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

Neubauer, Anna; Ruaud, Stéphanie; Waller, Manuel; Frangedakis, Eftychios; Li, Fay‐Wei; Nötzold, Svenja I.; Wicke, Susann; Bailly, Aurélien; Szövényi, Péter

doi:10.1002/aps3.11456

Cited by 13 publications

(9 citation statements)

References 31 publications

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“…In contrast, the endogenous AaEf1a promoter is more strongly expressed in new grown tissue of the Oxford isolate (Frangedakis, Waller, et al 2021), which we hypothesise to be more susceptible to Agrobacterium infection. Interestingly, the AaEf1a promoter shows high activity in protoplasts of A. agrestis Bonn , while CaMV 35S activity could not be detected (Neubauer et al 2022). Thus, we reasoned that driving expression of the hph under the AaEf1a promoter might improve transformation efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…Until recently, model organisms were only available for the mosses and the liverworts, while a tractable model system was lacking for the third group of bryophytes, the hornworts. To further our understanding of hornwort biology, Anthoceros agrestis was recently proposed as an experimental model system (Frangedakis, Shimamura, et al 2021; Szövényi et al 2015; Gunadi, Li, and Van Eck 2022; Neubauer et al 2022; Li et al 2020) with two geographic isolates (Oxford and Bonn) available. Axenic culturing methods for both isolates have been established, their genomes have been sequenced and an Agrobacterium -mediated transformation method has been developed (Frangedakis et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

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An optimised transformation protocol forAnthoceros agrestisand three more hornwort species

Waller

Frangedakis

Marron

et al. 2022

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An optimised transformation protocol forAnthoceros agrestisand three more hornwort species

Waller

Frangedakis

Marron

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the study of plant molecular biology processes, the establishment of a suitable protocol for protoplast transformation will enable a detailed analysis of early signs of protoplast regeneration (e.g., chloroplast division and cell wall reconstruction) and expand the prospects for functional studies of plants ( Neubauer et al., 2022 ). Fluorescent dye labeling and qPCR can also be used to examine the effect of abiotic stress on the expression of protoplast-related genes after enzymatic digestion ( Pasternak et al., 2005 ).…”

Section: Discussionmentioning

confidence: 99%

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

et al. 2022

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The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.

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“…In the study of plant molecular biology processes, the establishment of a suitable protocol for protoplast transformation will not only allow a detailed analysis of early signs of protoplast regeneration (e.g. chloroplast division and cell wall reconstruction), but also expand the prospects for functional studies of plants (Neubauer et al, 2022). Fluorescent dye labeling and the use of qPCR techniques can also be applied to protoplasts (Pasternak et al, 2005) for the analysis of abiotic stress on the expression of protoplast-related genes after enzymatic digestion.…”

Section: Discussionmentioning

confidence: 99%

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

Zhang

Wang

Xiao

et al. 2022

Preprint

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The protoplast experimental system has been becoming a powerful tool for functional genomics and cell fusion breeding. However, the physiology and molecular mechanism during enzymolysis is not completely understood and has become a major obstacle to protoplast regeneration. Our study used physiological, cytology, iTRAQ (Isobaric Tags for Relative and Absolute Quantification) -based proteomic and RT-PCR analyses to compare the young leaves of sugarcane (ROC22) and protoplasts of more than 90% viability. We found that oxidation product MDA content increased in the protoplasts after enzymolysis and several antioxidant enzymes such as POD, CAT, APX, and O2- content significantly decreased. The cytology results showed that after enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed many short rod-like structures in protoplasts. The proteomic results showed that 1,477 differential proteins were down-regulated and 810 were up-regulated after enzymolysis of sugarcane young leaves. The GO terms, KEGG and KOG enrichment analysis revealed that differentially abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which would allow protein biosynthesis or / degradation. The RT-PCR analysis revealed the expression of osmotic stress resistance genes such as DREB, WRKY, MAPK4, and NAC were up-regulated. Meanwhile, the expression of key regeneration genes such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA and GAUT were significantly down-regulated in the protoplasts. Hierarchical clustering, identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. We used a variety of methods to figure out how young sugarcane leaves react to enzymes.

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

Cited by 13 publications

References 31 publications

An optimised transformation protocol forAnthoceros agrestisand three more hornwort species

An optimised transformation protocol forAnthoceros agrestisand three more hornwort species

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

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