Biology subjects

van Gessel, N.

Publications and source records attributed to van Gessel, N..

7 recordsLinked to original sources

bioRxiv2025

Functional Characterization of Target of Rapamycin (TOR) Signalling in Physcomitrella

Target of rapamycin (TOR) is a conserved protein kinase and an important signalling hub in eukaryotes. The moss Physcomitrella (Physcomitrium patens) is a model organism for plant physiology, development, and evolution. However, little is known about TOR signalling in non-vascular plants, including Physcomitrella. Here, we report the effects of inhibiting TOR signalling in Physcomitrella. We identified and characterized Physcomitrella 12-kD FK506-binding protein (FKBP12), which binds TOR in the presence of rapamycin. Whereas the growth of wild-type protonema is unaffected by rapamycin, overexpressing endogenous FKBP12 rendered the plant susceptible to the inhibitor in a dose-dependent manner. Likewise, protonema growth was inhibited when the TOR-specific inhibitor AZD8055 was present in the culture. We show that rapamycin and AZD8055 have pleiotropic effects, as they delay cell cycle progression and development, induce chlorosis, inhibit photosynthesis, and alter total protein content. Additionally, we identified and characterized PpTOR, PpLST8, and PpRAPTOR, key components of TOR complex 1 (TORC1), whereas RICTOR and mSIN1 of TORC2 are not encoded by the P. patens genome. We found that PpLST8 substitutes its homolog in yeast to allow cell growth. Physcomitrella mutants were generated with a conditional downregulation of PpTOR, PpLST8, and PpRAPTOR, respectively. They were impaired in growth. Finally, we show TOR-dependent phosphorylation of a well-known TOR phospho-target, the ribosomal protein RPS6, via LC-MS/MS. Collectively, our results show that Physcomitrella growth and development is positively controlled by a conserved TOR kinase. We suggest to further dissect TOR signalling in Physcomitrella in order to elucidate signalling integration via TORC1 in plants. Key messageThe evolutionary conserved TOR kinase positively controls growth and development of the moss Physcomitrella, development and function of its chloroplasts, its protein synthesis and cell cycle progression.

plant biology↗

bioRxiv2025

CRISPR/Cas9 targeted genetic screening in Physcomitrella identifies novel cell division genes

Although plants share core cell division mechanisms with other eukaryotes, their unique features, such as acentrosomal spindle formation and cytokinesis via the phragmoplast, suggest the existence of plant-specific genes. This study used the model bryophyte Physcomitrium patens, commonly known as Physcomitrella, to uncover such genes and employed CRISPR/Cas9-based and localization-based screening to identify novel cell division genes. Co-expression data from known mitotic genes were used to create a pool of 216 candidate genes, which were targeted for CRISPR/Cas9 screening. Frameshift mutants with division defects were characterized using high-resolution imaging and fluorophore-based protein localization. Three novel gene families--CYR (Cytokinesis-Related), LACH (Lagging Chromosome), and SpinMi (Spindle and Phragmoplast Midzone)--were identified. CYR is linked to cytokinesis defects, LACH is essential for chromosome segregation, and SpinMi localizes to the spindle and phragmoplast midzone. None of these gene families has homologs in algae, suggesting their emergence during land colonization. These findings demonstrate the utility of co-expression-guided and targeted screening for discovering genes involved in specific cellular processes and provide insights into the evolution of the plant cell division machinery.

plant biology↗

bioRxiv2024

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are undesired on plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95% of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs. HighlightsMosses, such as Physcomitrella, encode six prolyl 4-hydroxylases in four subfamilies Computational analysis reveals specific environment for proline hydroxylation AlphaFold structure models and mass spectrometry reveal features of modification Quantitative proteomics and mutant analysis identify relevant modifying enzyme Modelling suggests relevant amino acids in active centre of the modifying enzyme

plant biology↗

bioRxiv2023

Characterization of GAPDH expression related to biotic stress responses in Physcomitrella

