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Brown algae (Phaeophyceae) are photoautotrophic marine organisms which belong to the phylum of Stramenopiles. This eukaryotic supergroup arose c. 1 billion yr ago on a secondary endosymbiotic event, by which a unicellular red alga was captured by an ancestral protist (Reyes-Prieto et al., 2007). In phylogenetic analyses, chromalveolates form a distinct group from animals and fungi (Opisthokonts) on the one hand, and red algae, green algae and plants (Archaeplastida) on the other (Baldauf, 2008). Thus, brown algae evolved complex multicellularity independently from Opisthokonts and Archaeplastida (Grosberg & Strathmann, 2007). As such, they represent an interesting outgroup to investigate the evolution of multicellularity in Eukaryotes. One key step in this evolutionary process was the development of an adherent extracellular matrix (ECM), allowing for the transition from cellular autonomy to cellular cooperation. Multicellular Eukaryotes share other common characteristics, such as the ability to strictly control cell differentiation in space and time (development), as well as the evolution of sophisticated defense systems (innate immunity). The ECM plays an important role in both of these functions, as a first boundary for nonself recognition and as the seat of cell–cell signaling (Brownlee, 2002).
The ECM of extant Eukaryotes is typically organized as a three-dimensional network of crystalline fibers embedded in amorphous components. In fungi and photosynthetic organisms, cells are bound by a rigid cell wall, essentially made up of polysaccharides, including crystalline β-1,3-d- and β-1,4-d-glucans, respectively. By contrast, the metazoan ECM is essentially made up of fibrillar proteins (collagens) interconnected by protein-bound sulfated polysaccharides, referred to as glycosaminoglycans (GAGs; Sugahara & Kitagawa, 2002). Brown algae have evolved a cell wall (Fig. 1) which shares features with both plants and animals and which also exhibits some unique characteristics (Kloareg & Quatrano, 1988). Like plants, brown algae produce cellulose, but these crystalline fibers account for only a small proportion of the cell wall, the amount of cellulose ranging between 1% and 8% of the dry weight of the thallus (Cronshaw et al., 1958). The main cell wall components are anionic polysaccharides, namely alginates and fucoidans (Kloareg & Quatrano, 1988). Alginates consist of two uronic acids, β-1,4-d-mannuronate and α-1,4-l-guluronate, arranged in blocks along the polysaccharide chain (Fig. 1a). Alginates are initially polymerized as mannuronan and the guluronate residues are made at the polymer level by the action of mannuronate C5-epimerases (MC5Es; Haug & Larsen, 1969). Alginates form gels in the presence of divalent cations and their self-assembling properties depend on the relative proportion of guluronate homopolymeric blocks (Haug et al., 1974).
Figure 1. Structures of the main polysaccharides typical of brown algae: (a) alginate; (b) sulfated fucan from Fucales; (c) sulfated fucan from Ectocarpales. (d) Hypothetical model of the biochemical organization of cell walls of brown algae (adapted from Kloareg et al., 1986).
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Fucoidans are sulfated polysaccharides containing α-l-fucose residues and occur in brown algae, but also in the body wall of sea cucumbers and in the egg jelly coat of sea urchins. Although echinoderm fucoidans are linear and repetitive polysaccharides (Pomin & Mourao, 2008), brown algal fucoidans encompass a continuous spectrum of highly ramified polysaccharides, ranging from high-uronic-acid, low-sulfate-containing polymers with significant proportions of d-xylose, d-galactose and d-mannose (xylo-fuco-glucuronan and xylo-fuco-glucans) to highly sulfated homofucan molecules (Mabeau et al., 1990). The sulfated fucans from Fucales contain long stretches of the disaccharide repeating unit (→4)-α-l-fucose-2,3-disulfate-(1→3)-α-l-fucose-2-sulfate-(1→) (Fig. 1b) (Chevolot et al., 2001; Colin et al., 2006). The sulfated fucans from Laminariales (Nishino et al., 1991) and Ectocarpales (Ponce et al., 2003) display a structure mainly based on 3-linked α-l-fucose residues mostly sulfated at C4 (Fig. 1c).
