Biology subjects

Sauret-Gueto, S.

Publications and source records attributed to Sauret-Gueto, S..

4 recordsLinked to original sources

bioRxiv2023

An Enhancer Trap system to track developmental dynamics in Marchantia polymorpha

A combination of streamlined genetics, experimental tractability and relative morphological simplicity compared to vascular plants makes the liverwort Marchantia polymorpha an ideal model system for studying many aspects of plant biology. Here we describe a transformation vector combining a constitutive fluorescent membrane marker with a nuclear marker that is regulated by nearby enhancer elements, and use this to produce a library of enhancer trap lines for Marchantia. Screening gemmae from these lines allowed for identification and characterization of novel marker lines, including markers for rhizoids and oil cells. The library allowed the identification of a margin tissue running around the thallus edge, highlighted during thallus development. Expression of this marker is correlated with auxin levels. We generated multiple markers for the meristematic apical notch region, which have different spatial expression patterns, reappear at different times during meristem regeneration following apical notch excision and have varying responses to auxin supplementation or inhibition. This reveals that there are proximodistal substructures within the apical notch that could not be observed otherwise. We employed our markers to study Marchantia sporeling development, observing meristem emergence as defining the protonema-to-prothallus stage transition, and subsequent production of margin tissue during the prothallus stage. Exogenous auxin treatment stalls meristem emergence at the protonema stage but does not inhibit cell division, resulting in callus-like sporelings with many rhizoids, whereas pharmacologically inhibiting auxin synthesis and transport does not prevent meristem emergence. This enhancer trap system presents a useful resource for the community and will contribute to future Marchantia research. Significance StatementThe liverwort Marchantia polymorpha is an emerging model species with huge potential for synthetic biology. We generated an enhancer trap library as a resource for Marchantia research and characterized marker lines for several important cell types (rhizoids, thallus margin, apical notch and oil cells). These marker lines can be used to track cellular dynamics in growing plants, exemplified by studying auxin responses and sporeling growth, revealing new insights into thallus development and cell fate specification.

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↗

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↗