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Fluorescence Activated Cell Sorting of Plant Protoplasts
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Measuring Sucrose Transporter Activities Using a Protoplast-Esculin Assay
Theresa Rottmann1, Ruth Stadler2
1Molecular Plant Physiology, Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|June 15, 2019
Summary
A novel in planta method using plant cells and a fluorescent sucrose analog allows for accurate analysis of sucrose transporter activity. This approach overcomes limitations of traditional methods, enabling better characterization of sugar transport proteins.
Area of Science:
- Plant molecular biology
- Biochemistry
- Membrane transport
Background:
- Sucrose transport is crucial for plant growth, mediated by SWEET and SUC proteins.
- Accurate characterization of sucrose transporter activity is essential for understanding sugar distribution.
Purpose of the Study:
- To develop and describe a new in planta method for analyzing sucrose transporter activity.
- To overcome limitations of heterologous expression systems in studying sucrose transport.
Main Methods:
- Utilized mesophyll protoplasts as an expression system.
- Employed the fluorescent sucrose analog esculin to monitor uptake.
- Applied confocal microscopy for visualization and quantitative analysis.
Main Results:
- Established a robust protoplast-esculin assay for measuring sucrose transporter function.
- Demonstrated the assay's utility for analyzing mutants, splice variants, and protein interactions.
- Provided a detailed protocol for construct design, experimental setup, and data evaluation.
Conclusions:
- The developed in planta assay offers a reliable alternative for studying sucrose transporters.
- This method enhances the accuracy of sucrose transport analysis, particularly when heterologous systems fail.
- Facilitates diverse research applications, including the study of transporter variations and interactions.
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