The Cell Walls of Green Algae: a Journey Through Evolution and Diversity

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MINI REVIEW ARTICLE published: 08 May 2012 doi: 10.3389/fpls.2012.00082 The cell walls of green algae: a journey through evolution and diversity

David S. Domozych1*, Marina Ciancia2, Jonatan U. Fangel 3, Maria Dalgaard Mikkelsen3, Peter Ulvskov 3 and William G.T.Willats 3

1 Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, NY, USA 2 Cátedra de Química de Biomoléculas, Departamento de Biología Aplicada y Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina 3 Department of Plant Biology and Biochemistry, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark

Edited by: The green algae represent a large group of morphologically diverse photosynthetic eukary- Jose Manuel Estevez, University of otes that occupy virtually every photic habitat on the planet. The extracellular coverings of Buenos Aires and Consejo Nacional de Investigaciones Cientíﬁcas y green algae including cell walls are also diverse. A recent surge of research in green algal Técnicas, Argentina cell walls fueled by new emerging technologies has revealed new and critical insight con- Reviewed by: cerning these coverings. For example, the late divergent taxa of the Charophycean green Malcolm ONeill, University of algae possess cell walls containing assemblages of polymers with notable similarity to the Georgia, USA cellulose, pectins, hemicelluloses, arabinogalactan proteins (AGPs), extensin, and lignin Zoë A. Popper, National University of Ireland, Ireland present in embryophyte walls. Ulvophycean seaweeds have cell wall components whose β β *Correspondence: most abundant ﬁbrillar constituents may change from cellulose to -mannans to -xylans David S. Domozych, Department of and during different life cycle phases. Likewise, these algae produce complex sulfated Biology and Skidmore Microscopy polysaccharides, AGPs, and extensin. Chlorophycean green algae produce a wide array of Imaging Center, Skidmore College, walls ranging from cellulose–pectin complexes to ones made of hydroxyproline-rich glyco- 815 North Broadway, Saratoga Springs, NY 12866, USA. proteins. Larger and more detailed surveys of the green algal taxa including incorporation e-mail: [email protected] of emerging genomic and transcriptomic data are required in order to more fully resolve evolutionary trends within the green algae and in relationship with higher plants as well as potential applications of wall components in the food and pharmaceutical industries.

Keywords: cell walls, scales, green algae, sulfated polysaccharides, glycoproteins

INTRODUCTION integral to growth and development, affords the alga physical pro- The emergence of green algae (Chlorophyta and Streptophyta, tection and defense against microbial attack, is involved in cell–cell Viridiplantae; sensu Leliaert et al., 2012) onto land roughly 470 and cell–substrate adhesion and in some cases,is involved in sexual million years ago represents one of the most important events in reproduction. Some green algae are covered by multiple layers of the history of life on the planet. Their successful colonization of intricately sculpted scales while others have crystalline glycopro- land and subsequent evolution into modern land plants signifi- tein coverings or thick multilaminate fibrillar cell walls. A few taxa cantly altered the atmosphere, changed terrestrial substrates and though have cell walls with remarkable structural and biochem- paved the way for the evolution of other biota. Today, humans ulti- ical similarity to cell walls found in land plants (Sørensen et al., mately depend on the evolutionary “offshoots” of green algae (i.e., 2010, 2011). How did these diverse extracellular coverings arise embryophytes or “land plants”) for food, textiles, building mate- and what are the evolutionary links between them? Many ana- rial, pharmaceuticals, and fuels. Yet these events and applications lytical approaches and technologies are now being used to study represent only parts of a much larger story of green algae. Contem- green algal ECMs and are providing new and critical insight into porary green algae are ubiquitous. They are important members of structure, chemistry, and evolution of these coverings (Table 1). the ocean’s phytoplankton, common and sometimes nuisance sea- Nevertheless, we are only in an infancy stage in our understand- weeds of coastal marine habitats,peculiar symbionts of lichens and ing of the green algal extracellular coverings. In this review, we flatworms, and inhabitants of just about any freshwater ecosystem describe some of these recent discoveries and comment on future ranging from ponds, rivers, lakes, wetlands, and snow banks. In directions for study of the cell walls of green algae. the 1.5 billion years since they first appeared (Lewis and McCourt, 2004; Becker and Marin, 2009; Finet et al., 2010; Leliaert et al., THE CURRENT STATE OF STUDY OF GREEN ALGAL CELL 2011; Wodnick et al., 2011), green algae have successfully adapted WALLS AND EXTRACELLULAR COVERINGS to virtually all photic zones of the planet. While the green algae display a large and diverse array of ECM- Similar to their land plant offspring, the vast majority of extant coverings, only a few taxa have been studied in detail. It is green algae today are covered by a very large assortment of types widely accepted that taxa of the Ulvophyceae and the Charo- of extracellular matrix (ECM). These external coverings are prod- phycean green algae (CGA) possess fibrillar cell walls (Popper et al., ucts of complex biosynthetic machineries that often make use of 2011) consisting of various polysaccharide and proteoglycan con- the bulk of the alga’s photosynthetically fixed carbon. The ECM is stituents while other taxa, especially those of the Prasinophyceae,

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Table 1 | Major methodologies used today in the study of green algal coverings.

