The cell wall polysaccharides of a photosynthetic relative of apicomplexans, Chromera velia.

Chromerids are a group of alveolates, found in corals, that show peculiar morphological and genomic features. These organisms are evolutionary placed in-between symbiotic dinoflagellates and parasitic apicomplexans. There are two known species of chromerids: Chromera velia and Vitrella brassicaformis. Here, the biochemical composition of the C. velia cell wall was analyzed. Several polysaccharides adorn this structure, with glucose being the most abundant monosaccharide (approx. 80%) and predominantly 4-linked (approx. 60%), suggesting that the chromerids cell wall is mostly cellulosic. The presence of cellulose was cytochemically confirmed with calcofluor white staining of the algal cell. The remaining wall polysaccharides, assuming structures are similar to those of higher plants, are indicative of a mixture of galactans, xyloglucans, heteroxylans, and heteromannans. The present work provides, for the first time, insights into the outermost layers of the photosynthetic alveolate C. velia.

arabinose

calcofluor white

fucose

galactose

glucose

mannose

rhamnose

standard error of the mean

xylose

Chromerids are photosynthetic unicellular eukaryotes of the superphylum Alveolata (Moore et al. 2008, Cavalier‐Smith 2018). Originally isolated from Australian scleractinian corals (Moore et al. 2008), the chromerids are also free‐living and are now known to be globally distributed (Mathur et al. 2018). There are two described species of chromerids: Chromera velia isolated from the coral Plesiastrea versipora in Sydney Harbor (Moore et al. 2008), and Vitrella brassicaformis from the Great Barrier Reef coral Leptastrea purpurea (Oborník et al. 2012).

When first discovered (Moore et al. 2008), Chromera was hailed as a missing link that represents a transition form between symbiotic dinoflagellates (with a photosynthetic plastid) and parasitic apicomplexans (with a relict, non‐photosynthetic plastid, referred to as an apicoplast; McFadden and Waller 1997, Okamoto and McFadden 2008, Janouškovec et al. 2010, Weatherby and Carter 2013). Indeed, analysis of C. velia and Vitrella   brassicaformis plastid and nuclear genomes confirms a common origin of apicomplexan, chromerid, and dinoflagellate (peridinin) plastids from a complex red algal endosymbiont (Janouškovec et al. 2010, Oborník and Lukeš 2013, Woo et al. 2015). Chromerids, together with colpodellids, constitute the Apicomonada, an important major group of alveolate protists (Cavalier‐Smith 2018), and they hold a key position in eukaryotic diversity.

Major groups of algae are characterized by signature morphologies (e.g., flagellar apparatuses and mitotic mechanisms), photosynthetic pigments (unique combinations of chlorophylls and accessory pigments), and their wall materials (e.g., cellulose, agar, carageenans, alginate, fucoidans, glycoproteins, calcium carbonate, silica, and others; Popper et al. 2011, Synytsya et al. 2015). Chromerids exhibit unique combinations of morphology (Oborník et al. 2011, 2016), pigments (Moore et al. 2008), and genome organizations (Janouškovec et al. 2010, Flegontov et al. 2015, Woo et al. 2015), but as yet there is no biochemical description of their cell walls. Here, we provide the first analysis of the chromerid cell walls with a monosaccharide linkage analysis, and cytochemical confirmation for the presence of a predominantly cellulosic wall.

To obtain a broad view of polysaccharides of cell wall preparations in Chromera velia, we applied monosaccharide linkage analysis to isolated cell walls (Pettolino et al. 2012). Chromera velia cultures (CCMP2878) were grown in f/2 medium under constant temperature (26°C), 12:12 h light: dark photoperiod cycle, and 30–50 μmol photons · m−2 · s−1 of light. Two replicates of 3 × 106 cells · mL−1 were used in the study to isolate cell walls. Cell aliquots were suspended in 0.6 M sorbitol, 20 mM Tris HCl pH 7.4, and 2 mM EDTA and processed (three cycles) in a French press at 35,000 psi. The homogenate was then centrifuged at 2,000g, and the supernatant spun at 17,000g on 10–50% Optiprep™ (Sigma‐Aldrich, Australia) gradient for 10 min. Specimens for transmission electron microscopy (TEM) were prepared and visualized as in Moore et al. 2008, and purity of the C. velia cell walls was confirmed microscopically (Fig. 1, B and C). After washing twice with 80% ethanol, cell walls were analyzed by monosaccharide linkage analysis as described by Pettolino et al. (2012).

