Step‐by‐step protocol for the isolation and transient transformation of hornwort protoplasts

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Abstract

Premise

A detailed protocol for the protoplast transformation of hornwort tissue is not yet available, limiting molecular biological investigations of these plants and comparative analyses with other bryophytes, which display a gametophyte‐dominant life cycle and are critical to understanding the evolution of key land plant traits.

Methods and Results

We describe a detailed protocol to isolate and transiently transform protoplasts of the model hornwort Anthoceros agrestis. The digestion of liquid cultures with Driselase yields a high number of viable protoplasts suitable for polyethylene glycol (PEG)‐mediated transformation. We also report early signs of protoplast regeneration, such as chloroplast division and cell wall reconstitution.

Conclusions

This protocol represents a straightforward method for isolating and transforming A. agrestis protoplasts that is less laborious than previously described approaches. In combination with the recently developed stable genome transformation technique, this work further expands the prospects of functional studies in this model hornwort.

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Most cited references 29

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Icy: an open bioimage informatics platform for extended reproducible research.

Fabrice de Chaumont, Stéphane Dallongeville, Nicolas Chenouard … (2012)

Current research in biology uses evermore complex computational and imaging tools. Here we describe Icy, a collaborative bioimage informatics platform that combines a community website for contributing and sharing tools and material, and software with a high-end visual programming framework for seamless development of sophisticated imaging workflows. Icy extends the reproducible research principles, by encouraging and facilitating the reusability, modularity, standardization and management of algorithms and protocols. Icy is free, open-source and available at http://icy.bioimageanalysis.org/.

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The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte

Mark Puttick, Jennifer L Morris, Tom Williams … (2018)

The evolutionary emergence of land plant body plans transformed the planet. However, our understanding of this formative episode is mired in the uncertainty associated with the phylogenetic relationships among bryophytes (hornworts, liverworts, and mosses) and tracheophytes (vascular plants). Here we attempt to clarify this problem by analyzing a large transcriptomic dataset with models that allow for compositional heterogeneity between sites. Zygnematophyceae is resolved as sister to land plants, but we obtain several distinct relationships between bryophytes and tracheophytes. Concatenated sequence analyses that can explicitly accommodate site-specific compositional heterogeneity give more support for a mosses-liverworts clade, "Setaphyta," as the sister to all other land plants, and weak support for hornworts as the sister to all other land plants. Bryophyte monophyly is supported by gene concatenation analyses using models explicitly accommodating lineage-specific compositional heterogeneity and analyses of gene trees. Both maximum-likelihood analyses that compare the fit of each gene tree to proposed species trees and Bayesian supertree estimation based on gene trees support bryophyte monophyly. Of the 15 distinct rooted relationships for embryophytes, we reject all but three hypotheses, which differ only in the position of hornworts. Our results imply that the ancestral embryophyte was more complex than has been envisaged based on topologies recognizing liverworts as the sister lineage to all other embryophytes. This requires many phenotypic character losses and transformations in the liverwort lineage, diminishes inconsistency between phylogeny and the fossil record, and prompts re-evaluation of the phylogenetic affinity of early land plant fossils, the majority of which are considered stem tracheophytes.

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Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts

Fay-Wei Li, Tomoaki Nishiyama, Manuel Waller … (2020)

Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB , a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants. Analyses of three high-quality genomes of Anthoceros hornworts place hornworts as a sister clade to the lineage including liverworts and mosses, and provide insights into the unique biological features of hornworts.

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Author and article information

Contributors

Anna Neubauer:

ORCID: https://orcid.org/0000-0002-5102-2491

anna.neubauer@uzh.ch

Péter Szövényi:

ORCID: https://orcid.org/0000-0002-0324-4639

peter.szoevenyi@systbot.uzh.ch

Journal

Journal ID (nlm-ta): Appl Plant Sci

Journal ID (iso-abbrev): Appl Plant Sci

Journal ID (doi): 10.1002/(ISSN)2168-0450

Journal ID (publisher-id): APS3

Title: Applications in Plant Sciences

Publisher: John Wiley and Sons Inc. (Hoboken )

ISSN (Electronic): 2168-0450

Publication date (Electronic): 11 February 2022

Publication date Collection: Mar-Apr 2022

Volume: 10

Issue: 2 , Methodologies in Gametophyte Biology ( doiID: 10.1002/aps3.v10.2 )

Electronic Location Identifier: e11456

Affiliations

[ ¹ ] Department of Systematic and Evolutionary Botany University of Zurich Zurich Switzerland

[ ² ] Zurich‐Basel Plant Science Center Zurich Switzerland

[ ³ ] Department of Plant Sciences University of Cambridge Cambridge United Kingdom

[ ⁴ ] Boyce Thompson Institute Ithaca New York USA

[ ⁵ ] Plant Biology Section Cornell University Ithaca New York USA

[ ⁶ ] Institute for Biology Humboldt University of Berlin Berlin Germany

[ ⁷ ] Späth‐Arboretum Humboldt University of Berlin Berlin Germany

[ ⁸ ] Department of Plant and Microbial Biology University of Zurich Zurich Switzerland

Author notes

[*] [* ] Correspondence Péter Szövényi and Anna Neubauer, Department of Systematic and Evolutionary Botany, University of Zurich, Zollikerstrasse 107, CH‐8008 Zurich, Switzerland.

Email: peter.szoevenyi@ 123456systbot.uzh.ch (P.S.) and anna.neubauer@ 123456uzh.ch (A.N.)

Author information

Anna Neubauer https://orcid.org/0000-0002-5102-2491

Stéphanie Ruaud https://orcid.org/0000-0001-7698-0605

Manuel Waller https://orcid.org/0000-0002-6060-0740

Eftychios Frangedakis https://orcid.org/0000-0002-3483-8464

Fay‐Wei Li https://orcid.org/0000-0002-0076-0152

Susann Wicke https://orcid.org/0000-0001-5785-9500

Aurélien Bailly https://orcid.org/0000-0001-5546-1945

Péter Szövényi https://orcid.org/0000-0002-0324-4639

Article

Publisher ID: APS311456

DOI: 10.1002/aps3.11456

PMC ID: 9039799

PubMed ID: 35495192

SO-VID: 8037ae27-682c-41a8-8794-29246258a7a6

License:

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

History

Date received : 15 August 2021

Date accepted : 23 November 2021

Page count

Figures: 5, Tables: 2, Pages: 10, Words: 5546

Custom metadata

source-schema-version-number 2.0

cover-date March-April 2022

details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.4 mode:remove_FC converted:26.04.2022

Keywords: anthoceros,hornworts,model organism,protoplasts,transient transformation

Data availability:

Keywords: anthoceros, hornworts, model organism, protoplasts, transient transformation

Step‐by‐step protocol for the isolation and transient transformation of hornwort protoplasts

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Abstract

Premise

Methods and Results

Conclusions

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G3: Genes|Genomes|Genetics

Most cited references 29

Icy: an open bioimage informatics platform for extended reproducible research.

The Interrelationships of Land Plants and the Nature of the Ancestral Embryophyte

Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts

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