Methodology Technical aspects Data obtained/status Reference

Biochemical Chemical and enzymatic fractionation; Monosaccharide composition, glycosidic linkage Popper et al. (2011), methylation analysis-GC/MS; NMR; composition, conformational studies, molecular weights Popper and Fry (2003), electrophoresis of various cell wall polysaccharides Estevez et al. (2009) Carbohydrate Sequential extraction of Early divergent CGA walls differ from late divergent taxa Sørensen et al. (2010, microarrays polysaccharides; immobilization onto walls; late divergent taxa possess HGA, RG-I, MLG, 2011), Moller et al. nitrocellulose, mAb probing various hemicelluloses, AGPs, extensins (2007) Immunocytochemistry Immunoﬂuorescence and Wall polymer mAbs may be used in live cell studies; Domozych et al. (2009, immunogold labeling of live cells and Coleochaete walls possess lignin-like epitopes 2011), Eder and sections of ﬁxed cells Lutz-Meindl (2010), Sørensen et al. (2011) FTIR IR spectral arrays obtained from Analysis of presence and distribution of polymers in the Estevez et al. (2009), microspectroscopy microscopically imaged covering cell wall of Ulvophyceae Fernández et al. (2011a), Carpita et al. (2001) Molecular Transcriptome and genome Genomes sequenced in Volvox carteri, Chlamydomonas Blanc et al. (2010), acquisition; annotation of genes reinhardtii, Micromonas sp. RCC299, Ostreococcus Timme and Delwiche tauri, and Ostreococcus lucimarinus (see (2010), Vannerum et al. http://bioinformatics.psb.ugent.be/plaza/) and Chlorella (2011), Timme et al. variabilis NC64; several transcriptomes analyzed (2012) mAb, monoclonal antibody; RG-I, rhamnogalacturonan-I; MLG, mixed linkage glucans; AGP, arabinogalactan protein; FTIR, Fourier transform infrared; CGA, Charophycean green algae.

Chlorodendrophyceae, and some taxa of the Chlorophyceae, pro- CHLORODENDROPHYCEAE: WALLS FROM A FUSION duce coverings that are structurally and biochemically unique. Taxa of the small Chlorodendrophyceae group of green algae Presently, extant green algae are classified into six distinct clades consist of motile or non-motile and sometimes stalked unicells (Delwiche and Timme, 2011; Leliaert et al., 2011, 2012). The fol- (Tetraselmis and Scherffelia) that are covered by a single cell wall lowing represent brief synopses of the current state of knowledge or layers of cell walls. Furthermore, the wall or theca consists concerning the ECM of these groups (see also Table 2). of regular repeating subunits and unlike the cell walls of other green algae, this wall is believed to be a product of fused scales. THE PRASINOPHYCEAEN MATRIX: SUBTLE TO THE The subunits of the theca are scale-like and are processed in the SPECTACULAR! Golgi apparatus like those of scaly prasinophytes. The acid sugars, The Prasinophyceae or prasinophytes represent a group of motile 2-keto-3-deoxy-d-manno-octulosonic acid, 5-O-methyl 2-keto- and non-motile unicells that are presently classified in four clades 3-deoxy-d-manno-octulosonic acid, and DHA comprise 60% of (Leliaert et al., 2012) and are most commonly found in marine the sugars present in the theca (Becker et al., 1991). habitats. In photic zones of oceans, picoplanktonic prasinophytes (perhaps the smallest extant eukaryotes; 0.8 μm cell size), like TREBOUXIOPHYCEAE: WALLS OF UNUSUAL POLYMERS Ostreococcus and Micromonas, exist in very large numbers. Previ- The Trebouxiophyceae consists of an assemblage of primarily ous microscopy-based research has shown that taxa like these are freshwater and terrestrial forms that exhibit diverse phenotypes either covered with scales or do not have any discernable matrix ranging from unicells to colonies to filaments as well as represent- at all (i.e., they are naked; Piganeau et al., 2011). However, recent ing most of the photobiont green algae of lichens (e.g., Trebouxia ). analysis of the Ostreococcus genome plus immunocytochemical Some are considered highly attractive candidate genera for use in investigations in our laboratories together raise questions as to algal biofuel production (e.g., Chlorella; Rodrigues and da Silva the nakedness of this picoalga. Other prasinophytes are signifi- Bon, 2011). Most members of this group possess cell walls but cantly larger and covered with layers of thousands of distinctly surprisingly little is known about their biosynthesis, composition, shaped scales coating both the cell and flagellar membrane surfaces or architecture. In Chlorella, the wall contains cellulose and in (Moestrup and Walne, 1979). Biochemical analyses have shown some species, the wall is coated by a highly resistant outer stra- that these scales are comprised primarily of neutral and acidic sug- tum consisting of “algaenan,”an aliphatic polymer containing long ars including 2-keto sugars such as 3-deoxy-lyxo-2-heptulosaric polymethylenic chains that are decorated with amide and N -alkyl acid (DHA; Becker et al., 1991, 1994). All scales of prasinophytes substituted pyrroles (Rodrigues and da Silva Bon, 2011). In Tre- are believed to be processed in the Golgi apparatus, packaged in bouxia isolated from lichens and grown separately from its fungal secretory vesicles and secreted to the cell surface near the flagellar partner, β-galactofuranan has been demonstrated, a polysaccha- apparatus or to vacuole-like scale reservoirs before release to the ride previously found in fungi but not known from green algae cell surface. (Cordeiro et al., 2006).

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Table 2 | Summary of the composition of extracellular coverings in green algae.

