Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical b1,2-core xylose, a1,3-fucose residues, as well as plant atypical terminal b1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact Nglycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.

For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRISPR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. This results in impaired polystyrene bead binding and transport but not gliding of cells on solid surfaces. Notably, assembly, intraflagellar transport, and protein import into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.

Chlamydomonas reinhardtii N-glycans carry plant typical β1,2-core xylose, α1,3-fucose residues as well as plant atypical terminal β1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact N-glycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.Significance StatementOur data provide novel insights into the function of XylT1B and FucT in C. reinhardtii as N-glycan core modifying enzymes. In the course of our study, different mutants were created by genetic crosses showing either varying or a lack of N-glycan core modification, enabling comparative analyses in relation to single N-glycan core epitope and overall protein function in C. reinhardtii.

Phytoplankton have short generation times, flexible reproduction strategies, large population sizes, and high standing genetic diversity, traits that should facilitate rapid evolution under directional selection. We quantified local adaptation of copper tolerance in a population of the diatom Skeletonema marinoi from a mining exposed inlet in the Baltic Sea and in a non-exposed population 100 km away. We hypothesized that mining pollution has driven evolution of elevated copper tolerance in the impacted population of S. marinoi. Assays of 58 strains originating from sediment resting stages revealed no difference in the average tolerance to copper between the two populations. However, variation within populations was greater at the mining site, with three strains displaying hyper-tolerant phenotypes. In an artificial evolution experiment, we used a novel intraspecific metabarcoding locus to track selection and quantify fitness of all 58 strains during co-cultivation in one control and one toxic copper treatment. As expected, the hyper-tolerant strains enabled rapid evolution of copper tolerance in the mining exposed population through selection on available strain diversity. Within 42 days, in each experimental replicate a single strain dominated (30-99% abundance) but different strains dominated the different treatments. The reference population developed tolerance beyond expectations primarily due to slowly developing plastic response in one strain, suggesting that different modes of copper tolerance are present in the two populations. Our findings provide novel empirical evidence that standing genetic diversity of phytoplankton resting stage allows populations to evolve rapidly (20-50 generations) and flexibly on timescales relevant for seasonal bloom progressions.

31For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins 32 on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar 33 surface adhesion. It is unknown whether the composition of N-glycans attached to respective 34 proteins is important for these processes. To this end, we examined several C. reinhardtii 35 insertional mutants and a CRIPSR/Cas9 knockout mutant of xylosyltransferase 1A, all 36 possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and 37 micropipette force measurements, our data revealed that reduction in N-glycan complexity 38 impedes the adhesion force required for binding the flagella to surfaces. In addition, polystyrene 39 bead binding and transport is impaired. Notably, assembly, Intraflagellar Transport and FMG-40 1B transport into flagella are not affected by altered N-glycosylation. Thus, we conclude that 41 proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto 42 surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.43