Fig. 1

(A) Transmission electron microscopy of a cross‐section of Chromera velia coccoid stage and zoom in to show the thick cell wall surrounding the cell. a = alveoli; cw = cell wall; mc = microtubules; mt = mitochondrion; n = nucleus; pm = plasma membrane. B) Transmission electron microscopy and C) light microscopy of isolated C. velia cell wall residues.

The cell wall comprised the following monosaccharides in decreasing abundance: glucose (Glc), galactose (Gal), mannose (Man), xylose (Xyl), fucose (Fuc), rhamnose (Rha), and arabinose (Ara; Table 1). Whereas glucose is overwhelmingly the dominant monosaccharide in the cell wall fraction (∼80%), a total sugar analysis of the ethanol extract of whole cells (data not shown) revealed that glucose constituted just 12% of Chromera velia soluble total sugars. This suggests that polymeric glucose is preferentially accumulated in the C. velia cell wall.

Table 1

Monosaccharide linkage composition (Mol %) of Chromera velia cell walls analyzed in duplicates.

Monosaccharide	Deduced linkage a	Mol%	SD
Araf	5‐	1.0	0.3
Fucp	terminal	1.0	0.1
	2‐	0.4	0
	4‐	0.3	0
	Total	1.7
Galp	terminal	2.7	0.4
	2‐	2.4	0.8
	4‐	2.4	0.3
	6‐	0.8	0.1
	Total	8.3
Glcp	terminal	9.1	0.7
	2‐	0.6	0.1
	3‐	0.2	0.1
	4‐	60.6	4.1
	2,3‐	0.9	0
	2,4‐	0.6	0.1
	3,4‐	1.0	0.3
	4,6‐	4.7	1.1
	3,4,6‐	0.5	0.2
	2,3,4,6‐	1.7	0.7
	Total	79.7
Manp	terminal	0.9	0.3
	2‐	0.5	0
	4‐	0.5	0.5
	3,6‐	0.5	0.4
	2,4,6‐	0.7	0.4
	Total	3.0
Rhap	2,4‐	0.4	0.5
Xylp	terminal	3.2	1.1
	4‐	2.0	0
	2,4‐	0.7	0.2
	Total	5.9

SD = standard deviation.

Linkages were deduced from 1,5‐di‐O‐acetyl‐6‐deoxy‐2,3,4‐tri‐O‐methyl fucitol; 1,2,5‐tri‐O‐acetyl‐6‐deoxy‐3,4‐di‐O‐methyl fucitol; 1,4,5‐tri‐O‐acetyl‐6‐deoxy‐2,3‐di‐O‐methyl fucitol; 1,2,4,5‐tri‐O‐acetyl‐6‐deoxy‐3‐O‐methyl rhamnitol; 1,4,5‐tri‐O‐acetyl‐2,3‐di‐O‐methyl arabinitol; 1,5‐di‐O‐acetyl‐2,3,4‐tri‐O‐methyl xylitol; 1,4,5‐tri‐O‐acetyl‐2,3‐di‐O‐methyl xylitol; 1,2,4,5‐tetra‐O‐acetyl‐3‐O‐methyl xylitol; 1,5‐di‐O‐acetyl‐2,3,4,6‐tetra‐O‐methyl mannitol; 1,2,5‐tri‐O‐acetyl‐3,4,6‐tri‐O‐methyl mannitol; 1,4,5‐tri‐O‐acetyl‐2,3,6‐tri‐O‐methyl mannitol; 1,3,5,6‐tetra‐O‐acetyl‐ 2,4‐di‐O‐methyl mannitol; 1,2,4,5,6‐penta‐O‐acetyl‐3‐O‐methyl mannitol; 1,5‐di‐O‐acetyl‐2,3,4,6‐tetra‐O‐methyl galactitol; 1,2,5‐tri‐O‐acetyl‐3,4,6‐tri‐O‐methyl galactitol; 1,4,5‐tri‐O‐acetyl‐2,3,6‐tri‐O‐methyl galactitol; 1,5,6‐tri‐O‐acetyl‐2,3,4‐tri‐O‐methyl galactitol; 1,5‐di‐O‐acetyl‐2,3,4,6‐tetra‐O‐methyl glucitol; 1,2,5‐tri‐O‐acetyl‐3,4,6‐tri‐O‐methyl glucitol; 1,3,5‐tri‐O‐acetyl‐2,4,6‐tri‐O‐methyl glucitol; 1,4,5‐tri‐O‐acetyl‐2,3,6‐tri‐O‐methyl glucitol; 1,2,3,5‐tetra‐O‐acetyl‐4,6‐di‐O‐methyl glucitol; 1,2,4,5‐tetra‐O‐acetyl‐3,6‐di‐O‐methyl glucitol; 1,3,4,5‐tetra‐O‐acetyl‐2,6‐di‐O‐methyl glucitol; 1,4,5,6‐tetra‐O‐acetyl‐2,3‐di‐O‐methyl glucitol; 1,3,4,5,6‐penta‐O‐acetyl‐2‐O‐methyl glucitol; and hexa‐O‐acetyl glucitol.

The monosaccharide linkage composition of Chromera velia cell wall, deduced from the linkage analysis, is reported in Table 1, with 4‐Glc being the predominant linkage type (61% of 80% total), which is indicative of a cell wall of C. velia is composed predominantly of cellulose, a β‐1,4‐linked‐Glc polymer that is also the predominant fibrillar polysaccharide of all plant species and some bacteria and fungi. To further confirm the cellulosic nature of C. velia cell wall, we used calcofluor white, a fluorescent stain indicative for β‐1,4 linkages, to probe the cell walls of intact cells. Chromerid cells were visualized with a Nikon A1R confocal laser scanning microscope (Nikon, Tokyo, Japan) with a 489 nm laser to detect chlorophyll autofluorescence, and a 409 nm laser to detect calcofluor white stained cell walls. The calcofluor white positive material surrounds the entire chromerid cell (Fig. 2), further suggesting the presence of cellulose in the C. velia wall, which is consistent with the thick cell wall shown by electron microscopy (Fig. 1A; Oborník et al. 2011). No cellulose synthase genes are currently annotated in the C. velia genome (www.cryptodb.org), but a thorough phylogenetic analysis of the genome for members of the cellulose synthesis machinery should be undertaken based on our biochemical evidence for a cellulosic cell wall.

Fig. 2

Bar plot of the glucose linkages (Glcp) in the cell wall of Chromera velia. Each bar represents the relative abundance molar percentage (Mol%) ± SEM of a glucosyl linkage. Microscopy images of control and calcofluor white (CW) staining of C. velia cell wall. TM = transmitted light microscopy. Chl a = algal chlorophyll autofluorescence in red. CW stain of the cellulosic C. velia wall is in blue. Chl a + CW = merged channels.

Cellulose is the main component of so‐called armored (walled) dinoflagellates, so identification of cellulose in the related Chromera   velia is not surprising. Dinoflagellate cellulose is deposited as ornate thecal plates (Fritz and Triemer 1985) within the alveolar sacs (variously known as amphiesma or alveolae or inner membrane complex) that are a defining feature of the superphylum Alveolata (Gould et al. 2008) and also characteristic of chromerids (Oborník et al. 2012). However, the thick cell wall of C. velia is clearly extracellular and not within its alveolae (Fig. 1), so clear differences in cell wall architecture and location are already apparent between dinoflagellates and C. velia. It will now be interesting to analyze the cell surface structures of the other chromerid V. brassicaformis and determine how cellulose has been utilized as a cell wall material in this branch of the eukaryotic tree.

AUTHOR CONTRIBUTIONS

G. Tortorelli: Data curation (equal); Methodology (equal); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). F. Pettolino: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (lead); Writing‐review & editing (equal). D.H. Lai: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Writing‐review & editing (equal). A. Tomčala: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal). A. Bacic: Conceptualization (equal); Funding acquisition (equal); Investigation (lead); Methodology (equal); Project administration (equal); Supervision (equal); Writing‐review & editing (equal). M. Oborník: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (lead); Methodology (equal); Project administration (equal); Supervision (equal); Writing‐review & editing (equal). J. Lukeš: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (lead); Methodology (equal); Project administration (equal); Supervision (equal); Writing‐review & editing (equal). G.I. McFadden: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (lead); Methodology (equal); Project administration (equal); Supervision (equal); Writing‐review & editing (equal).