Biology subjects

Reski, R.

Publications and source records attributed to Reski, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Zinc isotope fractionation in native and cloned mosses: implications for standardized pollution biomonitoring

Mosses are widely used as biomonitors of atmospheric trace-metal deposition, but metal retention by the moss itself may modify source-related isotope signatures. Here, controlled Zn adsorption experiments, stable Zn isotope measurements, surface acid-base characterization, and Zn K-edge X-ray absorption spectroscopy (XAS) were combined to investigate Zn2+ retention and isotope fractionation in four native moss taxa and in vitro-cultivated Sphagnum palustre clones. Zn2+ adsorption was strongly pH-dependent, consistent with progressive deprotonation of reactive surface sites. Moss-associated Zn2+ was slightly enriched in heavy isotopes relative to the coexisting solution, with {Delta}66Znmoss-solution generally ranging from +0.001 to +0.009 per cent, but no systematic relationship was observed between isotopic fractionation and the fraction of Zn2+ adsorbed. Linear-combination fitting of X-ray Absorption Near-Edge Structure (XANES) indicated predominantly cellulose-like, O-rich coordination environments, with variable phosphate and minor acetate contributions and only small nominal Zn-S fractions. Extended X-ray Absorption Fine Structure (EXAFS) showed first-shell Zn-O distances of approximately 1.96 - 2.05 Angstrom and coordination numbers of about 3.6 - 7.6, confirming substantial structural heterogeneity among samples. Importantly, {Delta}66Znmoss-solution was not correlated with either Zn-O distance or coordination number, indicating that the net isotope effect cannot be described by a single bulk structural parameter. Native and cultivated Sphagnum showed comparable isotopic offsets within analytical uncertainty despite differences in surface properties and adsorption capacity. These results indicate that Zn isotope fractionation in mosses reflects the integrated response of multiple binding environments and support the use of standardized Sphagnum clones in isotope-based biomonitoring.

plant biology↗

bioRxiv2025

Linking biosurface reactivity and photosynthesis to investigate Sphagnum mosses as peatland engineers

O_LISphagnum mosses regulate peatland carbon cycling, hydrology, nutrient dynamics, and structure. We investigated the link between Sphagnum surface chemistry and photosynthetic efficiency, and the combined effects of pH and waterlogging on photosynthesis, key mechanisms for peatland functioning but still poorly understood. C_LIO_LIWe quantified biosorption potential and functional groups in 20 field collected and in 4 axenically cloned Sphagnum species via titrations. Gas exchange and chlorophyll fluorescence measurements were performed in 5 representative species under submerged conditions across a pH gradient. C_LIO_LIThe 20 Sphagnum species share a basic surface chemistry, but they differ in surface charge and functional group abundance, with an overall greater biosorption potential in the subgenera Acutifolia and Sphagnum. Photosynthesis is species- and pH-dependent, influenced by surface functional-group composition, with phosphoryl and carboxyl groups enhancing CO2 assimilation. Sphagnum palustre emerges as a generalist, stress-tolerant species, maintaining stable photosynthesis and photoprotection across pH ranges. In-vitro cultivation reduces surface reactivity and interspecific differentiation but preserves fundamental Sphagnum surface chemistry. C_LIO_LIThese results highlight the interplay between chemical reactivity, photosynthetic performance, and ecological adaptation in Sphagnum. Sphagnum chemical traits and photosynthetic plasticity enable survival under variable pH, waterlogging, and nutrient conditions, with clones providing reliable models for research and applications. C_LI

plant biology↗

bioRxiv2025

Production of prolyl-hydroxylated human type III collagen in Physcomitrella photobioreactors

Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via protoplast transformation. Immunodetection identified collagen producing lines, and mass spectrometry validated the product and detected prolyl-hydroxylation on 23 sites. The presence of this important post-translational modification underscores the high biomimetic quality of the product. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions including CO2 supplementation, pH, and light intensity were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide in the culture supernatant after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens for cosmetic formulations. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements. Key messageScalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals.

