Abstract

We report for the first time that marine angiosperms (seagrasses) possess sulfated polysaccharides, which are absent in terrestrial and freshwater plants. The structure of the sulfated polysaccharide from the seagrass Ruppia maritima was determined. It is a sulfated D-galactan composed of the following regular tetrasaccharide repeating unit: [3-β-D-Gal-2(OSO3)-1→4-α-D-Gal-1→4-α-D-Gal-1→3-β-D-Gal-4(OSO3)-1→]. Sulfated galactans have been described previously in red algae and in marine invertebrates (ascidians and sea urchins). The sulfated galactan from the marine angiosperm has an intermediate structure when compared with the polysaccharides from these two other groups of organisms. Like marine invertebrate galactan, it expresses a regular repeating unit with a homogenous sulfation pattern. However, seagrass galactan contains the D-enantiomer of galactose instead of the L-isomer found in marine invertebrates. Like red algae, the marine angiosperm polysaccharide contains both α and β units of D-galactose; however, these units are not distributed in an alternating order, as in algal galactan. Sulfated galactan is localized in the plant cell walls, mostly in rhizomes and roots, indicative of a relationship with the absorption of nutrients and of a possible structural function. The occurrence of sulfated galactans in marine organisms may be the result of physiological adaptations, which are not correlated with phylogenetic proximity. We suggest that convergent adaptation, due to environment pressure, may explain the occurrence of sulfated galactans in many marine organisms.

Introduction

Sulfated polysaccharides comprise a complex group of macromolecules with a wide range of important biological properties. These anionic polymers are widespread in nature, occurring in a great variety of organisms. High amounts of sulfated galactans and sulfated fucans are found in marine algae (Painter, 1983; Pervical and McDowell, 1967). In the animal kingdom, sulfated glycosaminoglycans abound in vertebrate tissues (Cássaro and Dietrich, 1977; Mathews, 1975). Invertebrate species are also a rich source of sulfated polysaccharides with novel structures (Mourão and Perlin, 1987; Mourão et al., 1996; Pavão et al., 1998; Vieira et al., 1991; Vilela-Silva et al., 2002). However, sulfated polysaccharides have not been found in higher vascular plants (Cássaro and Dietrich, 1977; Yoon et al., 2002).

A curious observation is that evolutionary distant organisms that share the marine environment possess structurally related sulfated polysaccharides, mostly composed of sulfated fucose or sulfated galactose. Thus, the marine invertebrate ascidians (Chordata-Tunicata) contain high amounts of sulfated galactans as red algae (Mourão and Perlin, 1987; Pavão et al., 1989; Santos et al., 1992). In the same way, marine echinoderms possess sulfated fucans as brown algae (Alves et al., 1997; Mulloy et al., 1994; Ribeiro et al., 1994; Vilela-Silva et al., 2002). This raises an interesting question: Is the occurrence of sulfated polysaccharides an adaptation to marine life?

A possible approach to this question is to investigate the occurrence of sulfated polysaccharides in marine angiosperms or seagrasses. This is a polyphyletic assemblage of ∼60 marine species (Les et al., 1997), concentrated in the subclass Alismatidae (Cook, 1990; Cronquist, 1981; Tomlinson, 1982). They display several adaptations to the marine environment, such as a seawater submersed habitat, tolerance to salinity, hydrophily (water-pollinated), and an effective anchorage system. Marine angiosperms are commonly found in the vicinity of mangroves, estuaries, hypo- or hypersaline coastal lagoons and fish ponds (Creed, 2000), recognized as ecologically important habitats of the coast zone (Larkum and Hartog, 1989). These species provide food and habitat for associated animals (Hartog, 1970), enhancing near-shore productivity while buffering waves and currents; many species are uncovered in the meadows (Orth, 1992).

We now reported for the first time that marine angiosperms contain high amounts of sulfated polysaccharide in contrast with the terrestrial and freshwater species. The seagrass sulfated polysaccharide differs in its chemical structure from similar compounds found in red algae and marine invertebrates. This observation raises interesting questions, such as: Are the genes coding expression of enzymes involved in the sulfation of polysaccharides preserved during the evolution of vascular plants although not expressed or repressed in terrestrial and freshwater species? Did the seagrasses acquire the genes coding sulfotransferase expression by horizontal transfer? Is it plausible that the presence of sulfated galactan in these species is a convergent adaptation to the marine environment?

Results and discussion

Occurrence of sulfated galactans in the seagrasses

Species of marine angiosperms, namely, Ruppia maritima, Halodule wrightii, and Halophila decipiens, but not terrestrial angiosperms contain high amounts of sulfated polysaccharides (Table I).

Table I.

