BULLETIN OF MARINE SCIENCE, 71(3): 1171–1181, 2002
PHOTOSYNTHETIC PIGMENT COMPOSITION OF MARINE ANGIOSPERMS: PRELIMINARY CHARACTERIZATION OF MEDITERRANEAN SEAGRASSES
Gianna Casazza and Lucia Mazzella
ABSTRACT Pigments were investigated in the Mediterranean marine angiosperms Posidonia oceanica, Cymodocea nodosa, Zostera noltii and Halophila stipulacea. Samples were collected and analyzed from shallow and deep water habitats. Acetone extracts of leaves were analyzed by absorption spectroscopy, high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). The marine angiosperms examined pos- sessed: β,β-carotene, lutein, zeaxanthin, violaxanthin, neoxanthin and other minor pig- ments. Two unidentified pigments with the same retention time as two “siphonaxanthin- type” pigments were isolated from the green alga Flabella petiolata and are relatively abundant in the species P. oceanica and H. stipulacea. Pigment data indicate a possible biochemical adaptation to different light regimes in seagrass species that can colonize deeper habitats.
Seagrasses represent an outstanding example of adaptation of angiosperms to the ma- rine environment (Waycott and Les, 1996). They possess many specialized characteris- tics, such as hydrophilous pollination, morphological adaptations to submergence, nutri- ent assimilation by leaves and physiological adaptation to root anoxia (Den Hartog, 1970; Smith et al., 1988; Cox et al., 1992; Kraemer et al., 1997). Among the characteristics of shallow water marine environments where seagrasses grow, the variable and extremely reduced light regimes play a fundamental role. Seagrasses respond to these light reduced environments in a variety of morphological and physiological ways that have recently received considerable attention (Abal et al., 1994; Enriquez et al., 1995). Previous work has focused on growth and biomass measurements, morphological alterations, carbon balance and analysis of various photosynthetic parameters (Dennison and Alberte, 1986; Mazzella and Alberte, 1987; West, 1990; Zimmerman et al., 1994, 1995; Buia et al., 1992). The photosynthetic capabilities of plants are closely related to the structure of their photosynthetic apparatus. Chlorophyll a is common to all oxygen-producing photosyn- thetic organisms. Among all the photosynthetic organisms known, the greatest diversity of pigments is found in plants inhabiting the aquatic environments and particularly among the different groups of marine algae (Rowan, 1989). The role of the pigments in the depth distribution of algae, popularly known as ‘chromatic adaptation’ has been a controversial and greatly discussed topic for long time (Ramus et al., 1976; Ramus, 1983; Dring, 1981). Photosynthesis is strongly influenced by light quantity and quality. Although different photosynthetic performance of organisms can be ascribed to characteristics other than pigments, the light quality definitely plays a role in growth and development in deep water (Dring, 1981; Senger and Bauer, 1987). Seagrasses generally occur in shallow waters (Larkum and den Hartog, 1989) although reports indicate a wide range of depth limits for different species (Duarte, 1991). P. oceanica typically extends to 40 m depth in the Mediterranean Sea (Colantoni et al., 1982) while
1171 1172 BULLETIN OF MARINE SCIENCE, VOL. 71, NO. 3, 2002
Halophila engelmannii has been reported to 90 m depth in the Coral Sea (Duarte, 1991). Studies of marine angiosperms have shown that different seagrass species respond to changes in light availability over different time scales, reflecting variations in plant growth strategy and architecture (Abal et al., 1994; Duarte, 1991; Buia et al., 1992). Pigment adaptation plays a fundamental role in the success of submerged angiosperms in shaded aquatic environments (Dennison and Alberte, 1982; Frost-Christensen and Sand- Jensen, 1992) but data on the phosynthetic pigment composition of marine angiosperms are sparse and incomplete. Preliminary work (Ikemori and Arasaki, 1977) on Zostera marina and H. ovalis indicated that the pigment composition of seagrasses was identical to those of terrestrial vascular plants. Later studies of photosynthetic pigments focused on chlorophyll a/b contents and ratios (Drew, 1978; Wiginton and McMillan, 1979). More recently unidentified and putative UV blocking pigments have been reported (Trocine et al., 1981, 1982; Abal et al., 1994; Dawson and Dennison, 1996). To our knowledge, no other information on the photosynthetic pigments of marine angiosperms is available. The present work examines the pigment composition of four Mediterranean seagrasses: P. oceanica, Cymodocea nodosa, Z. noltii and H stipulacea, which represent different depth distributions. The aim was a comparison of the pigment composition of four seagrass species with: (1) a terrestrial plant, (2) two macroalgae inhabiting the same marine envi- ronment and (3) eventually a comparison of deep and shallow seagrass beds to ascertain whether pigment composition plays a role in the ability of some species to grow at lower light regimes hence colonizing greater depths.