plant biology↗

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↗

bioRxiv2025

Production of HPV-16 L1 virus-like particles in Physcomitrella photobioreactors

Virus-like particles (VLPs) are self-assembling nanoparticles composed of viral structural proteins which mimic native virions but lack viral DNA and infectivity. VLPs are a resourceful class of biopharmaceuticals applied as subunit vaccines or as delivery vehicles for drugs and nucleic acids. Similar to viruses, VLPs are diverse in structure, composition, and assembly, requiring a tailored production platform aligned with the intended application. The moss plant Physcomitrella (Physcomitrium patens) is an emerging expression system offering humanized N-glycosylation, scalability, and adaptability to existing industry settings. Here, we used Physcomitrella to produce human papillomavirus (HPV) 16 VLPs. HPV VLPs are composed of the major structural protein L1 and are used as vaccines against HPV infections which are the main causal agent of cervical and other anogenital cancers. We characterized Physcomitrella chloroplast transit peptides, which we used for targeting of moss-produced L1 to chloroplasts, leading to higher recombinant protein yield compared to nuclear or cytoplasmic localization. We confirmed subcellular localization with confocal laser scanning microscopy and found L1 to accumulate within the chloroplast stroma. Production in 5-liter photobioreactors yielded over 0.3 mg L1 per gram fresh weight. We established a purification protocol for moss-produced L1 using a combination of ammonium sulfate precipitation and cation exchange chromatography. Purified samples were subjected to a controlled dis- and reassembly, yielding fully assembled HPV-16 L1 VLPs. This is the first report of production, purification, and assembly of VLPs in a non-vascular plant.

plant biology↗

bioRxiv2024

Recombinant production of spider silk protein in Physcomitrella photobioreactors

Spider dragline silk stands out as a remarkable biomaterial, representing one of natures toughest fibres. Its strength rivals that of many synthetic fibres used commercially, rendering it applicable across various industrial and medical domains. However, its widespread utilisation requires cost-effective mass production. Biotechnology presents a promising avenue for achieving this goal, particularly through the production of recombinant dragline silk proteins in transgenic plant systems. This study aimed to assess the feasibility of producing one key protein component of dragline silk, MaSp1, from the western black widow spider, Latrodectus hesperus, (LhMaSp1) in the moss Physcomitrella (Physcomitrium patens). Here, we present the successful recombinant production of spider silk protein containing both the N- and C-terminal domains (NTD and CTD) of LhMaSp1 protein in moss cells. NTD and CTD are necessary domains for protein assembly in spider silk. The production of recombinant LhMaSp1 protein in Physcomitrella was performed in shake flasks and in five-litre photobioreactors and the correct synthesis of LhMaSp1 was proven via mass spectrometry. Key messageWe report the first successful plant-produced recombinant spider silk key protein component containing both the N- and the C-terminal domain.

plant biology↗

bioRxiv2024

A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins

Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. Additionally, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins. Key messageAnalysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear encoded, mitochondria-localized proteins. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/598444v3_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f31702org.highwire.dtl.DTLVardef@6b9ce6org.highwire.dtl.DTLVardef@109c041org.highwire.dtl.DTLVardef@12cf83e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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↗

bioRxiv2024

Differential GTP-dependent in-vitro polymerization of recombinant Physcomitrella FtsZ proteins

1.Bacterial cell division and plant chloroplast division require self-assembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed in E. coli and characterized their GTPase activity and polymerization in vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interact in vivo and in vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

plant biology↗

bioRxiv2023

An ancient role for the CYP73 gene family in t-cinnamic acid 4-hydroxylation, phenylpropanoid biosynthesis and embryophyte development

The phenylpropanoid pathway is a plant metabolism intimately linked to the transition to terrestrial life. It produces phenolic compounds that play essential roles in stress mitigation and ecological interactions. The pathway also provides the building blocks for hydrophobic polymers that form apoplastic diffusion barriers and make up a significant fraction of the land plant biomass. Despite its significance in embryophytes (i.e., land plants), the origin and evolutionary history of the phenylpropanoid pathway remain poorly understood. In particular, little is known about the organization and function of the pathway in bryophytes, the non-vascular embryophytes. In this study, we conducted a multidisciplinary analysis of the CYP73 gene family that encodes t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific enzyme in the pathway. Our results indicate that C4H activity originated with the emergence of the CYP73 gene family in an ancestor of land plants and was supported by an arginine residue that stabilizes its substrate in the active site. C4H deficiency in the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by the exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event for the development of the canonical plant phenylpropanoid pathway and underscores the deep-rooted conservation of the C4H enzyme function in embryophyte biology.

plant biology↗

bioRxiv2023

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular the GMP-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3UTRs (untranslated regions) of which 7,964 transcripts contain at least one intron. Over 91% of 3UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3UTRs, 14 terminator candidates were selected and characterized via transient Dual Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1,000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than 10-fold, demonstrating the stronger effect promoters play as compared to terminators. The number of polyadenylation sites as well as polyadenylation signals were found to be major determinants of terminator performance. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general. Key messageCharacterization of Physcomitrella 3UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming and indicates promoters and terminators to synergistically control gene expression.