Concentration of sulfated polysaccharide in species of marine and terrestrial angiosperms

Angiosperm
 
Species
 
Sulfated polysaccharide (µg/mg dry tissue)
 
Marine R. maritima 10.3 
 H. wrightii 8.5 
 H. decipiens 7.7 
Terrestrial Z. mays (maize) <0.001 
 P. vulgaris (bean) <0.001 
Angiosperm
 
Species
 
Sulfated polysaccharide (µg/mg dry tissue)
 
Marine R. maritima 10.3 
 H. wrightii 8.5 
 H. decipiens 7.7 
Terrestrial Z. mays (maize) <0.001 
 P. vulgaris (bean) <0.001 

Sulfated polysaccharides were extracted from the angiosperm species by protease digestion and their concentrations were determined based on the metachromatic assay using 1,9-dimethylmethylene blue (Farndale et al., 1986) and a standard curve with the purified sulfated galactan from R. maritima.

The sulfated polysaccharides extracted from the species R. maritima were studied in more details. They were initially partial purified by gel chromatography on Sephacryl 400HR, monitored for hexose, hexuronic acid, and metachromasia (data not shown). The fraction sensitive to hexuronic acid, possibly due to the presence of pectin, was discharged. Anion exchange chromatography on Mono Q–fast performance liquid chromatography (FPLC) of the pectin-free polysaccharides yielded three major fractions, denoted as F1, F2, and F3, eluted from the column with 1.0, 1.8, and 2.4 M NaCl, respectively (Figure 1A). Agarose gel electrophoresis of these fractions revealed F3 contains a single band (Figure 1B).

Fig. 1.

Purification of the sulfated galactan from R. maritima on a Mono Q-FPLC column (A) and analysis of the purified fractions by agarose gel electrophoresis (B). (A) Sulfated polysaccharides previously purified on a Sephacryl column (10 mg) were applied to a Mono Q-FPLC and eluted as described under Materials and methods. Fractions were assayed for metachromasia (closed circles) and NaCl concentration (dashed line). The fractions indicated by the horizontal bar were pooled, dialyzed against distilled water, and lyophilized. (B) The three fractions of sulfated polysaccharides (15 μg each) were applied to a 0.5% agarose gel in 50 mm 1,3-diaminopropane:acetate (pH 9.0) and run at 110 V for 1 h. Gel was fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution overnight, dried, and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:5:5, v/v).

Fig. 1.

Purification of the sulfated galactan from R. maritima on a Mono Q-FPLC column (A) and analysis of the purified fractions by agarose gel electrophoresis (B). (A) Sulfated polysaccharides previously purified on a Sephacryl column (10 mg) were applied to a Mono Q-FPLC and eluted as described under Materials and methods. Fractions were assayed for metachromasia (closed circles) and NaCl concentration (dashed line). The fractions indicated by the horizontal bar were pooled, dialyzed against distilled water, and lyophilized. (B) The three fractions of sulfated polysaccharides (15 μg each) were applied to a 0.5% agarose gel in 50 mm 1,3-diaminopropane:acetate (pH 9.0) and run at 110 V for 1 h. Gel was fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution overnight, dried, and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:5:5, v/v).

Chemical analysis of fraction F3 revealed the occurrence exclusively of galactose and sulfate. The trimethylsilylated (−)-2-butyl galactosides obtained from this polysaccharide had the same retention times and peaks areas as the standard D-galactose. Therefore, galactose occurs on the R. maritima galactan exclusively as the D-enantiomer.

Sulfated galactan from R. maritima has a regular tetrasaccharide repeating unit

We employed nuclear magnetic resonance (NMR) analysis to determine the structure of the sulfated galactan from R. maritima. The 1H 1D spectra of the native and desulfated galactan are shown in Figure 2.

Fig. 2.

1H NMR spectra at 400 MHz of native (A) and desulfated (B) galactan from R. maritima (fraction P3). The spectra were recorded at 60°C for samples in D2O solution. The residual water signal has been suppressed by presaturation. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm. The anomeric protons assigned by 1H/13C HMQC (see Figure 4A) are labeled in the native sample (A). The signal designated A1 refer to this produced by α(1→4) units, whereas those produced by β(1→3) residues are labeled B1 and C1. B2 and C4 are signals of protons bearing sulfation sites assigned to β-H2 and β-H4, respectively. The presence of a less intense C4 signal in the desulfated galactan (B) indicates that some 4-sulfate ester resists to the desulfation reaction. The integrals listed under the anomeric signals are normalized to the sum of all the anomeric protons.

Fig. 2.