Taxon Covering type Biochemical composition Reference

Prasinophyceae “Scales,” coatings 2-Keto sugars (e.g., DHA), mannans, glycoproteins Becker et al. (1991, 1994), Moestrup and Walne (1979) Chlorodendrophyceae Wall of fused scales 2-Keto sugars (e.g., DHA), proteins Becker et al. (1991) Trebouxiophyceae Cell walls Cellulose, algaenan, β-galactofuranan Rodrigues and da Silva Bon (2011), Cordeiro et al. (2006) Chlorophyceae Crystalline glycoprotein Hyp-rich glycoproteins, cellulose pectins, AGP,extensin Voigt et al. (2001, 2007), Kirk (1998), walls; ﬁbrillar cell walls Estevez et al. (2008) Ulvophyceae Cell walls Cellulose, β-mannans, β-xylans, sulfated (sometimes Ciancia et al. (2012), Estevez et al. pyruvylated) polysaccharides or sulfated (2009), Percival (1979), Lahaye and rhamnogalacturonans, AGP,extensin Robic (2007) Charophyceae-early Scales, cell walls 2-Keto sugars, cellulose, homogalacturonans, 1,3 Sørensen et al. (2011), Domozych et al. divergent clades β-glucans, AGP (1991) Charophyceae-late Cell walls Cellulose, homogalacturonans, RG-I xyloglucans, Sørensen et al. (2011, 2012), Popper divergent clades mannans, xylans, mixed linkage glucans, 1,3 β-glucans, and Tuohy (2010) AGP,extensin, lignin

For further detailed information, key references are provided. AGP,arabinogalactan proteins; Hyp, hydroxyproline.

CHLOROPHYCEAE: GLYCOPROTEINS AND CELLULOSE glycosyltransferases to deduce the function of the Arabidopsis The Chlorophyceae are the largest group of green algae and exhibit GT77 clade A genes. This proposition that the genes encode great morphological diversity ranging from motile unicells to extensin arabinosyltransferases,was later corroborated by extensin large filaments to blade-like thalli. The extracellular coverings of phenotypes of mutants knocked out in these genes (Velasquez the Chlorophycean algae are also very diverse and consist of a et al., 2011). The prolyl hydroxylases described in that paper also distinct assortment of “cell walls.” In Oedogonium, the cell wall have orthologs in the chlorophyte genomes and one from Chlamy- resembles those of higher plants in containing microfibrillar cel- domonas has been shown to be involved in cell wall biogenesis lulose, homogalacturonans and rhamnogalacturonan-I, extensin, (Keskiaho et al., 2007) and the CAZy-database classifies Chlamy- and arabinogalactan constituents (Estevez et al., 2008). Many domonas gene BAF46284 to family GT75. The encoded protein is of the polysaccharides that are common to embryophyte walls 68.6% identical to Arabidopsis At3g02230, one of the mutases that are thought to have evolved within the CGA, so these results catalyzes the interconversion of UDP-Arap and UDP-Araf (Raut- were unexpected. Further biochemical study and a much wider engarten et al., 2011), which we believe to be the donor substrate screening of this and other Chlorophycean taxa will be required also of these arabinosyl transferases. to ascertain the similarity of these polymers with those of the CGA and embryophytes. However, in the Chlamydomonas–Volvox ULVOPHYCEAE: SULFATED POLYSACCHARIDES, FIBRILLAR assemblage (i.e., volvocalean flagellates), the cell wall does not con- POLYMERS, AND GLYCOPROTEINS tain cellulose but is made of crystalline glycoproteins, specifically The Ulvophyceae area diverse assemblage of organisms including one based upon aggregates of hydroxyproline-rich glycoproteins the best known marine seaweeds with siphonocladous, sipho- (HRGPs) and glycine-rich glycoproteins (Imam et al., 1985; Adair nous, and filamentous forms (Leliaert et al., 2012). Some of them et al., 1987; Kirk, 1998; Voigt et al., 2001, 2007). Extensins of have cellulose as a major fibrillar component of their cell walls. plants are a group of cell wall glycoproteins that probably share However, others possess (1→4)-β-mannans or (1→3)-β-xylans at least some glycosylation motives and a common ancestry with (Percival and McDowell, 1981; Painter, 1983;Yamagakiet al., 1997; the HRGPs of Chlamydomonas–Volvox assemblage. The glycosy- Dunn et al., 2007; Estevez et al., 2009; Ciancia et al., 2012) and lation motives that govern extensin-type glycosylation comprise in some, the type of fibrillar polysaccharide is life cycle stage- the SPPPP sequence (i.e., serine-proline-proline-proline-proline) dependent (Huizing and Rietema, 1975; Wutz and Zetsche, 1976; usually occurring several times. The prolines are hydroxylated by Huizing et al., 1979; Dunn et al., 2007). prolyl hydroxylases prior to glycosylation. Showalter et al. (2010) These algae synthesize sulfated polysaccharides, an adaptation used SPPPSPPP to define the class of extensins in their bioin- to the marine habitats that is also observed in many other marine formatic classification of HRGPs. The genetic encoding of the organisms, including angiosperms and invertebrates (Aquino repetitive structures allows for substantial genetic drift without et al., 2011). These sulfated wall constituents may be classified loss of function (Kieliszewski and Lamport, 1994), this being the into one of two groups as originally designated by Percival (1979): reason why clear orthologies between individual vascular plant (1) uronic acid-rich polysaccharides also containing rhamnose, and chlorophyte extensins cannot be traced. The core arabinosy- xylose, and sometimes galactose, and (2) uronic acid-limited poly- lation machinery is clearly equivalent, however. Egelund et al. saccharides consisting of major quantities of galactose, arabinose (2007) used the phylogenetic distances between family GT77 and, in some cases, xylose. The first group is represented by Ulva,