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a housekeeping protein that plays an important role in the energy metabolism and is found in all kingdoms of life. While animals possess one GAPDH isoform, plants possess several GAPDHs. GAPA and GAPB are chloroplastic phosphorylating GAPDHs involved in the Calvin-Benson cycle. GAPC in the cytosol and GAPCp in plastids are glycolytic phosphorylating GAPDHs involved in the glycolysis pathway. In animals as well as in plants, GAPDH isoforms have various non-metabolic roles, e.g. in autophagy, apoptosis, and were identified as redox sensors. In plants, in addition to the variety of moonlight functions in abiotic stress, GAPDHs are also involved in biotic stress responses. Here, we identified 17 GAPDH isoforms from the moss Physcomitrella, belonging to the GAPA, GAPC, and GAPCp. We searched for protein and transcript evidences in publicly available proteome and transcriptome data, laying the focus on datasets of treatments with the phytohormones salicylic acid (SA) and methyl jasmonate (MeJA). We investigated the putative role of GAPDHs in plant immune response by identifying SA- and MeJA-inducible GAPAs and GAPC genes. The GAPAs Pp3c1_13170 and Pp3c11_15790 and the GAPC Pp3c18_15450 appear to be unresponsive to SA treatment. In contrast, we identified four MeJA-responsive genes. MeJA-treatment resulted in a 10- and 3-fold downregulation of the GAPAs Pp3c1_13170 and Pp3c11_15790, whereas expression of the two GAPC genes Pp3c18_15450 and Pp3c21_9380 showed an up to 7- and 33-fold upregulation after 4 hours of MeJA treatment, respectively. Simultaneously, a four-hour MeJA-treatment induced the remodeling of the Physcomitrella secretome, resulting in the production of antimicrobial compounds, which in turn led to a bacteriostatic growth inhibition of 26% of E. coli. These findings draw attention to the potential differential regulation of GAPDH genes in plant immune response as well as a potential role for GAPC in the defense response against necrotrophic pathogens.

plant biology↗

bioRxiv2022

Genome dynamics in mosses: Extensive synteny coexists with a highly dynamic gene space

BackgroundWhile genome evolutionary processes of seed plants are intensively investigated, very little is known about seed-free plants in this respect. Here, we use one of the largest groups of seed-free plants, the mosses, and newly generated chromosome-scale genome assemblies to investigate three poorly known aspects of genome dynamics and their underlying processes in seed-free plants: (i) genome size variation, (ii) genomic collinearity/synteny, and (iii) gene set differentiation. ResultsComparative genomic analyses on the model moss Physcomitrium (Physcomitrella) patens and two genomes of Funaria hygrometrica reveal that, like in seed plants, genome size change (approx. 140 Mbp) is primarily due to transposable element expansion/contraction. Despite 60 million years of divergence, the genomes of P. patens and F. hygrometrica show remarkable chromosomal stability with the majority of homologous genes located in conserved collinear blocks. In addition, both genomes contain a relatively large set of lineage-specific genes with no detectible homologs in the other species genome, suggesting a highly dynamic gene space fueled by the process of de novo gene birth and loss rather than by gene family diversification/duplication. ConclusionsThese, combined with previous observations suggest that genome dynamics in mosses involves the coexistence of a collinear homologous and a highly dynamic species-specific gene sets. Besides its significance for understanding genome evolution, the presented chromosome-scale genome assemblies will provide a foundation for comparative genomic and functional studies in the Funariaceae, a family holding historical and contemporary model taxa in the evolutionary biology of mosses.

genomics↗

bioRxiv2022

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in plant aging

Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.

plant biology↗

bioRxiv2020

Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrella patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding P. patens genes and their 87,533 annotated transcripts in a web application, physCO, for automatized codon-optimization. A thus optimized cDNA results in about eleven times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects also occur in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.

plant biology↗