1H NMR spectra at 400 MHz of native (A) and desulfated (B) galactan from R. maritima (fraction P3). The spectra were recorded at 60°C for samples in D2O solution. The residual water signal has been suppressed by presaturation. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm. The anomeric protons assigned by 1H/13C HMQC (see Figure 4A) are labeled in the native sample (A). The signal designated A1 refer to this produced by α(1→4) units, whereas those produced by β(1→3) residues are labeled B1 and C1. B2 and C4 are signals of protons bearing sulfation sites assigned to β-H2 and β-H4, respectively. The presence of a less intense C4 signal in the desulfated galactan (B) indicates that some 4-sulfate ester resists to the desulfation reaction. The integrals listed under the anomeric signals are normalized to the sum of all the anomeric protons.

The desulfated galactan shows two main anomeric resonance, one at 5.2 ppm (α-unit) and another at 4.8 ppm (β-unit) (Figure 2B). Peak integration shows 0.4:0.6 ratio. The spectrum of the native galactan (Figure 2A) indicates that the β-unit is in fact composed by two anomeric signals, B1 and C1, whereas the α-unit has a single signal at 5.21 ppm, A1. The integration of these peaks shows a 0.45:0.26:0.29 ratio for A:B:C. Beyond the signals ascribed to anomeric protons, the spectrum of the native galactan show two other signals with strong downfield shifts, as expected for sulfation sites, and noted as C4 and B2.

The assignment of peaks was achieved by analysis of 1H correlations spectroscopy (COSY) (Figure 3A), 1H total correlation spectroscopy (TOCSY) (Figure 3B and C), and 1H/13C heteronuclear multiple quantum coherence (HMQC) (Figure 4) spectra, giving the values presented in Table II. Both the β-H2 (denominated as B2) of residue B and β-H4 (C4) of residue C show strong downfield shifts (∼0.8 ppm), indicative of sulfation sites (Figures 3A, 3B, and 4A; Table II).

Table II.

1H and 13C chemical shifts for residues of galactose in native and desulfated galactan from the seagrass R. maritima and standard compounda

  1H chemical shifts (ppm)
 
     
Polysaccharide
 
Unit
 
H1
 
H2
 
H3
 
H4
 
H5
 
H6
 
Native galactan from R. maritima 5.21 4.18 4.41 4.35 4.35 3.03 
 5.01 4.69 4.25 4.10 ND 3.93 
 4.91 3.73 3.97 4.97 4.08 3.93 
Desulfated galactan from R. maritima α 5.21 ND 4.41 4.19 ND 3.93 
 β 4.84 3.85 4.36 4.19 ND 3.93 
Desulfated galactan from a red algaa 4-α-D-Gal-1 5.29 3.85 3.95 4.22 4.16 3.78 
 3-βD-Gal-1 4.40 3.78 3.75 4.12 3.73 3.78 
  1H chemical shifts (ppm)
 
     
Polysaccharide
 
Unit
 
H1
 
H2
 
H3
 
H4
 
H5
 
H6
 
Native galactan from R. maritima 5.21 4.18 4.41 4.35 4.35 3.03 
 5.01 4.69 4.25 4.10 ND 3.93 
 4.91 3.73 3.97 4.97 4.08 3.93 
Desulfated galactan from R. maritima α 5.21 ND 4.41 4.19 ND 3.93 
 β 4.84 3.85 4.36 4.19 ND 3.93 
Desulfated galactan from a red algaa 4-α-D-Gal-1 5.29 3.85 3.95 4.22 4.16 3.78 
 3-βD-Gal-1 4.40 3.78 3.75 4.12 3.73 3.78 
  13C chemical shits (ppm)
 
     

 

 
C1
 
C2
 
C3
 
C4
 
C5
 
C6
 
Native galactan from R. maritima 99.2 72.3 68.5 79.7 67.5 60.5 
 102.7 77.3 79.5 66.8 ND 60.5 
 103.5 71.8 72.8 77.4 68.7 60.5 
Desulfated galactan from R. maritima α 99.0 ND 69.0 79.6 ND 60.5 
 β 104.4 72.3 78.3 68.4 ND 60.5 
Desulfated galactan from a red algaa 4-α-D-Gal-1 102.9 71.5 70.9 80.9 73.9 63.0 
 3-β-D-Gal-1 105.3 72.3 82.7 70.4 77.5 63.0 
  13C chemical shits (ppm)
 
     

 

 
C1
 
C2
 
C3
 
C4
 
C5
 
C6
 
Native galactan from R. maritima 99.2 72.3 68.5 79.7 67.5 60.5 
 102.7 77.3 79.5 66.8 ND 60.5 
 103.5 71.8 72.8 77.4 68.7 60.5 
Desulfated galactan from R. maritima α 99.0 ND 69.0 79.6 ND 60.5 
 β 104.4 72.3 78.3 68.4 ND 60.5 
Desulfated galactan from a red algaa 4-α-D-Gal-1 102.9 71.5 70.9 80.9 73.9 63.0 
 3-β-D-Gal-1 105.3 72.3 82.7 70.4 77.5 63.0 

Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and to methanol for 13C. Values in bold and italic indicate sulfated and glycosilated positions, respectively.