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Monostroma, Gayralia, and Acetabularia (Ray, 2006; Chattopad- and Zygnematales, possess cell walls with notable similarity to hyay et al., 2007a; Dunn et al., 2007; Lahaye and Robic, 2007; those of land plants. Polysaccharides like cellulose,pectins (includ- Cassolato et al., 2008; Mao et al., 2008, 2009) and the second ing homogalacturonans and rhamnogalacturonan-I), β-(1-3) glu- one includes Caulerpa, Codium, and Bryopsis (Bilan et al., 2007; cans, and hemicellulosic polymers like xyloglucans, mannans, and Chattopadhyay et al., 2007b; Estevez et al., 2009; Ciancia et al., xylans are found in the cell wall of these advanced CGA. Mixed 2012). linkage glucans or MLGs that were once thought to be found Cell walls from the first group as represented by several Ulva only in members of the grass family of angiosperms, were also species comprise two major polysaccharide components, soluble found in Zygnematalean taxa, demonstrating a more widespread ulvans and cellulose, and two minor ones, an alkali-soluble linear occurrence in green plants. Also surprising is the presence of xyloglucan and a glucuronan. Ulvan is the family of sulfated poly- lignin-like compounds in the cell walls of several species of the saccharides that consist of large quantities of glucuronic acid and genus Coleochaete. rhamnose with the main repeating disaccharide being →4)-β-d- Arabinogalactan proteins and extensin have also been described GlcAp-(1→4)-α-l-Rhap-(1→. α-l-Iduronic acid or β-xylose can in many CGA taxa. Extensins surprisingly are not detected in replace glucuronic acid to a certain extent with the latter sugar also Nitella and Chara (Charales; Gotteli and Cleland, 1968). Ara- found as a side chain linked to C-2 of some rhamnose residues. binosyltransferases involved in extensin arabinosylation belong Sulfation appears mostly on C-3 of the rhamnose units and on C-2 to the GT77 A- and C-clades sensu Petersen et al. (2011) and of the glucuronic acid side chains. Distribution of these polymers it is remarkable that the Chara transcriptome (courtesy of Ger- in the cell walls of the bi-seriated thallus of Ulva was determined not Glöckner, Leibniz-Institute of Freshwater Ecology and Inland by cytochemical and physicochemical data (Lahaye and Robic, Fisheries, Berlin) as well as that of Nitella hyalina (Timme et al., 2007). These polymers are structurally related to glycosamino- 2012) each have a putative member in family GT77 clade D, but glycans from animal tissues, like chondroitin sulfate or heparin, none in clade A or C. The transcriptome of Nitella, but not that of but they have glucosamine units instead of rhamnose in their Chara comprises putative prolyl hydroxylases allowing for the exis- backbone, and the sulfation pattern is different. tence of AGPs even though extensins are missing. It thus appears The second group is represented by coenocytic Codium species, that members of Charales have evolved and separated themselves which produce a 4-linked β-D-mannan as fibrillar component. significantly from other members of the CGA, notably the Zygne- Three different sulfated polysaccharide structures were isolated matales and Coleochaetales which feature cell walls that more from C. vermilara including: (1) highly ramified sulfated and closely resemble that of vascular plants and which are known pyruvylated β-(1→3)-d-galactans, also present in other Codium to express enzymes involved in cell wall metabolism akin to that species (Bilan et al., 2007; Ciancia et al., 2007; Farias et al., 2008; observed in angiosperms (e.g.,Vannerum et al., 2011). These latter Ohta et al., 2009; Fernández et al., 2011a); (2) linear highly sulfated similarities suggest that late divergent taxa of the CGA (i.e., their β-(1→3)-l-arabinans, with the arabinose units in the pyranose ancestors 470 million years ago) may have possessed cell wall char- form (P. V. Fernandez, J. M. Estevez, A. S. Cerezo, and M. Cian- acteristics that pre-adapted them for successful emergence onto cia, unpublished results); and (3) linear β-(1→4)-d-mannans and life on land. partially sulfated mainly on C-2 of some of the mannose units (Fernández et al., 2011b). Also, HRGPs with characteristics simi- THE NEXT GOALS? lar to those of arabinogalactan proteins (AGPs) and extensins from More detailed characterization of the various taxa will be required vascular plants were detected in these cell walls (Estevez et al.,2009; before we can fully understand the evolution of extracellular cov- Fernández et al., 2011a). Distribution of β-mannans and HRGP- erings of green plants as well as adaptations to ECM chemistry like epitopes was shown to be found in two distinct cell wall layers, in response to life in marine, freshwater, and terrestrial habitats. whereas sulfated polysaccharides were distributed in the middle Some specific questions and areas of focus for future study include: area of the wall. (1) Insights into the polysaccharide biosynthetic machinery of CHAROPHYCEAE: CELL WALLS AND THE ORIGIN OF LAND CGA are and will at the same time offer insights into the evo- PLANTS lutionary events that accompanied adaptation to life on land. The Charophyceae or CGA are the extant group of green algae This will require full genomic sequencing as transcriptomic most closely related and ancestral to land plants. Over the past analysis is useful for demonstrating the functional expression decade, a surge of research activity focusing on the walls of CGA of genes, but not for the absence. This endeavor offers many taxa has revealed important insight into cell wall structure and challenges as many relevant CGA genomes are estimated to evolution (Popper and Fry, 2003; Domozych et al., 2007, 2009; be as big as if not bigger than that of Arabidopsis (Kapraun, Eder et al., 2008; Eder and Lutz-Meindl, 2010; Popper and Tuohy, 2007). 2010; Sørensen et al., 2010, 2011; Popper et al., 2011). First, the (2) Detailed analysis of the roles of cell wall polymers in the CGA cell walls of taxa of the early divergent CGA such as the Chloroky- is critical. Although the CGA share many cell wall constituents bales and Klebsormidiales, do not possess most of the polymers with their embryophyte descendents, it is not clear if they are commonly found in late divergent CGA and land plants. The used in equivalent roles. basal clade of CGA, the Mesostigmales, does not even have a cell (3) Sulfated polysaccharides from ulvophyte seaweeds have only wall but rather produces layers of scales (Domozych et al., 1991). recently been reexamined and have yet to be widely used Second, the late divergent clades, the Charales, Coleochaetales, in the hydrocolloid industry. Ulvan has been investigated as

