Biology subjects

Frangedakis, E.

Publications and source records attributed to Frangedakis, E..

12 recordsLinked to original sources

bioRxiv2026

Marchantia polymorpha as a simple platform for plant-based production of functional nanobodies

Recombinant proteins have transformative potential in biomedicine, but their production is often costly and carries contamination risks. Plants offer an attractive alternative, with low growth costs and reduced pathogen risk, yet their slow growth cycles limit their use for rapid protein engineering. Here, we establish Marchantia polymorpha, a genetically tractable liverwort with a short life cycle, as a new platform for recombinant protein production. Using stable Agrobacterium-mediated transformation, we expressed an anti-mCherry nanobody fused to the fluorescent protein mTurquoise2 with different purification tags. Expression levels reached up to [~]120 {micro}g/g fresh weight, and nanobody functionality was validated through a microscopy-based bead-binding assay. This yield rivals that of established systems such as Nicotiana benthamiana. Our results position M. polymorpha as a scalable, safe, and efficient chassis for protein engineering, with broad potential for applications in synthetic biology. This work opens the door to exploiting liverwort biotechnology for fast, cost-effective, and biosafe production of valuable recombinant proteins. Graphical Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/733889v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@aabc4aorg.highwire.dtl.DTLVardef@12f9fa4org.highwire.dtl.DTLVardef@199d3e0org.highwire.dtl.DTLVardef@2533a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Plant Biology↗

bioRxiv2025

A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha.

The liverwort Marchantia polymorpha is a widely used model organism for studying land plant biology, which has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has emerged as a transformative tool for precise genome modifications in M. polymorpha. However, a robust method for the simultaneous expression of multiple gRNAs, which is crucial for enhancing the efficiency and versatility of CRISPR/Cas9-based genome editing, has yet to be fully developed. In this study, we introduce an adaptation from the OpenPlant kit CRISPR/Cas9 tools, that facilitates expression of multiple gRNAs from a single transcript through incorporation of tRNA sequences. This approach significantly improves the efficiency and scalability of genome editing in M. polymorpha. Additionally, by combining this vector system with a simplified and optimized protocol for thallus transformation, we further streamline the generation of CRISPR/Cas9 mutants in M. polymorpha. The resulting gene- editing system offers a versatile, time-saving and straightforward tool for advancing functional genomics in M. polymorpha, enabling more comprehensive genetic modifications and genome engineering.

plant biology↗

bioRxiv2025

The ABCG1 transporter is required for terpene biosynthesis in the lumen of Marchantia polymorpha oil bodies.

Marchantia polymorpha oil bodies (OBs) are specialised cell structures housing a diverse array of C15-terpenes, called sesquiterpenes. These compounds are known for their roles as herbivore repellents, yet the enzymes responsible for the biosynthesis of their precursors (C5 isoprenoid units) remain poorly characterized. Discrepancies remain between enzyme localizations suggested by computational predictions and those observed in earlier experimental studies, complicating our understanding of terpene biosynthesis. We investigated the localization of isoprenoid biosynthetic enzymes using translational and transcriptional reporters, coupled with confocal microscopy. Most enzymes localized as predicted (e.g., cytosol, chloroplast and the endoplasmic reticulum), and OB cells were identified as the primary sites of terpene biosynthesis. To explore OBs as potential storage sites for terpenes, we attempted to produce exogenous but easily identifiable compounds in Marchantia, such as the diterpene taxadiene and the triterpene {beta}-amyrin. Targeting to OB cells resulted in measurable amounts of these compounds, but their yields remained unaffected by the over-expression of key precursor genes, underscoring challenges in redirecting metabolic flux. To further investigate terpene accumulation in OBs, we focused on ABCG1, an ABC transporter previously reported to localize at the OB membrane. Overexpression of ABCG1 in OB cells, alongside an exogenous sesquiterpene synthase, only increased the levels of endogenous sesquiterpenes, while CRISPR-mediated disruption of ABCG1 resulted in a dramatic reduction in sesquiterpene accumulation. These findings establish ABCG1 as a critical factor for sesquiterpene retention within OBs and provide new insights into the mechanisms governing terpene metabolism and storage in Marchantia polymorpha.

plant biology↗

bioRxiv2024

Semi-automated workflow for high-throughput Agrobacterium-mediated plant transformation

High-throughput experiments in plants are hindered by long generation times and high costs. To address these challenges, we present an optimized pipeline for Agrobacterium tumefaciens transformation and simplified a protocol to obtain stable transgenic lines of the model liverwort Marchantia polymorpha, paving the way for efficient high-throughput experiments for plant synthetic biology and other applications. Our protocol involves freeze-thaw Agrobacterium transformation method in 6-well plates that can be adapted to robotic automation. Using the Opentrons open-source platform, we implemented a semi-automated protocol showing similar efficiency compared to manual manipulation. Additionally, we have streamlined and simplified the process of stable transformation and selection of M. polymorpha, reducing cost, time, and manual labour without compromising transformation efficiency. The addition of sucrose in the selection media significantly enhances the production of gemmae, accelerating the generation of isogenic plants. We believe these protocols have the potential to facilitate high-throughput screenings in diverse plant species and represent a significant step towards the full automation of plant transformation pipelines. This approach allows testing [~]100 constructs per month, using conventional plant tissue culture facilities. We recently demonstrated the successful implementation of this protocol for screening hundreds of fluorescent reporters in Marchantia gemmae.

