Fluorescent reporters for functional analysis in rice leaves
| Autor: | Luginbuehl, Leonie H., El-Sharnouby, Sherif, Wang, Na, Hibberd, Julian M. |
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| Přispěvatelé: | Hibberd, Julian [0000-0003-0662-7958], Apollo - University of Cambridge Repository |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Cell type Fluorescence-lifetime imaging microscopy fluorescent proteins Plant Science 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) rice leaves 03 medical and health sciences Live cell imaging Gene expression Ecology Evolution Behavior and Systematics Original Research 030304 developmental biology 0303 health sciences Ecology Functional analysis Chemistry Botany food and beverages stable transformants 15. Life on land Protein subcellular localization prediction Fluorescence Transformation (genetics) QK1-989 Biophysics microparticle bombardment 010606 plant biology & botany |
| Zdroj: | Plant Direct Plant Direct, Vol 4, Iss 2, Pp n/a-n/a (2020) |
| ISSN: | 2475-4455 |
| DOI: | 10.1002/pld3.188 |
| Popis: | Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed analysis of processes ranging from gene expression and protein localization through to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualise fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee, and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins, mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato that are robustly expressed and visible in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolour imaging of different sub-cellular compartments. Overall, we conclude that mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato are suitable for high resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.One sentence summaryWe report five fluorescent reporters suitable for functional analysis in rice leaves. |
| Databáze: | OpenAIRE |
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