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↗

bioRxiv2023

Putative NAD(P)-binding Rossmann fold protein is involved in chitosan-induced peroxidase activity and lipoxygenase expression in Physcomitrella

Oxidative burst, the rapid production of high levels of reactive oxygen species (ROS) in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrella (new species name: Physcomitrium patens) mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in Physcomitrella, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO-lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes including alternative oxidase (AOX), lipoxygenase (LOX), NADPH-oxidase (NOX) and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO-lines than in wild-type plants after chitosan treatments. Expression of the LOX gene in Rossmann-fold KO-plants was significantly lower before and after chitosan treatment when compared to WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann-fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in Physcomitrella.

molecular biology↗

bioRxiv2023

Long-Distance Electrical and Calcium Signals Evoked by Hydrogen Peroxide in Physcomitrella

Electrical and calcium signals in plants are one of the basic carriers of information transmitted over a long distance. Together with reactive oxygen species (ROS) waves, electrical and calcium signals can participate in cell-to-cell signaling, conveying information about different stimuli, e.g. abiotic stress, pathogen infection, or mechanical injury. There is no information on the ability of ROS to evoke systemic electrical or calcium signals in the model moss Physcomitrella and on the relationships between these responses. Here, we show that external application of hydrogen peroxide evokes electrical signals in the form of long-distance changes in the membrane potential, which transmit through the plant instantly after stimulation. The responses were calcium dependent, since their generation was inhibited by lanthanum, a calcium channel inhibitor (2 mM) or EDTA, a calcium chelator (0.5 mM). The electrical signals were partially dependent on glutamate receptor ion channels (GLR), since the knockout of GLR genes only slightly reduced the amplitude of the responses. The basal part of the gametophyte, which is rich in protonema cells, was the most sensitive to hydrogen peroxide. The measurements carried out on the protonema expressing fluorescent calcium biosensor GCaMP3 proved that. We also demonstrate upregulation of a stress-related gene which appears in a distant section of the moss 8 minutes after H2O2 treatment. The results help to understand the importance of both types of signals in the transmission of information about the appearance of ROS in the plant cell apoplast.

plant biology↗

bioRxiv2022

Structural modelling of human complement FHR1 and two of its synthetic derivatives provides insight into their in-vivo functions

Human complement is the first line of defence against invading pathogens and is involved in tissue homeostasis. Complement-targeted therapies to treat several diseases caused by a dysregulated complement are highly desirable. Despite huge efforts invested in their development, only very few are currently available, and a deeper understanding of the numerous interactions and complement regulation mechanisms is indispensable. Two important complement regulators are human Factor H (FH) and Factor H-related protein 1 (FHR1). MFHR1 and MFHR13, two promising therapeutic candidates based on these regulators, combine the dimerization and C5-regulatory domains of FHR1 with the central C3-regulatory and cell surface-recognition domains of FH. Here, we used AlphaFold2 to model the structure of these two synthetic regulators. Moreover, we used AlphaFold-Multimer (AFM) to study possible interactions of C3 fragments and membrane attack complex (MAC) components C5, C7 and C9 in complex with FHR1, MFHR1, MFHR13 as well as the best-known MAC regulators vitronectin (Vn), clusterin and CD59, whose experimental structures remain undetermined. AFM successfully predicted the binding interfaces of FHR1 and the synthetic regulators with C3 fragments and suggested binding to C3. The models revealed structural differences in binding to these ligands through different interfaces. Additionally, AFM predictions of Vn, clusterin or CD59 with C7 or C9 agreed with previously published experimental results. Because the role of FHR1 as a MAC regulator has been controversial, we analysed possible interactions with C5, C7 and C9. AFM predicted interactions of FHR1 with proteins of the terminal complement complex (TCC) as indicated by experimental observations, and located the interfaces in FHR11-2 and FHR4-5. According to AFM predictions, FHR1 might partially block the C3b binding site in C5, inhibiting C5 activation, and block C5b-7 complex formation and C9 polymerization, with similar mechanisms of action as clusterin and vitronectin. Here, we generate hypotheses and provide the basis for the design of rational approaches to understand the molecular mechanism of MAC inhibition, which will facilitate the development of further complement therapeutics.

immunology↗

bioRxiv2022

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte abortion

O_LIThe auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens) three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. C_LIO_LIWe established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. C_LIO_LIPpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Further, the pinC mutants have a higher portion of differentially expressed genes (DEGs) related to spermatogenesis, increased fertility, and an increased abortion rate of premeiotic sporophytes. C_LIO_LIHere, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore, while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring. C_LI

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↗