Fig. 3.

COSY (A) and TOCSY (B, C) spectra of native (A, B) and desulfated (C) galactan from R. maritima at 400 MHz, 60°C, in D2O.

Fig. 3.

COSY (A) and TOCSY (B, C) spectra of native (A, B) and desulfated (C) galactan from R. maritima at 400 MHz, 60°C, in D2O.

Fig. 4.

1H/13C HMQC spectra of native (A) and desulfated (B) galactan from R. maritima at 400 MHz, 60°C, in D2O. The assignment was based on TOCSY and COSY spectra. The values of chemical shifts reported in Table II are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and methanol for 13C. The anomeric signals were identified by the characteristic carbon chemical shifts.

Fig. 4.

1H/13C HMQC spectra of native (A) and desulfated (B) galactan from R. maritima at 400 MHz, 60°C, in D2O. The assignment was based on TOCSY and COSY spectra. The values of chemical shifts reported in Table II are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and methanol for 13C. The anomeric signals were identified by the characteristic carbon chemical shifts.

The position of the glycosidic linkage was not easily deduced from the 1H chemical shifts, but the 13C chemical shifts of the desulfated galactan displayed two signals, α-C4 and β-C3 (Figure 4B, Table II), which are ∼10 ppm downfield shifts. This indicates glycosilated positions (Falshaw and Furneaux, 1994). Methylation analysis using the desulfated galactan yielded equimolar proportions of 2,3,6-tri-O-methylgalactose (49%) and 2,4,6-tri-O-methylgalactose (51%), as expected. Therefore, α-units and β-units are 4-linked and 3-linked, respectively.

The distribution of the different residues in the native sulfated galactan was deduced from the nuclear Overhauser enhancements spectroscopy (NOESY) spectrum (Figure 5). A strong NOE is seen between residue B and A and between residue A and C. Because residue A forms a single type of unit and peak integration shows 0.45:0.26:0.29 (A:B:C) ratio, we deduced that two residues A are linked to each other. An interresidue NOE can be visualized between residues C and B. Overall these results indicate the sequence -B-A-A-C-, as shown in Figure 6B.

Fig. 5.

NOESY spectrum of the sulfated galactan from R. maritima at 400 MHz, 60°C, in D2O. The spectrum shows signals connecting residues A-C, C-B, and B-A. This indicates the following sequence of residues: -B-A-A-C-.

Fig. 5.

NOESY spectrum of the sulfated galactan from R. maritima at 400 MHz, 60°C, in D2O. The spectrum shows signals connecting residues A-C, C-B, and B-A. This indicates the following sequence of residues: -B-A-A-C-.

Fig. 6.

Phylogenetic relationships and divergence times of marine red algae, angiosperms and invertebrates and structure of their sulfated galactans. (A) The sulfated galactans from marine red algae have a repeating structure (-4-α-D-Gal-1→3-β-D-Gal-1→)n, with a variable sulfation pattern. In the species B. occidentalis, ∼ one-third of the units are 2,3-disulfated and another third are 2-sulfated (Farias et al., 2000). (B) The sulfated galactan from the marine angiosperm R. maritima has the repeating structure (3-β-D-Gal-2(OSO3)-1→4-α-D-Gal-1→4-α-D-Gal-1→3-β-D-Gal-4(OSO3)-1→)n. (C) In the case of tunicates, the sulfated galactans have 3-sulfated, 4-linked α-L-galactose units, and in several species nonsulfated L-galactose residues occur as branched units (Mourão and Perlin, 1987; Pavão et al., 1989; Santos et al., 1992). (D) Sulfated galactan from sea urchin has a regular repeating structure, with a single sulfated monosaccharide unit, composed of 2-sulfated, 3-linked α-L-galactose (Alves et al., 1997). Land angiosperms and vertebrates do not contain sulfated galactan.

Fig. 6.

Phylogenetic relationships and divergence times of marine red algae, angiosperms and invertebrates and structure of their sulfated galactans. (A) The sulfated galactans from marine red algae have a repeating structure (-4-α-D-Gal-1→3-β-D-Gal-1→)n, with a variable sulfation pattern. In the species B. occidentalis, ∼ one-third of the units are 2,3-disulfated and another third are 2-sulfated (Farias et al., 2000). (B) The sulfated galactan from the marine angiosperm R. maritima has the repeating structure (3-β-D-Gal-2(OSO3)-1→4-α-D-Gal-1→4-α-D-Gal-1→3-β-D-Gal-4(OSO3)-1→)n. (C) In the case of tunicates, the sulfated galactans have 3-sulfated, 4-linked α-L-galactose units, and in several species nonsulfated L-galactose residues occur as branched units (Mourão and Perlin, 1987; Pavão et al., 1989; Santos et al., 1992). (D) Sulfated galactan from sea urchin has a regular repeating structure, with a single sulfated monosaccharide unit, composed of 2-sulfated, 3-linked α-L-galactose (Alves et al., 1997). Land angiosperms and vertebrates do not contain sulfated galactan.