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potential dietary ﬁber for human diet (Lahaye and Robic, The study of green algal cell walls and other coverings has now 2007) due to its medical properties and potentially prof- entered a truly exciting phase whereby new methodologies espe- itable extraction yields from harvested specimens. In addition, cially from the biochemical and molecular fronts are allowing for ulvans have some interesting biological activities, including detailed resolution of wall polymers. While evolutionary and cell acting as antioxidants, modifying certain macrophage activi- biology-based studies have been and will continue to be driving ties, and serving as potential anti-hyperlipidemic agent (Wije- forces for this, the study of green algal coverings represents a criti- sekara et al., 2011). Sulfated polysaccharides from the Bryop- cal step for emerging applied technologies as green algae are being, sidales are not obtained in large yields (Ciancia et al., 2007, or will be, used as food sources for humans and domesticated 2012), but have piqued interest as bioactive compounds with animals, hydrocolloids for the food and pharmaceutical industry, several potential pharmacological applications (Ohta et al., bioactive compounds for medicinal use and starting material for 2009; Ciancia et al., 2010; Costa et al., 2010; Lee et al., 2010). biofuels. More detailed characterization of these molecules is needed in order to fully recognize their potential. ACKNOWLEDGMENTS (4) Comprehensive chemical and functional screening of the large This work was supported by grants from CONICET (PIP but virtually unknown polysaccharide complexes secreted 559/2010) and ANPCYT (PICT 2008-0500) of Argentina to outside the cell walls of many Zygnematalean taxa (Domozych Marina Ciancia, the Danish Research Council (FTP-09-066624) et al., 2005) will be important for understanding their to Peter Ulvskov, the National Science Foundation (USA) grant physiology and importance to ecosystem dynamics. MCB-0919925 to David S. Domozych.