MATERIALS AND METHODS
SAMPLE COLLECTION.—Leaf samples of P. oceanica, C. nodosa, Z. noltii were collected using SCUBA, from different locations around the Island of Ischia (Gulf of Naples). H. stipulacea was collected in the Levante Bay of Vulcano Island (Eolian Islands, Sicily). For comparison, material of the Chlorophyceae Flabella petiolata and Chaetomorpha aerea and of the terrestrial plant Spinacia olearacea, as one of the most used species in photosynthetic pigment studies, was obtained. All samples were collected in shallow waters, at depths ranging between 3 and 5 m. Z. noltii and the macroalga C. aerea are limited to these depths; C. nodosa, H. stipulacea P. oceanica and the macroalga F. petiolata were also collected at the maximum depth observed, for these species, in the investigated areas. C. nodosa was found at a maximum depth of 13 m, H. stipulacea at 27 m and P. oceanica at a depth of 37 m. The epiphytes were manually removed from the leaves and thalli with a razor blade; tissues were either weighed and processed immediately for pigment extraction, or frozen at −20°C for further analyses. Three samples from each species and depth were pooled together. Tissues were repeatedly cold extracted with 100% acetone using an Ultra Turrax homogenizer. Acetone extracts were transferred to diethyl ether 1:1, with the addition of 10% cold NaCl, according to Jeffrey (1968). Samples were concentrated by drying under a stream of nitrogen gas in reduced light to minimize photoxidation. PIGMENT CHARACTERIZATION.—The pigment composition of all samples was analysed by High Performance Liquid Cromatography (HPLC), Thin Layer Chromatography (TLC) and absorption spectroscopy. A Beckman ultrasphere RP18 column (ODS 5µm , 250 × 4.6 mm, IP) HPLC system was used with a two solvent mixture for separation: A) methanol:water:P solution (80:10:10) and B) methanol:ethyl acetate (80:20), as specified in Brunet et al. (1993). Pigments were detected both spectrophotometrically (440 nm, with Beckman Gold system, model 166) and fluorometrically (excitation 345 to 510 nm, emission 610 to 650 nm, with a Gilson, Model 121). Sigma chl a, chl b, β,β-carotene and “V.K.I. - Water Quality Institute - International Agency for 14C Determination”, CASAZZA AND MAZZELLA: PHOTOSYNTHETIC PIGMENT COMPOSITION 1173
Denmark, were used as standard pigments. Carotenoid quantification was obtained as relative amounts of total carotenoids, based on integrated peak areas of the chromatographs, from three separated pigment determination. Thin layer chromatography was used to separate pigments. The chromatographic systems used were: I) precoated silica gel plates (Sigma), with two developing solvent systems, a) n-propanol (4−10%) in petroleum ether (40°−60°) and b) 30% acetone in petroleum ether (Sherma and Lippstone, 1969); II) high performance, reverse phase, TLC plate (HPTLC Merck, RP-8), with developing system 90% methanol (Vesk and Jeffrey, 1987). For the identification of the isolated pigments, the pigmented zones were scraped from the plate with a razor blade and eluted into ethanol (100%, v/v) for carotenoids and acetone (100%, v/v) for chlorophylls. The absorption spectra of the pigments were analysed; some carotenoid spectra were examined after reactions with NaBH4, for the presence of specific chemical structure, i.e. conju- gated ketogroups (Walton et al., 1970). Absorption spectra of the acetone extracts of the macrophytes and the carotenoid ethanol ex- tracts of the separated pigments were obtained using a UV-VIS Varian CARY 1E spectrophotom- eter.
RESULTS
SPECTROPHOTOMETRIC ANALYSES.—Absorption spectra of acetone extracts of the four seagrasses, collected in the same depth range (3−5 m), are shown in Figure 1. All spectra were normalized to the chl a absorption maximum at long wavelength. Differences are evident in the blue portion of the spectrum (400−600 nm), where the Soret band of chl b and the carotenoids absorb (Jeffrey et al. 1997). In particular the P. oceanica and H. stipulacea extracts reveal an enhanced absorption in the Soret band for chl b (ca 450 nm) and a red shift in the Soret peak from 451 nm to 456 nm in H. stipulacea. This shift is most likely due to a change in the carotenoids composition.