Sulfated galactan is located in the cell walls of the seagrass

To find out the tissue localization of the sulfated galactan in the marine seagrass R. maritima, we obtained semithin sections from three different regions of the plant, which were stained with toluidine blue and visualized by optical microscopy (Figure 7A–C). The sulfated galactan is mostly localized in the plant cell wall, like the sulfated polysaccharides from marine algae. High amounts of the sulfated galactans were detected in the rhizomes of the seagrass, low proportion in the roots and practically absent in the leaves as demonstrated by the different staining intensities at the microscopy experiments. Biochemical analysis (Figure 7D) indicates that although at lower proportion, leaves of R. maritima also possess sulfated polysaccharides. This observation suggests that the sulfated galactans have an osmotic function in the marine angiosperms, as already reported for the sulfated polysaccharides in marine algae. In this way, sulfated galactan is more concentrated in the rhizome and root and less in the leaves of the seagrass, which are compartments responsible for the nutrient absorption from the sea water and, thus, more vulnerable to the salinity gradient. The histological analysis also indicates that the sulfated polysaccharides are more concentrated in the external than in the internal regions of the plant. This is especially noted in the root (Figure 7C). These observations suggest that the sulfated polysaccharides could perform similar functions as in algae, selecting the molecules that enter into the cells and also maintaining the ion balance between cytoplasm and environment (Kloareg and Quatrano, 1988). In addition, the roots and rhizomes are the regions that anchor the plant to the substrate, indicating that the presence of sulfated polysaccharide could also have a structural function, allowing the plant to survive in a high-kinetic-force environment like the sea coast.

Fig. 7.

Concentration of sulfated polysaccharide in different regions of the seagrass R. maritima, determined by histological (AC) and biochemical (D) analysis. Optical microscopy images of the leaf (A), rhizome (B), and root (C) of the seagrass show differences in the metachromasia intensity of toluidine blue. The metachromasia observed in these compartments were restricted to cell walls, whereas rhizomes and roots had presented more labeling intensity compared with leaves. Original magnification was 400× for all images. These observations were confirmed by biochemical determination of the sulfated polysaccharide content in the different regions of the plant (D).

Fig. 7.

Concentration of sulfated polysaccharide in different regions of the seagrass R. maritima, determined by histological (AC) and biochemical (D) analysis. Optical microscopy images of the leaf (A), rhizome (B), and root (C) of the seagrass show differences in the metachromasia intensity of toluidine blue. The metachromasia observed in these compartments were restricted to cell walls, whereas rhizomes and roots had presented more labeling intensity compared with leaves. Original magnification was 400× for all images. These observations were confirmed by biochemical determination of the sulfated polysaccharide content in the different regions of the plant (D).

Variety of chemical structures found in sulfated galactans from marine organisms

Sulfated galactans have been described in several organisms, which share the marine environment but belong to phylogenetic distant groups, such as red algae (Farias et al., 2000; Painter, 1983) and the invertebrate ascidians (Albano and Mourão, 1986; Pavão et al., 1989; Santos et al., 1992) and sea urchins (Alves et al., 1997). We report for the first time that seagrasses, a group of angiosperms adapted to the marine environment, also contain high amounts of sulfated galactan. The presence of sulfated galactans in seagrasses that evolved separately indicates that the occurrence of sulfated polysaccharide is an important adaptation to the marine environment.

The structure of the sulfated galactans found in these marine organisms varies significantly. In the marine red algae, the sulfated galactans (also known as carrageenans) have a backbone composed of [4-α-D-Gal-1→3-β-D-Gal-1] but with a heterogeneous sulfation pattern (Farias et al., 2000; Painter, 1983) (Figure 6A). Small amounts of L-enantiomer of galactose and 3,6-anhydro-galactose are also reported in the polysaccharide from red algae (Painter, 1983). Sulfated galactans from marine invertebrates have regular and repetitive chemical structures. They are 3-sulfated, 4-linked (Mourão and Perlin, 1987; Pavão et al., 1989; Santos et al., 1992) (Figure 6C) and 2-sulfated, 3-linked (Alves et al., 1997) (Figure 6D) in ascidian and sea urchin, respectively. In the invertebrate polysaccharides, galactose occurs exclusively as the unusual L-enantiomer (Mourão and Perlin, 1987; Pavão et al., 1989). Furthermore, sulfated galactans from some species of ascidians are highly branched polysaccharides (Mourão and Perlin, 1987; Pavão et al., 1989). The sulfated galactan from marine angiosperm has an intermediate structure. Like marine invertebrate polysaccharide, it exhibits a regular repeating sequence with a homogenous sulfation pattern. However, seagrass galactan contains the D-enantiomer of galactose instead of the L-isomer found in marine invertebrates. Like red algae galactan, the marine angiosperm polysaccharide is composed of α and β units of D-galactose. These units, however, are not distributed in an alternating order, as in the algal polysaccharide.

