RNA editing changes posttranscriptionally single nucleotides in chloroplast-encoded transcripts. Although much work has been done on mechanistic and functional aspects of plastid editing, little is known about evolutionary aspects of this RNA processing step. To gain a better understanding of the evolution of RNA editing in plastids, we have investigated the editing patterns in ndhB and rbcL transcripts from various species comprising all major groups of land plants. Our results indicate that RNA editing occurs in plastids of bryophytes, fern allies, true ferns, gymnosperms, and angiosperms. Both editing frequencies and editing patterns show a remarkable degree of interspecies variation. Furthermore, we have found that neither plastid editing frequencies nor the editing pattern of a specific transcript correlate with the phylogenetic tree of the plant kingdom. The poor evolutionary conservation of editing sites among closely related species as well as the occurrence of single speciesspecific editing sites suggest that the differences in the editing patterns and editing frequencies are probably due both to independent loss and to gain of editing sites. In addition, our results indicate that RNA editing is a relatively ancient process that probably predates the evolution of land plants. This supposition is in good agreement with the phylogenetic data obtained for plant mitochondrial RNA editing, thus providing additional evidence for common evolutionary roots of the two plant organellar editing systems.

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-Omethylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of 18 O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants. Molecular & Cellular

N-Glycosylation, a major co-and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the Nglycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc 3 Man 9 GlcNAc 2 -PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the ␣-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.