REFERENCES Carpita, N. C., Defernez, M., Findlay, A., Estevez, J. M., and Cerezo, Streptophyta). Asymmetry in a sym- Adair, W. S., Steinmetz, S. A., Matt- K., Wells, B., Shoue, D. A., Catch- A. S. (2007). Polysaccharides from metric world. J. Phycol. 45, 879–893. son, D. M., Goodenough, U. W., pole, G.,Wilson, R. H., and McCann, the green seaweeds Codium fragile Domozych, D. S., Serfis, A., Kiemle, and Heuser, J. E. (1987). Nucleated M. C. (2001). Cell wall architecture and C. vermilara with controversial S. N., and Gretz, M. R. (2007). assembly of Chlamydomonas and of the elongating maize coleoptile. effects on hemostasis. Int. J. Biol. The structure and biochem- Volvox cell walls. J. Cell Biol. 105, Plant Physiol. 127, 551–565. Macromol. 41, 641–649. istry of charophycean cell walls. 2373–2382. Cassolato, J. E. F., Noseda, M. D., Pujol, Cordeiro, L. M., Carbonero, E. I. Pectins of Penium mar- Aquino, R. S., Grativol, C., and C. A., Pellizzari, F. M., Damonte, E. R., Sassaki, G. L., Reis, R. A., garitaceum. Protoplasma 230, Mourão, P. A. S. (2011). Rising from B., and Duarte, M. A. R. (2008). Stocker-Worgotter, E., Gorin, P. 99–115. the sea: correlations between sul- Chemical structure and antiviral A. J., and Iacomini, M. (2006). Domozych, D. S., Wells, B., and Shaw, fated polysaccharides and salinity activity of the sulfated heterorham- A fungus-like β-galactofuranan P. J. (1991). The basket scales of the in plants. PLoS ONE 6, e18862. nan isolated from the green seaweed in the cultivated Trebouxia pho- green alga Mesostigma viride: chem- doi:10.1371/journal.pone.0018862. Gayralia oxysperma. Carbohydr. Res. tobiont of the lichen Ramalina istry, immunology and ultrastruc- Becker, B., Becker, D., Kamerling, J. P., 343, 3085–3095. gracilis. FEMS Microbiol. Lett. 244, ture. J. Cell Sci. 100, 397–407. and Melkonian, M. (1991). 2-Keto- Chattopadhyay, K., Mandal, P., Lerouge, 193–198. Dunn, E. K., Shoue, D. A., Huang, sugar acids in green flagellates: a P., Driouich, A., Ghosal, P., and Ray, Costa, L. S., Fidelis, G. P., Cordeiro, S. L., X., Kline, R. E. L., MacKay, A. chemical marker for prasinophycean B. (2007a). Sulphated polysaccha- Oliveira, R. M., Sabry, D. A., Cámara, L., Carpita, N. C., Taylor, I. E. P., scales. J. Phycol. 27, 498–504. rides from Indian samples of Entero- R. B. G., Nobre, L. T. D. B,. Costa, and Mandoli, D. F. (2007). Spectro- Becker, B., and Marin, B. (2009). morpha compressa (Ulvales, Chloro- M. S. S. P., Almeida-Lima, J., Farias, scopic and biochemical analysis of Streptophyte algae and the origin phyta): isolation and structural fea- E. H. C., Leite, E. L., and Rocha, H. regions of the cell wall of the uni- of embryophytes. Ann. Bot. 103, tures. Food Chem. 104, 928–935. A. O. (2010). Biological activities of cellular ‘mannan weed’, Acetabularia 999–1004. Chattopadhyay, K., Adhikari, U., Ler- sulfated polysaccharides from tropi- acetabulum. Plant Cell Physiol. 48, Becker, B., Marin, B., and Melkonian, M. ouge, P., and Ray, B. (2007b). Poly- cal seaweeds. Biomed. Pharmacother. 122–133. (1994). Structure, composition, and saccharides from Caulerpa race- 64, 21–28. Eder, M., and Lutz-Meindl, U. (2010). biogenesis of prasinophyte cell cov- mosa: purification and structural Delwiche, C. F., and Timme, R. E. Analyses and localization of pectin- erings. Protoplasma 181, 233–244. features. Carbohydr. Polym. 68, (2011). Plants. Cur. Biol. 21, R417– like carbohydrates in cell wall and Bilan, M. I., Vinogradova, E. V., 407–415. R423. mucilage of the green alga Netrium Shashkov, A. S., and Usov, A. I. Ciancia, M., Alberghina, J., Arata, P. Domozych, D. S., Brechka, H., Britton, digitus. Protoplasma 243, 25–38. (2007). Structure of a highly pyruvy- X., Benavides, H., Leliaert, F., Ver- A., and Toso, M. (2011). Cell wall Eder, M., Tenhaken, R., Driouich, lated galactan sulfate from the pacific bruggen, H., and Estevez, J. M. growth and modulation dynamics A., and Lutz-Meindl, U. (2008). green alga Codium yezoense (Bry- (2012). Characterization of cell wall in a model unicellular green alga – Occurrence and characterization opsidales, Chlorophyta). Carbohydr. polysaccharides of the coenocytic Penium margaritaceum:livecell of arabinogalactan-like proteins Res. 342, 586–596. green seaweed Bryopsis plumosa labeling with monoclonal antibod- and hemicelluloses in Micraste- Blanc, G., Duncan, G., Agarkova, I., (Bryopsidaceae, Chlorophyta) from ies. J. Bot. doi:10.1155/2011/632165 rias (Streptophyta). J. Phycol. 44, Borodovsky, M., Gurnon, J., Kuo, the Argentine coast. J. Phycol. 48, Domozych, D. S., Kort, S., Benton, S., 1221–1234. A., Lindquist, E., Lucas, S., Pangili- 326–335. and Yu, T. (2005). The extracellu- Egelund, J., Obel, N., Ulvskov, P., Geshi, nan, J., Polle, J., Salamov, A., Terry, Ciancia, M., Quintana, I., and Cerezo, lar polymeric substance of the green N., Pauly, M., Bacic,A., and Petersen, A., Yamada, T., Dunigan, D. D., A. S. (2010). Overview of anticoagu- alga Penium margaritaceum and its B. L. (2007). Molecular characteriza- Grigoriev, I. V., Claverie, J. M., lant activity of sulfated polysaccha- role in biofilm formation. Biofilms 2, tion of two Arabidopsis thaliana gly- and Van Etten, J. L. (2010). The ridesfromseaweedsinrelationto 1–16. cosyltransferase mutants, rra1 and Chlorella variabilis NC64A genome their structures, focusing on those of Domozych, D. S., Lambiasse, L., Kiemle, rra2, which have a reduced resid- reveals adaptation to photosym- green seaweeds. Cur. Med. Chem. 17, S. N., and Gretz, M. R. (2009). ual arabinose content in a polymer biosis, coevolution with viruses, 2503–2529. Cell-wall development and bipo- tightly associated with the cellulosic and cryptic sex. Plant Cell 22, Ciancia, M., Quintana, I., Vizcargüé- lar growth in the desmid Penium wall residue. Plant Mol. Biol. 64, 2943–2955. naga, M. I., Kasulin, L., de Dios, margaritaceum (Zygnematophyceae, 439–451.

www.frontiersin.org May 2012 | Volume 3 | Article 82 | 5 Domozych et al. Cell walls of green algae