Alginates, fucoidans and cellulose are in an average weight ratio of 3 : 1 : 1 in mature intertidal brown algae (Kloareg & Mabeau, 1987). In addition, brown algal cell walls contain phlorotannins, which consist of halogenated and/or sulfated phenolic compounds (Vreeland et al., 1998; Schoenwaelder & Wiencke, 2000) and c. 5% of proteins (Quatrano & Stevens, 1976). Microchemical imaging analyses have recently demonstrated that the cell walls of Laminariales bind huge amounts of iodine (Verhaeghe et al., 2008), probably through the involvement of apoplastic, vanadate-dependent iodoperoxidases (Colin et al., 2005). In the presence of halides, vanadate haloperoxidases also catalyze the cross-linking of alginates with phlorotannins (Berglin et al., 2004), suggesting an important role of the halogenated and phenolic compounds in cell wall cohesion. Alginates, sulfated fucans and phlorotannins are synthesized in the Golgi, transported in vesicles to the plasma membranes and secreted into the expanding cell wall (Callow et al., 1978; Schoenwaelder & Wiencke, 2000). Cellulose microfibrils are produced and deposited in situ by cellulose synthase complexes (terminal complexes) localized in the plasma membrane (Peng & Jaffe, 1976). In contrast with plants, however, terminal complexes do not form rosettes in brown algae, but single rows, comprising between 10 and 100 subunits. This linear arrangement results in a flat ribbon-like shape of cellulose microfibrils, with a uniform thickness of c. 2.6 nm and a variable width in the range 2.6–30 nm (Tamura et al., 1996; Tsekos, 1999). Another difference from terrestrial plants is that the terminal complexes are guided by F-actin filaments, and not by microtubules (Bisgrove & Kropf, 2001). Cell morphogenesis and cell wall deposits have been essentially studied in Fucales zygotes (Kropf et al., 1988; Fowler & Quatrano, 1997). At germination, tip growth is initiated at a determined site at the surface of the zygote and a rhizoid emerges. The apical cell of the rhizoid will then elongate by tip growth, whereas cells in the thallus will elongate by diffuse growth (Bisgrove & Kropf, 2001). As in plants, cell expansion is driven by turgor pressure in brown algae, but the biochemical mechanisms underlying cell wall relaxation are currently unknown.
The molecular bases of cell wall biogenesis in brown algae have received little attention so far. Our current knowledge is essentially limited to the terminal step of alginate biosynthesis, the conversion of d-mannuronate into l-guluronate. Two full-length cDNAs, homologous to bacterial MC5E, were isolated from Laminaria digitata (Nyvall et al., 2003) and their expression pattern confirmed that these cDNAs encode functional MC5E. Based on phylogenetic analyses, it was proposed that MC5E, and probably the entire pathway for the biosynthesis of alginates, was acquired by brown algae from an ancestral alginate-producing bacterium (Nyvall et al., 2003). MC5Es form a large multigenic family of at least 45 genes in L. digitata, which can be divided into several subgroups according to their expression profile, suggesting a fine tuning of alginate structure in response to developmental and environmental conditions (Roeder et al., 2005; Tonon et al., 2008).
Ectocarpus siliculosus is a filamentous brown alga from the order Ectocarpales (Charrier et al., 2008), a sister group of Laminariales which includes the most complex brown algae (Bartsch et al., 2008). The sequencing of its complete, 214-Mbp genome yielded a wealth of data that provide inferences on the adaptation of this organism to the intertidal environment and on the evolution of muticellularity, such as the presence of a rich array of signal transduction genes, including a family of receptor kinases specific to brown algae (Cock et al., 2010). The genome sequence also provides an in-depth, comprehensive view of the carbohydrate metabolism of brown algae (Michel et al., 2010). In this article, we reconstruct the metabolic pathways for the biosynthesis and remodeling of cellulose, alginate and sulfated fucans in brown algae, and discuss the origin and evolution of ECM polysaccharides in photosynthetic Eukaryotes.
