer at the level of the cell wall. In higher plants, phenylpropanoid and flavonoid compounds usually accumulate in the central vacuoles of guard cells and epidermal cells as well as subepidermal cells of leaves (Weissenböck et al., 1986; Schnabl et al., 1989). Furthermore, some compounds were found to be covalently bound to plant cell walls (Strack et al., 1988; Schnitzler et al., 1996). In many Phaeophytae, phenols have been found in the cortical cells in special organelles called physodies around the cell wall. Autofluorescence observations revealed a homogeneous distribution of THC in the two layers. The phenolic compounds are structural components integrated in the cell wall of the brown algal cells, as also observed for the green alga D. vermicularis, and they play a very important role in its formation. Evidence of this were observed in the plug formation in cells of Dasycladus and another genera of siphonous green algae (Caulerpales) in relation to fertilization and injury (Menzel, 1979, 1980). Microscopic observations revealed rapid coumarin accumulation at the site of injury in parallel with high peroxidase activities indicating an active formation of the plug.
From the disposition of high concentrations of THC in the cell in specific layers, it could be thought that two in vivo UV screens are present in D. vermicularis. The transmittance of UV radiation estimated for the two UV-screening layers supports this hypothesis. The outer layer, at the level of the cell wall was able to absorb almost 90% of the incident radiation at the maximum peak of absorbance for THC in the UVA region (346 nm). Thus, the cytoplasm is significantly protected from UV, and damage to DNA, chloroplast and other organelles, structures which are described as UV radiation targets, are minimized. Measurements of the screening potential of THC for solar radiation by using biological dosimeters indicated a reduction of the numbers of MEDs (minimal erythematic doses) per day in the field. Similar results on the protection to the organism against UV radiation has previously been reported for different MAAs. Riegger and Robinson (1997) reported for various phytoplankton species from Antarctica that the presence of MAAs caused absorption of up to 50% of harmful UV quanta before reaching cytoplasmic molecular targets in diatom cells, and up to 72% for Phaeocystis antarctica colonies. Ishikura et al. (1997) measured maximum MAAs concentrations in the outermost surface layer of the siphonal mantle in the giant clam Tridacna crocea. These authors calculated that the sunscreen capacity of the measured MAAs was sufficient to absorb 87% of 310 nm radiation and 90% of 320 nm radiation before reaching 0.2 mm depth in the siphonal mantle.
THC within a cell could also protect against oxidative damage produced by an excess of radiation due to its antioxidant property (Pérez-Rodríguez et al., 2001). The coumarins are phenolic derivates characterized by a benzopyrone nucleus (Murray et al., 1982). The presence of ortho-dihydroxyl groups (as is the case for the THC from D. vermicularis) confers strong antioxidant properties to the molecule (Payá et al., 1992a; Foti et al., 1996). It has been demonstrated that THC of D. vermicularis has an antioxidant strength similar to that of ascorbic acid (Pérez-Rodríguez et al., 2001). Coumarins have been proposed as the main substrate in the peroxidase reaction involved in the formation of wound plugs in the Dasycladales and other siphonalean green algae (Menzel, 1979, 1980); and as another phenols, they are particularly effective as antioxidants in inhibiting lipid peroxidation as well as in scavenging superoxide/hydroxyl radicals and hypochloric acid (Payá et al., 1992a, b, 1993; Liu et al., 1999). Thus, an efficient scavenging system against the production of free radicals may be advantageous in environments subject to high solar radiation and temperature. However, further investigation is necessary to elucidate whether the THC of Dasycladus could play the same antioxidant role in vivo as that measured in vitro since it is accumulated in specific layers within the cell.
Another possibility arises from recent publications on natural populations of D. vermicularis describing a significant release of THC to the seawater by this species under different sources of stress like high solar radiation and temperature (Gómez et al., 1998; (Pérez Rodriguez et al., 1998, 2001). This THC can accumulate in intertidal pools and might protect other algae from harmful UV radiation at low tide. Field experiments demonstrated a potential role of THC as a UV screen for other algae; the reduction of the effective quantum yield of photosystem II was significant when the algae were exposed to filtered UV solar radiation using different concentrations of dissolved THC.
Thus, the special accumulation of high contents of the UV-absorbing trihydroxycoumarin in D. vermicularis, as well as its antioxidant ability may play an important role in the photoprotection of this species exposed to rapid and drastic changes in the environmental light conditions.
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Acknowledgements |
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The study was supported by grants from the European Union (Environmental and Climate Program, CT96-ENV4-0188) and the Spanish Ministerio de Educación y Cultura (CICYT, AMB97-1021-CO2-01 and FEDER, 1FD97-0824). The authors are indebted to the Cellular Biology and Zoology Departments for their help in the histological preparations and to L García for her help with the epifluorescence microscopy techniques.
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