Estevez, J. M., Leonardi, P. I., and Kieliszewski, M. J., and Lamport, D. Percival, E. (1979). The polysaccha- Sørensen, I., Pettolino, F. A., Bacic, A., Alberghina, J. S. (2008). Cell T. A. (1994). Extensin: repetitive rides of green, red, and brown sea- Ralph,J.,Lu,F.,O’Neill,M. A.,Fei,Z., wall carbohydrate epitopes in the motifs, functional sites, posttransla- weeds: their basic structure, biosyn- Rose, J. K. C., Domozych, D. S., and green alga Oedogonium bharuchae tional codes and phylogeny. Plant J. thesis and function. Br. Phycol. J. 14, Willats, W. G. T. (2011). The Charo- F. minor (Oedogoniales, Chloro- 5, 157–172. 103–117. phycean green algae provide insights phyta). J. Phycol. 44, 1257–1268. Kirk, D. L. (1998). Volvox.NewYork: Percival, E., and McDowell, R. H. into early origin of plant cell walls. Estevez, J. M, Kasuli, L., Fernandez, Cambridge University Press. (1981). “Algal walls. Composition Plant J. 153, 366–372. P. V., Dupree, P., and Ciancia, M. Lahaye, M., and Robic, A. (2007). and biosynthesis,” in Encyclopedia of Sørensen, I., Rose, J. K. C., Doyle, J. J., (2009). Chemical in situ character- Structure and functional proper- Plant Physiology, Vol. 13B, eds W. Domozych, D. S., and Willats, W. G. ization of macromolecular compo- ties of ulvan, a polysaccharide from Tanner and F. A. Loewus (Berlin: T. (2012). The Charophycean green nents of the complex cell walls from green seaweeds. Biomacromolecules Springer), 277–316. algae as model systems to study plant the coenocytic green alga Codium 8, 1766–1774. Petersen, B. L., Faber, K., and Ulvskov, cell walls and other evolutionary fragile. Glycobiology 18, 250–259. Lee, J.-B., Ohta, Y., Hayashi, K., and P. (2011). “Glycosyltransferases adaptations that gave rise to land Farias, E. H. C., Pomin, V. H., Valente, Hayashi, T. (2010). Immunostimu- of the GT77 family,” in Annual plant. Plant Signal. Behav. 7, 1–3. A. P., Nader, H. B., Rocha, H. A. lating effects of a sulfated galactan Plant Reviews: Plant Polysaccharides, Timme, R. E., Bachvaroff, T. R., and O., and Mourão, P. A. S. (2008). from Codium fragile. Carbohydr. Res. Biosynthesis, and Bioengineering,ed. Delwiche, C. F. (2012). Broad phy- A preponderantly 4-sulfated, 3- 345, 1452–1454. P. Ulvskov (New York: Blackwell), logenomic sampling and the sis- linked galactan from the green alga Leliaert, F., Smith, D. R., Moreau, H., 305–320. ter lineage of land plants. PLoS Codium isthmocladum. Glycobiology Herron, M., Verbruggen, H., Del- Piganeau, G., Grimsley, N., and Moreau, ONE 7, e29696. doi: 10.1371/jour- 18, 250–259. wiche, C. F., and De Clerck, O. H. (2011). Genome diversity in nal.pone.0029696 Fernández, P. V., Ciancia, M., and (2012). Phylogeny and molecular the smallest marine photosynthetic Timme, R. E., and Delwiche, C. Estevez, J. M. (2011a). Cell wall vari- evolution of the green algae. Crit. eukaryotes. Res. Microbiol. 62, 570- F. (2010). Uncovering the evo- ability in the green seaweed Codium Rev. Plant Sci. 31, 1–46. 577 lutionary origin of plant mol- vermilara (Bryopsidales Chloro- Leliaert, F., Verbruggen, H., and Zech- Popper, Z. A., and Fry, S. C. (2003). ecular processes: comparison of phyta) from the Argentine coast. J. man, F. W. (2011). Into the deep: Primary cell wall composition of Coleochaete (Coleochaetales) and Phycol. 47, 802–810. new discoveries at the base of the bryophytes and charophytes. Ann. Spirogyra (Zygnematales) transcrip- Fernández, P.V., Ciancia, M., Miravalles, green plant phylogeny. Bioessays 33, Bot. 91, 1–12. tomes. BMC Plant Biol. 10, 96. A. B., and Estevez, J. M. (2011b). 683–692. Popper, Z. A., Michel, G., Herve, C., doi:10.1186/1471-2229-10-96 Cell-wall polymer mapping Lewis,L. A.,and McCourt,R. M. (2004). Domozych, D. S., Willats, W. G. Vannerum, K., Huysman, M. J. J., De in the coenocytic macroalga Green algae and the origin of land T., Tuohy, M. G., Kloareg, B., and Rycke, R., Vuylsteke, M., Leilaert, F., Codium vermilaria (Bryopsidales, plants. Am.J.Bot.91, 1535–1556. Stengel, D. B. (2011). Evolution and Pollier, J., Lutz-Meindl, U., Gillard, Chlorophyta). J. Phycol. 46,456–465. Mao, W., Li, H., Li, Y., Zhang, H., Qi, diversity of plant cell walls: from J., DeVeylder, L., Goossens, A., Inze, Finet, C., Timme, R. E., Delwiche, C. X., Sun, H., Chen, Y., and Guo, S. algae to flowering plants. Annu. Rev. D., and Vyverman, W. (2011). Tran- F., and Marlétaz, F. (2010). Multi- (2009). Chemical characteristic and Plant Biol. 62, 567–590. scriptional analysis of cell growth gene phylogeny of the green lineage anticoagulant activity of the sul- Popper, Z. A., and Tuohy, M. G. (2010). and morphogenesis in the unicellu- reveals the origin and diversifica- fated polysaccharide isolated from Beyond the green: understanding the lar green alga Micrasterias (Strepto- tion of land plants. Cur. Biol. 20, Monostroma latissimum (Chloro- evolutionary puzzle of plant and phyta), with emphasis on the role of 2217–2222. phyta). Int. J. Biol. Macromol. 44, algal cell walls. Plant Physiol. 153, expansin. BMC Plant Biol. 11, 128. Gotteli, L. B., and Cleland, R. (1968). 70–74. 373–383. doi:10.1186/1471-2229-11-128 Differences in the occurrence and Mao, W.-J., Fang, F., Li, H.-Y., Qi, Rautengarten, C., Ebert, B., Herter, T., Velasquez,S. M.,Ricardi,M. M.,Dorosz, distribution of hydroxyproline- X.-H., Sun, H.-H., Chen, Y., and Petzoldc, C. J., Ishii, T., Mukhopad- J. G., Fernandez, P. V., Nadra, A. proteins among the algae. Am. J. Guo, S.-D. (2008). Heparinoid- hyay, A., Usadel, B., and Schellera, D., Pol-Fachin, L., Egelund, J., Gille, Bot. 55, 907–914. active two sulfated polysaccharides H. V. (2011). The interconversion S., Harholt, J., Ciancia, M., Verli, Huizing, H. J., and Rietema, H. (1975). isolated from marine green algae of UDP-Arabinopyranose and UDP- H., Pauly, M., Bacic, A., Olsen, Xylan and mannan as cell wall con- Monostroma nitidum. Carbohydr. Arabinofuranose is indispensable C. E., Ulvskov, P., Petersen, B. L., stituents of different stages in the Polym. 74, 834–839. for plant development in Arabidop- Somerville, C., Iusem, N. D., and life-histories of some siphoneous Moestrup, O., and Walne, P. L. (1979). sis. Plant Cell 23, 1373–1390. Estevez, J. M. (2011). O-glycosylated green algae. Br. Phycol. J. 10, 13–16. Studies on scale morphogenesis in Ray, B. (2006). Polysaccharides from cell wall proteins are essential in Huizing, H. J., Rietema, H., and Sietsma, the Golgi apparatus of Pyramimonas Enteromorpha compressa: isolation, root hair growth. Science 332, J. H. (1979). Cell wall constituents tetrarhynchus (Prasinophyceae). J. purification and structural features. 1401–1403. of several siphoneous green algae in Cell Sci. 36, 437–459. Carbohydr. Polym. 66, 408–416. Voigt, J., Liebich, I., Kieß, M., and relation to morphology and taxon- Moller, I., Sørensen, I., Bernal, A. J., Rodrigues, M. A., and da Silva Frank, R. (2001). Subcellular distri- omy. Br. Phycol. J. 14, 25–32. Blaukopf, C., Lee, K., Øbro, J., Pet- Bon, E. P. (2011). Evaluation of bution of 14-3-3 proteins in the uni- Imam S. H., Buchanan, M. J., Shin, tolino, F., Roberts, A., Mikkelsen, J., Chlorella (Chlorophyta) as source cellular green alga Chlamydomonas H.-C., and Snell, W. J. (1985). The Knox, J. P., Bacic, T., and Willats W. of fermentable sugars via cell wall reinhardtii. Eur. J. Biochem. 268, Chlamydomonas cell wall: character- G. T. (2007). High-throughput map- enzymatic hydrolysis. Enzyme Res. 6449–6457. ization of the wall framework. J. Cell ping of cell wall polymers within and doi:4061/2011/405603 Voigt, J., Woestemeyer, J., and Frank, R. Biol. 101, 1599–1607. between plants using novel microar- Showalter, A. M., Keppler, B., Lichten- (2007). The chaotrope-soluble gly- Kapraun, D. F. (2007). Nuclear DNA rays. Plant J. 50, 1118–1128. berg, J., Gu, D., and Welch, L. R. coprotein GP2 is a precursor of the content estimates in green algal Ohta, Y., Lee, J.-B., Hayashi, K., and (2010). A bioinformatics approach insoluble glycoprotein framework of lineages: Chlorophyta and Strepto- Hayashi, T. (2009). Isolation of sul- to the identification, classification, the Chlamydomonas cell wall. J. Biol. phyta. Ann. Bot. 99, 677–701. fated galactan from Codium fragile and analysis of hydroxyproline-rich Chem. 282, 30381–30392. Keskiaho, K., Hieta, R., Sormunen, R., and its antiviral effect. Biol. Pharm. glycoproteins. Plant Physiol. 153, Wijesekara, I., Pangestuti, R., and Kim, and Myllyharju, J. (2007). Chlamy- Bull. 32, 892–898. 485–513. S.-K. (2011). Biological activities domonas reinhardtii has multiple Painter, T. J. (1983). “Algal polysaccha- Sørensen, I., Domozych, D., and Willats, and potential health benefits of sul- prolyl 4-hydroxylases, one of which rides,” in The Polysaccharides, Vol. 2, W. G. T. (2010). How have plant cells fated polysaccharides derived from is essential for proper cell wall ed. G. O. Aspinall (New York, NY: walls evolved? Plant Physiol. 153, marine algae. Carbohydr. Polym. 84, assembly. Plant Cell 19, 256–269. Academic Press), 827–837. 366–372. 14–21.