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Very few studies have looked at the evolution of extracellular matrices across the whole eukaryotic tree (Niklas, 2004), and those that have been carried out were focused essentially on the biosynthesis of cellulose and hemicelluloses (Nobles et al., 2001; Nakashima et al., 2004; Yin et al., 2009) and chitin (Ruiz-Herrera et al., 2002). Brown algae are the only organisms in the Stramenopile lineage to have evolved complex multicellularity, thus providing a new angle to address the relationships between the development of multicellular organisms and the differentiation of their ECM. As brown algae are marine organisms, and as life first evolved in the sea, these seaweeds also provide a reference to better understand the specific traits that were developed by plants to colonize terrestrial environments. We have predicted the metabolic routes which lead to the synthesis of main brown algal ECM polysaccharides, cellulose, hemicelluloses, fucans and alginates, based on the gene content of the recently released genome of Ectocarpus siliculosus, the first available for a multicellular Stramenopile (Cock et al., 2010). Nonetheless, the exact function of the diverse candidate genes needs to be rigorously tested by future functional approaches. Below, we discuss the possible origins of these carbohydrates and propose various evolutionary scenarios to account for the evolution of the ECM cement and fiber polysaccharides in multicellular photosynthetic Eukaryotes.
Origin and speciation of cell wall polysaccharides in brown algae
Sulfated fucans are of ancestral origin Ectocarpus possesses the complete pathway for the de novo production of GDP-fucose from GDP-mannose. The existence of a salvage pathway is less certain, although candidate genes for FK and GFPP have been identified (Fig. 5). The GTs involved in the polymerization and branching of sulfated fucans are currently unknown. Ectocarpus contains four fucosyltransferases, from families GT10, GT23 and GT65. However, in other eukaryotic phyla, these enzymes are usually involved in protein glycosylation. Therefore, it is difficult to predict whether these fucosyltransferases are genuine fucan synthase(s) or whether brown algae have evolved novel GTs which cannot be detected by sequence comparisons with the currently known activities. By contrast, the sulfation and desulfation of fucans very probably are catalyzed by the six STs and the nine fgSs, which are related to the STs and fgS involved in the biosynthesis and remodeling of animal GAGs (Fig. 5). Note that the distant homologs of plant STs may not necessarily encode enzymes specific for sulfate transfer onto carbohydrates (Fig. 7). Yet, considering the widespread distribution of sulfated polysaccharides in archaeplastidal algae (see paragraph on land colonization), and based on the occurrence in the Ectocarpus genome of animal orthologs for both the synthesis of fucose and the addition or the removal of sulfate groups onto/from polysaccharide chains, the metabolism of sulfated fucans in brown algae is probably an ancestral pathway.
The Ectocarpus GTs from the GT14, GT47 and GT64 families are conserved in both animals and plants, indicating that these enzymes are also of ancestral origin. The animal GTs are involved in GAG biosynthesis (Gotting et al., 2000; Sugahara & Kitagawa, 2002), whereas the plant enzymes catalyze the branching of xyloglucans and pectins (Iwai et al., 2002; Madson et al., 2003) and the synthesis of β-1,4-galactan branches in pectins (Bown et al., 2007). The polyspecificity of these GT families makes it difficult to predict their exact function in brown algae. They may participate in the synthesis of the xyloglycan ramifications of sulfated fucans or in the synthesis of hemicellulose-like polymers.
Cellulose biosynthesis was acquired from the red algal endosymbiont In plants, the GT2 family comprises genuine cellulose synthases (CESA), but also CSL proteins, which are mainly involved in hemicellulose biosynthesis and have been classified into several subfamilies (from CSLA to CSLH) (Lerouxel et al., 2006). Molecular evidence suggests that plants acquired cellulose synthases and most CSL families from cyanobacteria (Nobles & Brown, 2004). The families CSLA and CSLC have a different origin and are most closely related to a CSL gene specific to green algae (Yin et al., 2009). A survey of sequenced algal genomes indicates that cellulose synthases are absent from diatoms and from the red microalga C. merolae (Yin et al., 2009). However, a CESA gene has been cloned recently from the red macroalga Porphyra yezoensis and clustered with cellulose synthases from Phytophthora, suggesting that Stramenopiles acquired CESA genes from their red algal endosymbiont (Roberts & Roberts, 2009). Our own phylogenetic analysis (Fig. 2) retrieved a topology similar to that observed previously (Roberts & Roberts, 2009; Yin et al., 2009). Ectocarpus contains four proteins, which emerge within the large group comprising the characterized cellulose synthases from Porphyra yezoensis, cyanobacteria, plants and Oomycetes. This predicts that Esi0004_0105, Esi0120_0014, Esi0231_0017 and Esi0231_0020 are genuine cellulose synthases. However, the exact position of Ectocarpus CESAs within this clade must be taken with caution. The brown algal sequences are unexpectedly closer to cyanobacterial cellulose synthases (c. 30% sequence identity) than to the CESAs from Porphyra yezoensis and Oomycetes (c. 25% sequence identity). This odd phylogenetic relationship may be explained by the lack of genomic data for a typical red alga, as C. merolae lacks a cell wall (Matsuzaki et al., 2004). A more representative, multicellular red alga probably would contain several CSL proteins, as observed for plants and brown algae. If this is the case, Oomycetes and brown algae may have conserved distinct red algal CSL proteins, as found for trehalose-phosphate synthases (Michel et al., 2010). Altogether, the most parsimonious scenario is that the common ancestor of brown algae and Oomycetes acquired the pathway for cellulose biosynthesis from their rhodobiont.