synthetic biology↗

bioRxiv2023

Synergistic control of chloroplast biogenesis by MYB-related and Golden2-like transcription factors

Chloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors, but glk mutants contain residual chlorophyll and therefore other proteins must also be involved. Here we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwort Marchantia polymorpha and angiosperm Arabidopsis thaliana. In both species, double mutant alleles in MYB-Related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in mutants of GLK. In M. polymorpha MYB-related genes act upstream of GLK, while in A. thaliana this relationship has been rewired. In both species, genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins whilst those allowing CO2 fixation, photorespiration and photosystem assembly and repair require the MYB-related proteins. Thus, MYB-related and GLK genes have overlapping as well as distinct targets. We conclude that together MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.

plant biology↗

bioRxiv2023

Mapping the landscape of transcription factor promoter activity during vegetative development in Marchantia

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterising the transcriptional activity of TF genes in space and time is a critical step towards understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot-apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only [~]450 TF genes, Marchantia polymorpha is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested in planta reporter fusions for all the TF promoters in the collection and systematically analysed expression patterns in Marchantia gemmae. This allowed us to build a map of precise expression domains and identify a unique set of TFs expressed in the stem-cell zone, providing new insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that the promoter elements characterised here will be useful for cell-type specific expression, synthetic biology applications, and functional genomics.

plant biology↗

bioRxiv2023

Streamlined regulation of chloroplast development in the liverwort Marchantia polymorpha

Photosynthesis in eukaryotic cells takes place in chloroplasts that develop from undifferentiated plastids in response to light. In angiosperms, after perception of light de-repression allows Elongated Hypocotyl 5 (HY5) transcription factor to initiate photomorphogenesis, and two families of transcription factors known as Golden2-like (GLK) and GATA are considered master regulators of chloroplast development. The MIR171-targeted SCARECROW-LIKE (SCL) GRAS transcription factors also impact on chlorophyll biosynthesis. The extent to which these proteins carry out conserved roles in non-seed plants is not known. Here we report in the model liverwort Marchantia polymorpha that GLK controls chloroplast biogenesis and HY5 shows a small conditional effect on chlorophyll content. In contrast, GATA and SCL have no detectable roles in this fundamental process. ChIP-SEQ and RNA-SEQ revealed that MpGLK regulates many photosynthetic and chloroplast development-related genes, but also has a broader set of targets than previously reported in angiosperms. This implies GLK carries out a conserved role relating to chloroplast biogenesis in land plants but also supports extensive divergence between its targets in M. polymorpha and flowering plants. The data support the hypothesis that regulation of chloroplast biogenesis in M. polymorpha is streamlined compared with angiosperms and allows us to present a core regulatory network for chloroplast biogenesis in land plants.

plant biology↗

bioRxiv2022

An optimised transformation protocol for Anthoceros agrestis and three more hornwort species

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology.

plant biology↗

bioRxiv2022

Loss of plastid developmental genes coincides with a reversion to monoplastidy in hornworts

The first plastid evolved from an endosymbiotic cyanobacterium in the common ancestor of the Archaeplastida. The transformative steps from cyanobacterium to organelle included the transfer of control over developmental processes; a necessity for the host to orchestrate, for example, the fission of the organelle. The plastids of almost all embryophytes divide independent from nuclear division, leading to cells housing multiple plastids. Hornworts, however, are monoplastidic (or near-monoplastidic) and their photosynthetic organelles are a curious exception among embryophytes for reasons such as the occasional presence of pyrenoids. Here we screened genomic and transcriptomic data of eleven hornworts for components of plastid developmental pathways. We find intriguing differences among hornworts and specifically highlight that pathway components involved in regulating plastid development and biogenesis were differentially lost in this group of bryophytes. In combination with ancestral state reconstruction, our data suggest that hornworts have reverted back to a monoplastidic phenotype due to the combined loss of two plastid division-associated genes: ARC3 and FtsZ2.

evolutionary biology↗

bioRxiv2021

A simple Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the {beta}-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

plant biology↗

bioRxiv2020

Comparative analysis of early divergent land plants and construction of DNA tools for hyper-expression in Marchantia chloroplasts

Chloroplast genes are present at high ploidy in plants, and capable of driving very high levels of gene expression if mRNA production and stability are properly regulated. Marchantia polymorpha is a simple model plant that allows rapid transformation studies, however post-transcriptional regulation in plastids is poorly characterized in this liverwort. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 early-divergent plant species, and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilisation. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplasmic transformed plants. We have produced novel DNA tools for protein hyper-expression in a facile plant system that is a test-bed for chloroplast engineering.

plant biology↗

bioRxiv2020

Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha.

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

synthetic biology↗