An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue
Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that requ...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-12-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/10/12/197 |
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| author | Gabriele Panicucci Sergio Iacopino Elisa De Meo Pierdomenico Perata Daan A. Weits |
| author_facet | Gabriele Panicucci Sergio Iacopino Elisa De Meo Pierdomenico Perata Daan A. Weits |
| author_sort | Gabriele Panicucci |
| collection | DOAJ |
| description | Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead. This article reports the design, test, and optimization of a hypoxia-signaling reporter, based on five-time repeated hypoxia-responsive promoter elements (HRPE) driving the expression of different reporter proteins. Specifically, this study aimed to improve its performance as a reporter of hypoxic conditions by testing the effect of different untranslated regions (UTRs) at the 5′ end of the reporter coding sequence. Next, we characterized an optimized version of the <i>HRPE</i> promoter (<i>HRPE-Ω</i>) in terms of hypoxia sensitivity and time responsiveness. We also observed that severe oxygen deficiency counteracted the reporter activity due to inhibition of GFP maturation, which requires molecular oxygen. To overcome this limitation, we therefore employed an oxygen-independent UnaG fluorescent protein-coupled to an O<sub>2</sub>-dependent mCherry fluorophore under the control of the optimized <i>HRPE-Ω</i> promoter. Remarkably, this sensor, provided a different mCherry/UnaG ratiometric output depending on the externally imposed oxygen concentration, providing a solution to distinguish between different degrees of tissue hypoxia. Moreover, a ubiquitously expressed UnaG-mCherry fusion could be used to image oxygen concentrations directly, albeit at a narrow range. The luminescent and fluorescent hypoxia-reporters described here can readily be used to conduct studies that involve anaerobiosis in plants. |
| first_indexed | 2024-03-10T14:21:48Z |
| format | Article |
| id | doaj.art-d54ee37913734c83b291d87d30dee82d |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-10T14:21:48Z |
| publishDate | 2020-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-d54ee37913734c83b291d87d30dee82d2023-11-20T23:19:01ZengMDPI AGBiosensors2079-63742020-12-01101219710.3390/bios10120197An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant TissueGabriele Panicucci0Sergio Iacopino1Elisa De Meo2Pierdomenico Perata3Daan A. Weits4Biology Department, University of Pisa, 56126 Pisa, ItalyBiology Department, University of Pisa, 56126 Pisa, ItalyDepartment of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, ItalyInstitute of Life Sciences, Scuola Superiore Sant’Anna, 56127 Pisa, ItalyInstitute of Life Sciences, Scuola Superiore Sant’Anna, 56127 Pisa, ItalyOxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead. This article reports the design, test, and optimization of a hypoxia-signaling reporter, based on five-time repeated hypoxia-responsive promoter elements (HRPE) driving the expression of different reporter proteins. Specifically, this study aimed to improve its performance as a reporter of hypoxic conditions by testing the effect of different untranslated regions (UTRs) at the 5′ end of the reporter coding sequence. Next, we characterized an optimized version of the <i>HRPE</i> promoter (<i>HRPE-Ω</i>) in terms of hypoxia sensitivity and time responsiveness. We also observed that severe oxygen deficiency counteracted the reporter activity due to inhibition of GFP maturation, which requires molecular oxygen. To overcome this limitation, we therefore employed an oxygen-independent UnaG fluorescent protein-coupled to an O<sub>2</sub>-dependent mCherry fluorophore under the control of the optimized <i>HRPE-Ω</i> promoter. Remarkably, this sensor, provided a different mCherry/UnaG ratiometric output depending on the externally imposed oxygen concentration, providing a solution to distinguish between different degrees of tissue hypoxia. Moreover, a ubiquitously expressed UnaG-mCherry fusion could be used to image oxygen concentrations directly, albeit at a narrow range. The luminescent and fluorescent hypoxia-reporters described here can readily be used to conduct studies that involve anaerobiosis in plants.https://www.mdpi.com/2079-6374/10/12/197oxygenhypoxiaERF-VIIUnaGanoxiareporter |
| spellingShingle | Gabriele Panicucci Sergio Iacopino Elisa De Meo Pierdomenico Perata Daan A. Weits An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue Biosensors oxygen hypoxia ERF-VII UnaG anoxia reporter |
| title | An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue |
| title_full | An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue |
| title_fullStr | An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue |
| title_full_unstemmed | An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue |
| title_short | An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue |
| title_sort | improved hrpe based transcriptional output reporter to detect hypoxia and anoxia in plant tissue |
| topic | oxygen hypoxia ERF-VII UnaG anoxia reporter |
| url | https://www.mdpi.com/2079-6374/10/12/197 |
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