Polysaccharide pathways unique to brown algae In the alginate synthesis pathway shown in Fig. 3, steps 1–3 are not exclusive to alginate metabolism. They are common to all pathways which utilize activated mannose (e.g. protein glycosylation). The reactions specific for alginate biosynthesis are steps 4–6, from the conversion of the GDP-mannose into GDP-mannuronate to the final C5-epimerization of the polymannuronan chain into alginic acid. Brown algae are essentially the only Eukaryotes that possess alginate, although the presence of alginate has been reported in some calcareous red algae of the family Corallinaceae (Okasaki et al., 1982; Usov et al., 1995). However, the alginate pathway in Corallinaceae remains uncharacterized, at both the biochemical and gene levels. In Laminaria digitata, the last step of alginate biosynthesis is catalyzed by a large multigenic family of enzymes homologous to bacterial MC5Es (Nyvall et al., 2003). It was proposed that red algae acquired the alginate pathway from cyanobacteria via the primary plastid endosymbiosis and that, subsequently, this metabolism was passed on to brown algae via the secondary endosymbiosis (Nyvall et al., 2003). However, this evolutionary scenario suffers from several weaknesses: extant cyanobacteria do not contain MC5E genes; this scenario would require multiple losses of the alginate pathway in green algae, in plants and in the vast majority of the red algae (Archaeplastida), but also in the Stramenopiles other than brown algae, diatoms and Oomycetes.
On the basis of phylogenetic relationships (Fig. 4b), only the PMM Esi0149_0031 (step 2, Fig. 3) may have been acquired from Cyanobacteria, via the rhodobiont. By contrast, the first step of alginate biosynthesis is catalyzed by an ancient eukaryotic protein family (MPI, Fig. 4a). The MPG (step 3), present in bacteria and most Eukaryotes, is not encoded by the Ectocarpus and diatom genomes. Nevertheless, the presence of GDP-mannose and MPG activity has been demonstrated experimentally in the brown alga Fucus gardneri (Lin & Hassid, 1966). Therefore, brown algae and diatoms have lost the classical MPG genes, which are conserved in Oomycetes, but they are likely to possess a novel family of enzymes which catalyze the synthesis of GDP-mannose.
The GMD (step 4) and MC5E (step 6) of brown algae are conserved with a few bacteria only, including Gamma-proteobacteria and Actinobacteria. For both of these activities, as well as for the two candidate mannuronan synthases in Ectocarpus, Esi0010_0147 and Esi0086_0005, the closest orthologs are found in Frankia species (Actinobacteria). Therefore, we propose that the biogenesis of alginate in brown algae involves a hybrid pathway, with the initial, central carbon metabolism steps having an ancient eukaryotic origin, whereas the subsequent, alginate-specific steps were acquired by horizontal gene transfer (HGT) from an actinobacterium. In terrestrial environments, extant Frankia species occupy two distinct ecological niches, the soil and plant root nodules (Benson & Silvester, 1993). Similar associations may have existed between marine Actinobacteria and ancestral brown algae that promoted the HGT of alginate biosynthesis into the host. In the absence of genomic data for Corallinales, the origin of alginate in red algae is uncertain. We now favor a scenario more parsimonious than acquisition through the primary plastid endosymbiosis (Nyvall et al., 2003), namely an independent HGT with an alginate-producing bacterium limited to the ancestor of calcareous red seaweeds. Such more recent, independent HGTs have been observed, e.g. for amoeba and animals producing cellulose (Blanton et al., 2000; Nakashima et al., 2004).