Evolutionarily distant species contain sulfated galactan: what's the meaning?

The observation that in contrast with terrestrial and freshwater plants the marine angiosperms contain high amounts of sulfated polysaccharides (like other marine species) raises puzzling questions concerning the evolution of these organisms, such as described next.

Are the genes coding expression of enzymes involved in the sulfation of polysaccharides present but not expressed or repressed in terrestrial and fresh water plants?

A possible explanation for the occurrence of sulfated galactans in marine but not in terrestrial and freshwater angiosperms could be that the genes coding for the enzyme involved in the sulfation of these macromolecules remains preserved in plants although not expressed or repressed in the terrestrial or freshwater species. If this hypothesis held true, the marine environment would induce the expression of these genes. Our search indicated that the known genomic sequences of two species of terrestrial plants have no homology to sulfotransferase genes. Thus, genes coding enzymes involved in sulfation of polysaccharides are not conserved during the evolution of terrestrial plants or have been extensively mutated in higher plants and hence are no homologous to the sulfotransferase genes.

A horizontal gene transfer from another marine organism?

The occurrence of sulfated galactan correlates more with physiological adaptation than with phylogenetic distance, and hence fits a selective scenario. A possible explanation for this event is that the marine angiosperms acquire the genes for the enzymes involved in the sulfation of polysaccharides from other marine organism, such as red algae. It is conceivable that marine angiosperms could assimilate the genes for biosynthesis of the sulfated galactan from marine alga or invertebrate through microorganism infection and use them as an adaptive device to the marine environment. In a similar way, Lidholt et al. (1994) reported that a bacterial glycosyltransferase involved in the biosynthesis of a capsular polysaccharide mimics the highly elaborate substrate specificity of the corresponding enzymes necessary for heparin biosynthesis. As a plausible explanation for this result, the authors speculated that the microorganism had assimilated the gene for glycosyltransferases from an infected mammalian host and used it to generate a protective capsule. However, the structural differences among the sulfated galactans from marine angiosperms, invertebrates, and red algae (Figure 6) do not favor a horizontal transfer of genes coding enzymes for the biosynthesis of the sulfated galactan, because in that case we would expect higher homology between the galactans. Furthermore, seagrasses make up a polyphyletic group, which makes the horizontal gene transfer even more improbable. Finally, the transfer of gene is an unusual event during the evolutionary process and has not been clearly demonstrated during the evolution of animals, plants, and fungi (Cho et al., 1998; Goddard and Burt, 1999), although it has been reported in flowering plants (Bergthorsson et al., 2003).

A convergent adaptation?

A more plausible explanation for the presence of related polysaccharides, the sulfated galactans, in evolutionary distant organisms that share the marine environment is a convergent adaptation due to environmental selective pressure. It has been reported previously in seagrasses, related with the hydrophily of these plants (Les et al., 1997). Convergent adaptation (or convergent evolution) is a common biological event, when similar-appearing structures evolved in entirely unrelated groups of organisms, such as the classical examples of the vertebrate eye and the cephalopod eye, the bivalve shells of mollusks and of brachiopods, and so on (Brusca and Brusca, 1990). However, convergent evolution has not yet been suggested for molecules found in the extracellular matrix of phylogenetic unrelated organisms, as in the present study.

Major conclusions

We reported the occurrence of sulfated galactan in the marine angiosperm. This polysaccharide has a unique structure, with a regular repeating tetrasaccharide sequence, composed of [3-β-D-Gal-2(OSO3)-1→4-α-D-Gal-1→4-α-D-Gal-1→3-β-D-Gal-4(OSO3)-1→]. The occurrence of sulfated galactan in living organisms correlates more with physiological adaptation than with phylogenetic distance and hence fits a selective scenario. We suggest that a convergent adaptation due to environmental pressure may explain the occurrence of high concentration of sulfated polysaccharide in marine organisms.