Figure 1. Absorption spectra of acetone (100%) extracts of four seagrass species: Posidonia oceanica, Cymodocea nodosa, Zostera noltii and Halophila stipulacea from shallow waters (3−5 m). Spectra are normalized to equal height of the chl a long wavelength maximum. 1174 BULLETIN OF MARINE SCIENCE, VOL. 71, NO. 3, 2002
Figure 2. Normalized absorption spectra of acetone (100%) extracts of three seagrass species: Posidonia oceanica, Cymodocea nodosa and Halophila stipulacea collected at different depths. For each species, the absorption spectra of tissue from shallow water is compared with that from the maximum depth limit.
For each seagrass species the absorption characteristics of the tissue extracts of shal- low and deep plants were compared (Fig. 2). No differences were observed in deep and shallow tissues of C. nodosa (Fig. 2a), while enhanced blue (400−460 nm) absorption was observed in H. stipulacea and P. oceanica tissues collected from deep water (Fig. 2b,c). The enhanced absorption in the Soret region suggests enhanced levels of chl b and possibly of carotenoids. PIGMENT COMPOSITION.—The HPLC analyses are shown in Figures 3 and 4. Pigment identities and distribution among the different macrophytes analyzed (seagrasses and Chlorophyceae) are summarized in Table 1. CASAZZA AND MAZZELLA: PHOTOSYNTHETIC PIGMENT COMPOSITION 1175
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Figure 3. HPLC elution patterns of the total pigment extracts of four seagrasses. Detection was at 440 nm. Peak identities are given in Table 1. CASAZZA AND MAZZELLA: PHOTOSYNTHETIC PIGMENT COMPOSITION 1177
Figure 4. HPLC elution patterns of the total pigment extracts of a terrestrial plant (a) and two green algae (b, c). Detection was at 440 nm. Peak identities are given in Table 1.
The HPLC system employed separates about 12 individual pigments, with a clear baseline separation of all pigments except for zeaxanthin, which eluted together with lutein (Figs. 3,4, peak 7). The pigments neoxanthin, violaxanthin, lutein and zeaxanthin, chlorophyll b, chlorophyll a and β,β-carotene were common to all the four seagrass spe- cies. In addition five other pigments were observed: traces of taraxanthin and 1178 BULLETIN OF MARINE SCIENCE, VOL. 71, NO. 3, 2002
antheraxanthin were found in all the seagrasses with the exception of Z. noltii, and α- carotene was detected only in P. oceanica; two unknown carotenoids were detected in all the seagrasses analyzed (Fig. 3, peak 2 and 8, referred as unknown A and B in Table 1). The two unknown pigment amounts were consistent in P. oceanica and H. stipulacea where they showed a slight increase in the samples from deeper beds (data not shown) Their presence was also observed by TLC, revealed as pale orange bands. Identification of these two carotenoids by their absorption spectra was not possible, due to contamina- tion from other pigments: unknown A from violaxanthin, unknown B from the lutein/ zeaxanthin zone. However their spectra, after reaction with NaBH4, showed a blue ab- sorption shift indicating the occurence of a chemical reduction, while the spectra of the isolated violaxantin and lutein/zeaxantin pigments were not altered at all by the reagent. Two pigments with the same retention times (both in HPLC and in TLC) as the seagrass unknown A and B were clearly separated and identified from the acetone extracts of the Chlorophyta F. petiolata. They were also present, as traces, in the macroalga C. aerea (Table 1) while they were not detected at all in the HPLC separation of the terrestrial plant S. olearacea (Fig. 4). Finally an evaluation of the relative quantities, determined by HPLC, of the major carotenoids in the seagrass samples was approached. Total carotenoid amounts range from 32% of the total pigment content in C. nodosa and Z. noltii to 36−40% in P. oceanica and H. stipulacea. The most abundant carotenoids in all seagrasses are lutein\zeaxanthin (40−45%), followed by α and β,β-carotenes (17−24%) and violaxanthin (12−15%). Un- knowns A and B in Z. noltii and C. nodosa are present only at very low levels while in P. oceanica and H. stipulacea they account for 8−11%, unknown A, and 7−8%, unknown B, of total carotenoids. Neoxanthin regards for 6−7% of total carotenoids in all the seagrasses but H. stipulacea where it is much less abundant (3−4%).