The evidence for an HGT between Actinobacteria and brown algae is not limited to alginate metabolism. Phylogenetic analyses in the first part of this study (Michel et al., 2010) suggest that the capacity of brown algae to synthesize mannitol was also acquired from Actinobacteria. In addition, Ectocarpus contains a subfamily of GT2 CSL proteins which are conserved with Actinobacteria, fungi and two Phycodnaviruses only (Fig. 2). These proteins emerge as an independent clade robustly rooted with the actinobacterial representatives of this family-2 glycosyltransferase (Fig. 2), suggesting that brown algae and fungi independently acquired these CSLs by HGT from an ancestral actinobacterium. The Phycodnaviruses, which are double-stranded DNA viruses infecting algae (Dunigan et al., 2006), probably obtained these genes from brown algae or related Stramenopiles.
The uniqueness of brown algae is further highlighted by their cell wall remodelling enzymes. Ectocarpus does not feature any homologs of known cellulases, XTHs, alginate lyases or fucanases. The different families of plant expansins are also absent. Only the carbohydrate sulfatases potentially involved in the modification of sulfated fucans are found in the Ectocarpus genome. After the acquisition of cellulose from red algae, then of alginate and hemicellulosic material from Actinobacteria, brown algae may have evolved different molecular mechanisms for wall stress relaxation and expansion. More likely, they have evolved novel cellulases, hemicellulases and alginate lyases which are too derived to be detected by comparative genomics. If this is the case, a variety of new CAZY families and/or activities awaits to be discovered in the brown algae.
Insights into the origin and evolution of ECM polysaccharides in Eukaryotes
The loss of ester-sulfate substituents in the plant matrix polysaccharides, a consequence of land colonization All multicellular marine algae feature sulfated polysaccharides as major ECM components, such as ulvans and sulfated galactans in green algae (Lahaye & Robic, 2007; Farias et al., 2008), sulfated galactans in red algae and sulfated fucans in brown algae (Kloareg & Quatrano, 1988). Irrespective of their phylogenetic position, unicellular marine algae also produce extracellular sulfated polysaccharides in large amounts, including green microalgae (Sieburth et al., 1999), red microalgae (Simon-Bercovitch et al., 1999), diatoms (Hoagland et al., 1993) and haptophytes (Fichtinger-Schepman et al., 1981). Fossil data support that the first plants appeared on emerged land c. 500 million yr ago, diverging from a pioneer green alga related to extant Charales (Bateman et al., 1998). The ancestors of land plants faced several new constraints. Terrestrial environments provide no buoyancy. In their competition for light, land plants thus have evolved several specific characters, such as secondary cell wall reinforcement by lignins (Martone et al., 2009) and stronger cellulose fibers. The arrangement of the terminal complexes in rosettes, from the Characean algae and onwards in all of the terrestrial Archaeplastida, is notably recognized as an important innovation in the evolution of upright, nonaquatic plants (Graham et al., 2000).
Another major environmental change was the lower salinity, including a dramatic decrease in the availability of sulfate ions. The concentration of sulfate in seawater is high, 28 mM, whereas the concentration of this anion varies from 0.09 to 1.40 mM in freshwater and soil interstitial waters (Schmidt-Nielsen, 1997). In contrast with marine algae, the cell walls of freshwater and land plants do not contain any sulfated polysaccharides. Here, we have shown that the synthesis of sulfated polysaccharides is an ancestral metabolism in Eukaryotes. Notably, brown algae possess carbohydrate STs, FGEs and fgSs. In Archaeplastida, the presence of FGEs and fgSs has been established in the green microalga C. reinhardtii (Merchant et al., 2007). Volvox carterii is also known to express at least one fgS gene (Hallmann & Sumper, 1994), probably involved in the metabolism of its ECM sulfated glycoproteins (Ertl et al., 1989). By contrast, FGEs, fgSs and carbohydrate-specific STs are completely absent from the available genomes of land plants. We propose that FGE and fgS, as well as carbohydrate-specific STs, were present in the common ancestor of volvocine algae and land plants, and that these enzymes, and hence the capacity to synthesize and remodel sulfated polysaccharides, were lost on adaptation to sulfate-scarce freshwater and terrestrial environments. However, it is difficult to determine whether this process was completed before or after the transition from freshwater to the land. A similar, convergent evolution probably occurred within the Stramenopiles: the matricial polysaccharides of terrestrial Oomycetes, essentially β-(1,3)- and β-(1,6) glucans (Bartnicki-Garcia, 1968), are not sulfated and the Phytophthora genomes do not contain FGE and fgS genes.