Materials and methods

Sulfated galactans from seagrasses

Extraction

The seagrasses R. maritima and H. wriight were collected at Ilha do Japonês, Cabo Frio, and H. decipiens at Urca, Rio de Janeiro, Brazil. They were carefully separated from other organisms and sun-dried. The dried tissue (20 g) was crushed with a blender (Oster, Super Deluxe), suspended in 400 ml 0.1 M sodium acetate (pH 6.0), containing 2 g papain (Merck, Darmstadt, Germany), 5 mM ethylenediamine tetra-acetic acid, and 5 mM cysteine. After incubation for 24 h at 60°C, the mixture was filtrated and the supernatant saved. The sulfated polysaccharides in the solution were precipitated with 800 ml absolute ethanol. After 24 h at 4°C, the precipitate formed was collected by centrifugation (2560 × g for 20 min at 5°C). The final precipitate was dried at 60°C for 12 h. Approximately 1.6 g (dry weight) of crude polysaccharide was obtained after these procedures. The sulfated galactan was purified by a combination of gel filtration and anion exchange chromatography, as described next.

Purification

The crude polysaccharide (100 mg) was applied to a Sephacryl 400 HR column (200 × 3.5 cm), equilibrated with 0.2 M sodium bicarbonate (pH 6.0), and eluted with the same solution. The flow rate of the column was 5 ml/h and fractions of 4.0 ml were collected in regular intervals. Fractions were checked for hexose and hexuronic acid by the Dubois reaction (Dubois et al., 1956) and carbazol reaction (Dische, 1947), respectively, and also by metachromatic assay using 1,9-dimethylmethylene blue (Farndale et al., 1986). Fractions containing the sulfated galactan (indicated by the positive metachromatic reaction) and free of contaminants such as pectins (indicated by positive carbazole reaction) or a yellow pigment were pooled, dialyzed against distilled water, and lyophilized.

The sulfated galactan obtained from the Sephacryl column (10 mg) was applied to a Mono Q-FPLC column (HR 5/5, Amersham Pharmacia Biotech, Little Chalfont, UK), equilibrated with 20 mM Tris–HCl (pH 8.0), containing 10 mM ethylenediamine tetra-acetic acid. The column was developed by a linear gradient of 1–4 M NaCl in the same solution. The flow rate of the column was 0.5 ml/min and fractions of 0.5 ml were collected and assayed by metachromasia. The fractions containing the sulfated galactan were pooled, dialyzed against distilled water, and lyophilized.

Agarose gel electrophoresis

Sulfated galactans were analyzed by agarose gel electrophoresis as described (Alves et al., 1997; Vieira et al., 1991). Samples (∼15 μg) were applied to a 0.5% agarose gel and run for 1 h at 110 V in 50 mM 1,3-diaminopropane:acetate buffer (pH 9.0). The sulfated polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:5:5, v/v).

Chemical analysis

After acid hydrolysis (6.0 M trifluoroacetic acid 100°C for 5 h) of the sulfated polysaccharide, the hexose was identified by paper chromatography in n-butanol:pyridine:water (3:2:1, v/v) for 48 h on Whatman No. 1 paper, followed by staining with silver nitrate and by gas-liquid chromatography (GLC) of derived alditol acetates (Kircher, 1960). Sulfate was measured by the BaCl2/gelatin method (Saito et al., 1968).

Determination of the D or L configuration of galactose

The enantiomeric form of the galactose was assigned based on analysis of the trimethylsilylated (−)-2-butyl glycoside, as described (Gerwig et al., 1978). The polysaccharide from R. maritima (1 mg) was mixed with 0.5 ml (−)-2-butanol, containing 1 M HCl (Aldrich, Milwaukee, WI). After butanolysis for 18 h at 80°C, the solution was neutralized with Ag2CO3, and the supernatant was concentrated and dissolved in 50 ml. Thereafter, we added 50 ml bis(trimethylsilyl)trifluoro acetamide (Sigma, St. Louis, MO) and kept the solution for 30 min at room temperature. The butanolyzed and trimethylsilylated derivatives were analyzed on a DB-5 GLC column. The temperature was programmed from 120°C to 240°C at 2°C/min. The injector and detector temperatures were 220°C and 260°C, respectively. Appropriate controls of trimethylsilylated (−)-2-butyl-D- and L-galactosides were analyzed under the same conditions.

Desulfation and methylation of the sulfated galactan

Desulfation of the sulfated galactan was performed as described (Mourão and Perlin, 1987; Vieira et al., 1991). The sulfated polysaccharide (10 mg) was dissolved in 5 ml distilled water and mixed with 1 g (dry weight) of Dowex 50-W (H1, 200–400 mesh). After neutralization with pyridine, the solution was lyophilized. The resulting pyridinium salt of the sulfated galactan was dissolved in 2.5 ml dimethyl sulfoxide:methanol (9:1,v/v). The mixture was heated at 80°C for 4 h, and the desulfated product was exhaustively dialyzed against distilled water and lyophilized.

