Biology subjects

Eberhard, S.

Publications and source records attributed to Eberhard, S..

5 recordsLinked to original sources

bioRxiv2025

Arabidopsis GALACTURONOSYLTRANSFERASE (GAUT) 1 synthesizes a homogalacturonan tightly bound to the cell wall and required for cell expansion

Arabidopsis GALACTURONOSYLTRANSFERASE1 (GAUT1) synthesizes homogalacturonan (HG), the most abundant pectin in growing plant cells. GAUT1 has the greatest in vitro enzyme activity of the six confirmed Arabidopsis HG biosynthetic GAUTs, but its biological activity remains elusive. Here we show that Arabidopsis GAUT1 homozygous mutants have a severe dwarfed seedling phenotype, survive several weeks as 2 to 3 mm seedlings, and have severely reduced shoot and root growth and hypocotyl epidermal, cortex and endodermal cell size. gaut1-1 pollen tubes are shorter than WT with increased bursting. Complementation of homozygous gaut1-1 with GAUT1 coding sequence driven by the GAUT1 promoter restored WT-like growth. The extreme dwarf phenotype of homozygous gaut1-1 seedlings precluded their use for detailed cell wall analysis, thus suspensions cultures were produced from callus generated from mutant and WT seedlings. Homozygous gaut1-1 suspension cells were smaller than WT with [~]30% reduced wall GalA content compared to WT. Sequential extraction of the walls with increasingly harsh solvents and sugar composition analysis revealed reduced GalA content in only the 4M KOH post-chlorite fraction, indicating that GAUT1-synthesized HG was held tightly in the wall by direct or indirect hydrogen bonding and/or oxidation-sensitive linkages. Treatment of wall fractions with endopolygalacturonase to hydrolyze HG and gel electrophoretic separation of hydrolysates exposed an HG-associated doublet band markedly downregulated in the homozygous gaut1-1 4M KOH post-chlorite fraction and to a lesser extent in 4M KOH and sodium chlorite fractions. NMR analysis identified the band as rhamnogalacturonan (RG)-II. Super resolution microscopy using anti-HG antibodies showed that, compared to WT, the homozygous gaut1-1 hypocotyl epidermal and callus cells had reduced content and length of HG nanofilaments, HG fibers associated with cell expansion in Arabidopsis. The results demonstrate that GAUT1-synthesized HG resides in a tightly-cell-wall-bound, RG-II-containing polymer required for HG nanofilament formation and seedling cell expansion.

plant biology↗

bioRxiv2023

Telomerase-independent survival leads to a mosaic of complex subtelomere rearrangements in Chlamydomonas reinhardtii

Telomeres and subtelomeres, the genomic regions located at chromosome extremities, are essential for genome stability in eukaryotes. In the absence of the canonical maintenance mechanism provided by telomerase, telomere shortening induces genome instability. The landscape of the ensuing genome rearrangements is not accessible by short-read sequencing. Here, we leverage Oxford Nanopore Technologies long-read sequencing to survey the extensive repertoire of genome rearrangements in telomerase mutants of the model green microalga Chlamydomonas reinhardtii. In telomerase mutant strains grown for [~]700 generations, most chromosome extremities were capped by short telomere sequences that were either recruited de novo from other loci or maintained in a telomerase-independent manner. Other extremities did not end with telomeres but only with repeated subtelomeric sequences. The subtelomeric elements, including rDNA, were massively rearranged and involved in breakage-fusion-bridge cycles, translocations, recombinations and chromosome circularization. These events were established progressively over time and displayed heterogeneity at the subpopulation level. New telomere-capped extremities composed of sequences originating from more internal genomic regions were associated with high DNA methylation, suggesting that de novo heterochromatin formation contributes to restore chromosome end stability in C. reinhardtii. The diversity of alternative strategies to maintain chromosome integrity and the variety of rearrangements found in telomerase mutants are remarkable and illustrate genome plasticity at short timescales.

genomics↗

bioRxiv2022

Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii

Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.

genomics↗

bioRxiv2021

Architecture and evolution of subtelomeres in the unicellular green alga Chlamydomonas reinhardtii

In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyze their structure and infer how they evolved. Here, we use recent and forthcoming genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is an array of 1 to 46 tandem copies of Sultan elements adjacent to the telomere and followed by a transcribed centromere-proximal Spacer sequence, a G-rich microsatellite and a region rich in transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Comparison of genomic sequences of three laboratory strains and a wild isolate of C. reinhardtii shows that the overall subtelomeric architecture was already present in their last common ancestor, although subtelomeric rearrangements are on-going at the species level. Analysis of other green algae reveals the presence of species-specific repeated elements, highly conserved across subtelomeres and unrelated to the Sultan element, but with a subtelomere structure similar to C. reinhardtii. Overall, our work uncovers the complexity and evolution of subtelomere architecture in green algae.

genomics↗

bioRxiv2019

Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. They shorten with each cell division because of the end-replication problem. Expression of the reverse transcriptase telomerase allows for extension of telomeric repeats to counteract telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly non-degenerate and that the telomeres form a protective structure, ending with a 3' overhang. While telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that mutants of this gene display an \"ever shortening telomere\" phenotype and eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

plant biology↗