The evolution towards complex multicellularity involved the expansion of ancestral matrix polysaccharides and the acquisition of skeletal fibers Based on the phylogenetic relationships of the various ECM polysaccharides depicted above, the ECM polysaccharide components of archetypal Eukaryotes probably consisted of sulfated polysaccharides and relatively short-chained anionic or neutral glycans. The latter probably included extracellular β-1,3-glucans, which are widespread in extant Eukaryotes, and which were retained as intracellular carbon storage compounds in the Stramenopiles (Michel et al., 2010). β-1,3-Glucans are capable of self-assembling into triple helices, but these fibers provide weaker biomechanical support than cellulose fibers (Stone & Clarke, 1992; Burgert, 2006). Hence, the ECM matrix of early Eukaryotes probably was made up of relatively flexible material, which could not support the building of organisms with complex morphology (Fig. 8).
Figure 8. Schematic flow chart illustrating the origin and evolution of the main extracellular matrix polysaccharides in the Archaeplastida and Stramenopiles. The origins of plant pectins and diatom chitin are unclear and these polysaccharides have not been included. Endosymbiosis events are indicated by dotted lines. PE, plastid primary endosymbiosis; SE, plastid secondary endosymbiosis; HGT, horizontal gene transfer; sulfated polysac., sulfated polysaccharides.
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In Archaeplastida and Stramenopiles, the acquisition of cellulose through the primary and secondary plastid endosymbioses, respectively, brought the capacity to build longer and stronger crystalline fibers. This gave rise to denser and stiffer ECM materials, which made it possible to erect more rigid and complex organisms (Fig. 8). This speciation probably also involved a marked expansion of the matrix polysaccharides from the common stock inherited from the hosts, in order to provide the various cements embedding and cross-linking of the cellulose fibers. Extant multicellular marine algae and land plants indeed feature a large variety of noncrystalline polysaccharides, which is reflected in the high diversity of CAZYmes and CAZYme families in the Arabidopsis (Henrissat et al., 2001) and Ectocarpus (Michel et al., 2010) genomes. Similar conclusions have been drawn, at the ECM matrix protein level, for the green algae of the lineage of Volvocines, an interesting example of a relatively recent (c. 50 million yr) transition towards multicellularity (Kirk, 2005).
Brown algae provide another interesting case study, in which the evolution towards complex multicellularity involved an independent HGT event with an ancestral actinobacterium. Here, we show that this HGT, which must have occurred after the divergence of the ancestor of brown algae from diatoms and Oomycetes, resulted in the acquisition of alginate, the main gel-forming polysaccharide in this lineage (Fig. 8), as well as new hemicellulose-like material. This provided a wealth of new molecular combinations to bridge the cellulose microfibrils with the other wall components, including alginate and sulfated fucans (as sketched in Fig. 1), as well as molecular bases for nonself recognition following pathogen attack (Küpper et al., 2001, 2002). The importance of alginate in the general biology of extant brown algae is reflected by the large number of MC5E genes: 28 in E. siliculosus, a filamentous alga (this study), and a minimum of 45 in the more complex, parenchymatous kelps (Roeder et al., 2005; Tonon et al., 2008). Another possible illustration of the key biological functions of alginate is the high frequency in the Ectocarpus genome of WSC protein domains, which may be involved in alginate–protein interactions. Finally, the HGT from Actinobacteria also provided a crucial storage compound, mannitol (Michel et al., 2010), the carbon translocation form in extant brown algae. Altogether, this HGT event was a turning point in the evolution of brown algae towards the acquisition of complex multicellularity.