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Wodnick, S., Brinkmann, H., Glock- Yamagaki, T., Maeda, M., Kanazawa, could be construed as a potential con- This article was submitted to Frontiers in ner, G., Heidel, A. J., Philippe, K., Ishizuka, Y., and Nakanishi, H. flict of interest. Plant Physiology, a specialty of Frontiers H., Melkonian, M., and Becker, (1997). Structural clarification of in Plant Science. B. (2011). Origin of land plants: Caulerpa cell wall β-(1→3)-xylan by Received: 10 March 2012; accepted: 12 Copyright © 2012 Domozych, Ciancia, do conjugating green algae hold NMR spectroscopy. Biosci. Biotech- April 2012; published online: 08 May Fangel, Mikkelsen, Ulvskov and Willats. the key? BMC Evol. Biol. 11, 104. nol. Biochem. 61, 1077–1080. 2012. This is an open-access article distributed doi:10.1186/1471-2148-11-104 Citation: Domozych DS,Ciancia M,Fan- under the terms of the Creative Commons Wutz, M., and Zetsche, K. (1976). gel JU, Mikkelsen MD, Ulvskov P and Attribution Non Commercial License, Zur Biochimie und Regulation des Conflict of Interest Statement: The Willats WGT (2012) The cell walls of which permits non-commercial use, dis- Heteromorphen Generationswech- authors declare that the research was green algae: a journey through evolution tribution, and reproduction in other sels der Grünalge. Planta 129, conducted in the absence of any com- and diversity. Front. Plant Sci. 3:82. doi: forums, provided the original authors and 211–216. mercial or financial relationships that 10.3389/fpls.2012.00082 source are credited.

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