Determination of protoplast growth properties using quantitative single-cell tracking analysis
Abstract Background Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cell...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-05-01
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| Series: | Plant Methods |
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| Online Access: | https://doi.org/10.1186/s13007-022-00895-x |
| _version_ | 1828323341301186560 |
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| author | Jonathan Dawson Saurabh Pandey Qiuju Yu Patrick Schaub Florian Wüst Amir Bahram Moradi Oleksandr Dovzhenko Klaus Palme Ralf Welsch |
| author_facet | Jonathan Dawson Saurabh Pandey Qiuju Yu Patrick Schaub Florian Wüst Amir Bahram Moradi Oleksandr Dovzhenko Klaus Palme Ralf Welsch |
| author_sort | Jonathan Dawson |
| collection | DOAJ |
| description | Abstract Background Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions. Results High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4). Conclusions AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis. |
| first_indexed | 2024-04-13T18:51:47Z |
| format | Article |
| id | doaj.art-726d4be6ec09449c9d66e56d5f7cf380 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-04-13T18:51:47Z |
| publishDate | 2022-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-726d4be6ec09449c9d66e56d5f7cf3802022-12-22T02:34:24ZengBMCPlant Methods1746-48112022-05-0118111510.1186/s13007-022-00895-xDetermination of protoplast growth properties using quantitative single-cell tracking analysisJonathan Dawson0Saurabh Pandey1Qiuju Yu2Patrick Schaub3Florian Wüst4Amir Bahram Moradi5Oleksandr Dovzhenko6Klaus Palme7Ralf Welsch8Institute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgInstitute of Biology II, Faculty of Biology, Albert-Ludwigs-University of FreiburgAbstract Background Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions. Results High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4). Conclusions AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.https://doi.org/10.1186/s13007-022-00895-xBAGCell expansionCell trackingProtoplastsSingle cellTobacco |
| spellingShingle | Jonathan Dawson Saurabh Pandey Qiuju Yu Patrick Schaub Florian Wüst Amir Bahram Moradi Oleksandr Dovzhenko Klaus Palme Ralf Welsch Determination of protoplast growth properties using quantitative single-cell tracking analysis Plant Methods BAG Cell expansion Cell tracking Protoplasts Single cell Tobacco |
| title | Determination of protoplast growth properties using quantitative single-cell tracking analysis |
| title_full | Determination of protoplast growth properties using quantitative single-cell tracking analysis |
| title_fullStr | Determination of protoplast growth properties using quantitative single-cell tracking analysis |
| title_full_unstemmed | Determination of protoplast growth properties using quantitative single-cell tracking analysis |
| title_short | Determination of protoplast growth properties using quantitative single-cell tracking analysis |
| title_sort | determination of protoplast growth properties using quantitative single cell tracking analysis |
| topic | BAG Cell expansion Cell tracking Protoplasts Single cell Tobacco |
| url | https://doi.org/10.1186/s13007-022-00895-x |
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