DISCUSSION
The data here reported are the first submerged angiosperm data determining the pho- tosynthetic pigment composition of seagrasses and possibly pigment variations among various species inhabiting different light regimes. All the specific carotenoids contained in the acetone extracts of seagrasses, besides the chlorophylls, were identified. The four seagrass species shared all the photosynthetic pigments possessed by terres- trial vascular plants with a dominance of chl a and b and the presence of the major plant carotenoids. Other pigments, less commom carotenoids, were also determined: α-carotene, antheraxanthin and taraxanthin, in addition to trace levels of two unknown yellow-or- ange pigments. HPLC retention times of the unknowns were similar to those of a ‘siphonaxanthin-like’ pigment and siphonein, found in our chromatographic analysis of the green algae F. petiolata and Codium sp. In these Chlorophyceae three orange pigments were isolated: siphonaxanthin, a siphonaxanthin-like pigment, (as its absorption spectrum is similar to that of siphonaxanthin but its retention time is slightly different both in HPLC and in TLC) and siphonein. Unknowns A and B from the seagrass samples coeluted in HPLC with the fractions of the siphonaxanthin-like and siphonein pigments isolated from Codium sp., when run together. CASAZZA AND MAZZELLA: PHOTOSYNTHETIC PIGMENT COMPOSITION 1179
The presence of conjugated ketogroups in the chemical structure of the unknown pig- ments was suggested by the specific chemical reduction observed under absorption spec- troscopy. From all these information we suggest the unknonw pigments belonging to the siphonaxanthin type of carotenoids. Compared to terrestrial plants, marine angiosperms are subjected to very low light regimes, more typical to that of macrophytic algae. Light availability decreases with depth and its spectral quality is dramatically changed in deeper habitats, depending on water clarity. In oligotrophic open ocean water, blue light penetrates to the greatest depth, while in coastal water, with high scattering, green light reaches the greatest depths (Anderson, 1983; Kirk, 1983). Seagrasses inhabit mostly shallow waters but their depth distribution varies signifi- cantly world-wide, varing with species and localities (Duarte, 1991). The fact that some species can colonize deeper habitats could be ascribed to biochemical changes for adap- tation to light quality and quantity variations. The seagrass habitats is shared by the green algae whose pigment composition is very similar to that found in the photosynthetic tissues of higher plants, i.e., they contain β,β-carotene (often with α-carotene), neoxanthin, lutein and the xanthophyll cycle carotenoids, violaxanthin, antheraxanthin and zeaxan- thin (Young and Britton, 1993). Other carotenoids are also present and occasionally at high concentrations. The best known are siphonaxanthin and siphonein (Kleinig, 1969; Smith and Alberte, 1994), which can occur instead of, or as well as, lutein, the dominant xanthophyll of most green algae and vascular plants. These xanthophylls have been re- ported as characteristic of most deep-water green algae where they function as accessory pigments in light harvesting for photosystem II, extending the absorption into the green portion of the spectrum (Yokoama et al., 1977, 1992; Anderson, 1983; Smith and Alberte, 1994). The fact that the two unknown pigments, most probably belonging to the siphonaxanthin-type of carotenoids, were detected in higher quantities in the two seagrass species P. oceanica and H. stipulacea which have depth ranges extending to deeper habi- tats, and that both pigment amounts increase with depth (unpubl. data) suggests a bio- chemical adaptation to the light quality and quantity regimes found at depth, for these species. Adaptation to available light at the level of pigment composition among submerged angiosperms has been claimed (Frost-Christensen and Sand-Jensen, 1992) but no data other than chl b and chl a/b ratios have been reported before (Wiginton and McMillan, 1979; Dennison and Alberte, 1982, 1985). Our data on the presence of these novel caro- tenoids in seagrasses, particularly in those species which can colonize deeper habitats, support this hypothesis. Moreover the preliminary data on carotenoid amounts variations in seagrasses at dif- ferent depths, suggest that seagrass adaptation to littoral marine environments, character- ized by reduced light availability, could be not only in terms of increased levels of chlo- rophyll b, as found in higher plants, but also in utilization of other pigments for light harvesting, as typical in algae (Anderson, 1983; Owens et al., 1987). More work is needed to better characterize the chemical structure of the novel caro- tenoids in seagrasses, quantify the different pigment weights and possibly their functions in the photosynthetic apparatus of the the marine angiosperms. 1180 BULLETIN OF MARINE SCIENCE, VOL. 71, NO. 3, 2002
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