The desulfated galactan (5 mg) was subjected to three rounds of methylation as described previously (Ciucanu and Kerek, 1984) with the modifications suggested by Patankar et al. (1993). The methylated polysaccharide was hydrolyzed in 6 M trifluoroacetic acid for 5 h at 100°C and reduced with borohydride. The alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) (Kircher, 1960). The alditol acetates of the methylated sugar was dissolved in chloroform and analyzed in a gas chromatography–mass spectrometer (DB-1 capillary column).

NMR spectroscopy

1H and 13C spectra of the native and desulfated galactan were recorded using a Bruker DRX 400 apparatus with a triple-resonance probe. About 4 mg of each sample was dissolved in 0.5 ml 99.9% D2O (Cambridge Isotope Laboratory, Andover, MA). All spectra were recorded at 60°C with HOD suppression by presaturation. COSY, TOCSY, NOESY, and 1H/13C HMQC spectra were recorded using states-time proportion phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with 4046 × 400 points with a spin-lock field of ∼10 kHz and a mixed time of 80 ms. HMQC spectra were run with 1024 × 256 points and globally optimized alternating phase rectangular pulses for decoupling. NOESY spectra were run with a mixing time of 100 ms. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H and to methanol for 13C.

Sulfated polysaccharides localization by optical microscopy

Samples of the seagrass R. maritima were collected in the same area described, transported to the laboratory in plastic bags with sea water, and cleaned from other organisms. Fragments of 5 mm diameter were cut from the leaves, roots, and rhizomes and immediately fixed in 2.5% glutaraldehyde (Merck EM grade) buffered with 0.2 M sodium cacodylate (pH 7.2) in sea water for 2 h at room temperature. The fragments were rinsed in the same buffer, dehydrated with acetone graded series, and embedded in Spurr resin. Semithin sections (2 µm) were obtained in a microtome (Sorvall, Asheville, NC) and stained with 1% toluidine blue (Sigma), pH 4.4, for 3 min at 40°C. This stain indicates the presence of sulfated polysaccharides by metachromasia. The slides of the three compartments of the seagrass were observed in a Nikon Eclipse, and digital images were acquired using the software Image Pro Plus using the same conditions for all slides.

GenBank database analysis

To look for homologous sequences of the sulfotransferase gene, we conducted a search on GenBank database of two terrestrial plants with known genomic sequences. Because various enzymes act on the sulfation of diverse compounds, such as alcohols, phenols, and steroids, we had to compare the sequences of genes for an enzyme specific for sulfation of polysaccharide between several species. Thus, we tested a consensus sequence of eight 2-O-sulfotransferases (the seagrass polysaccharide has a sulfate at the position 2). The sulfotransferase sequences were identified using an in silico approach. The screening for 2-O-sulfotransferase sequences was performed on the National Center for Biotechnology Information GenBank sequence database (accession numbers: BC059008.1, BC025443.1, NM_204481.1, NM_012262.2, AB093516.1, NM_011828.2, AF169243, AB024568.1) and translated in all six frames using the Expert Protein Analysis System translate tool. A highly conserved consensus sequence (MFRKMGLLRIMMPPKHWLQLLAVVAFAVAMLFLENQIQKLEESRAKLERAIARHEVREIEQRHTMDGPRQDATLDEEEDIIIIYNRVPKTASTSFTNIAYDLCAKNRYHVLHINTTKNNPVMSLQDQVRFVKNITTWNEMKPGFYHGHISYLDFAKFGVKKKPIYINVIRDPIERLVSYYYFLRFGDDYRPGLRRRKQGDKKTFDECVAEGGSDCAPEKLWLQIPFFCGHSSECWNVGSRWAMDQAKSNLINEYFLVGVTEELEDFIMLLEAALPRFFRGATDLYRTGKKSHLRKTTEKKLPTKQTIAKLQQSDIWKMENEFYEFALEQFQFIRAHAVREKDGDLYILAQNFFYEKIYPKSN) was obtained after alignment using CLUSTAL W (Thompson et al., 1994). tBLASTn searches against the plant genome data base of Arabidopsis thaliana and Oryza sativa using the consensus sequence of 2-O-sulfotranferase as a query to identify a plant homologous sequence to polysaccharide sulfotranferase.

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. We thank Adriana A. Piquet for technical assistance, Dr. Orlando B. Martins for help on the search of homology sequences of sulfotransferase genes in plant species, and Hailton G. Aquino and Antonia G. S. Aquino for their help in the collection of seagrasses. This work was submitted as part of a Ph.D. thesis by R.S. Aquino to the Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro.

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Author notes

2Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brasil; 3Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brasil; 4Centro de Ressonância Nuclear Magnética de Macromoléculas, Universidade Federal do Rio de Janeiro; and 5Laboratório de Biomineralização, Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro