Classification, naming and evolutionary history of glycosyltransferases from sequenced green and red algal genomes

Abstract

The Archaeplastida consists of three lineages, Rhodophyta, Virideplantae and Glaucophyta. The extracellular matrix of most members of the Rhodophyta and Viridiplantae consists of carbohydrate-based or a highly glycosylated protein-based cell wall while the Glaucophyte covering is poorly resolved. In order to elucidate possible evolutionary links between the three advanced lineages in Archaeplastida, a genomic analysis was initiated. Fully sequenced genomes from the Rhodophyta and Virideplantae and the well-defined CAZy database on glycosyltransferases were included in the analysis. The number of glycosyltransferases found in the Rhodophyta and Chlorophyta are generally much lower then in land plants (Embryophyta). Three specific features exhibited by land plants increase the number of glycosyltransferases in their genomes: (1) cell wall biosynthesis, the more complex land plant cell walls require a larger number of glycosyltransferases for biosynthesis, (2) a richer set of protein glycosylation, and (3) glycosylation of secondary metabolites, demonstrated by a large proportion of family GT1 being involved in secondary metabolite biosynthesis. In a comparative analysis of polysaccharide biosynthesis amongst the taxa of this study, clear distinctions or similarities were observed in (1) N-linked protein glycosylation, i.e., Chlorophyta has different mannosylation and glucosylation patterns, (2) GPI anchor biosynthesis, which is apparently missing in the Rhodophyta and truncated in the Chlorophyta, (3) cell wall biosynthesis, where the land plants have unique cell wall related polymers not found in green and red algae, and (4) O-linked glycosylation where comprehensive orthology was observed in glycosylation between the Chlorophyta and land plants but not between the target proteins.

Figure 1. The red algal ECM.

The ECM of red algae consists of fibrillar components and gel-like polysaccharides. (A) Porphyra is a common sheet-like red alga found in coastal waters throughout the world. Scale bar = 5 cm. (B) The walls of Polysiphonia are multilayered consisting of alternating layers of fibrils. Scale bar = 200 nm.

Figure 2. Variation in extracellular coverings of green algae.

(A) Mesostigma viride (CGA) and many members of the Prasinophyceae (Chlorophyta) have a very distinct extracellular matrix (ECM; arrow). DIC image. Scale bar = 5 µm. (B) Close examination of the Mesostigma ECM reveals regular repeating units (arrow). DIC image. Scale bar = 175 nm. (C) The repeating units of the ECM represent the large body scales aligned upon the outer surface. TEM image. Scale bar = 100 nm. (D) In addition to the large outer body scales, two other body scale layers (arrow) are part of the inner regions of the ECM. TEM image. Scale bar = 50 nm. (E) In Chlorokybus (CGA), the ECM consists of a gel-like wall that holds cells together in sarcinoid packets (arrow). DIC image. Scale bar = 25µm. (F) The Chlorokybus sheath labels with JIM13 (arrow), a mAb with specificity toward arabinogalactan protein epitopes of land plants. Fluorescence microscopy. Scale bar = 30 µm. (G) In desmids like Penium (CGA), the ECM consists of a cell wall and extracellular polymeric substance or EPS. DIC image. Scale bar = 8 µm. (H) Penium’s cell wall is highlighted by a pectin-rich outer wall layer (arrow) as highlighted by JIM5 labeling. Fluorescence image. Scale bar = 4.5 µm. (I) The EPS of Penium is extensive as it covers the surface of cells after they stop gliding (arrow). The EPS was labeled with an anti-EPS antibody. Fluorescence image. Scale bar = 17 µm. (J) Thalloid CGA like Coleochaete have cell walls. DIC image. Scale bar = 25 µm. (K) The cell wall of Coleochaete also contains epitopes of wall polysaccharides that are similar to those found in land plants. Here, JIM5, with specificity toward pectin, labels the junction zone of three cells (arrow). Scale bar = 75 nm.

Figure 3. The distribution of glycosyltransferases in analyzed genomes.

Arabidopsis is included as reference genome.

Figure 4. Schematic illustration of the N-linked glycan before its transfer onto the protein.

The linkages and name of biosynthetic enzyme is presented in the figure along with an overview of CAZy family for the different glycosyltransferases.

Figure 5. Illustration of the most common Hyp arabinosides so far found in Chlorophytes, Charyophytes and plants.

Structure 1–3 is shared among viridiplantae (Lamport and Miller, 1971, Harholt, Petersen and Ulvskov, unpublished). Structure 4 has so far only been observed in Charophycean green algae and plants (Lamport and Miller, 1971, Harholt, Petersen and Ulvskov, unpublished). Structure 5 and 6 is found in at least C. reinhardtii but not been reported in plants . Structure 5 and 6 can be methylated at either C-6 of the galactose or C-3 at the ultimate arabinose .

Figure 6. CAZy family GT77.

Characterized members of GT77 are involved in extensin biosynthesis as arabinosyltransferases (A- and C-clades) and rhamnoglacturonan II biosynthesis as xylosyltransferases (B-clade. Not shown – no algal members). Chlorophyte orthologs to the extensin arabinosyltransferases RRA and XEG113 from arabidopsis could be identified (At1g75120: AtRRA1, At1g75110: AtRRA2, At1g19360: AtRRA3 and At2g35610: XEG113). Additionally, rhodophyte members could be identified belonging to the GT77D clade with unknown activity. Rice XEG113 orthologs have been identified, but was omitted from the tree due to apparent annotation mistakes in the protein sequence. The scale bar indicates the average number of amino acid substitutions per site.