Reporter genes and systems play essential roles in the biological sciences. β-glucuronidase (GUS), luciferase (LUC), and green fluorescent protein (GFP) are extensively used as reporters or selectable markers in plants. These compounds all require the use of special equipment or substrates, whereas RUBY produces the red pigment betalain, which is visible to the naked eye. However, production of betalain in leaves or other tissues is irreversible and may interfere with various biological processes, including photosynthesis. It is therefore important to develop more efficient, convenient, and widely usable reporter genes for plants.
With this in mind, researchers modified a GFP-like protein, eYGFPuv, that shows no harmful effects on plant biological processes. Its strong green fluorescence can be observed by the naked eye in real time under UV light in both transient expression and stable transformation systems regardless of the shape, size, and location of the target samples. It requires neither a fluorescence microscope nor specific substrates. Researchers successfully observed eYGFPuv green fluorescence in two herbaceous plant species (Arabidopsis and tobacco) and two woody plant species (poplar and citrus). This work demonstrates the feasibility of using 1× eYGFPuv in transformation studies with diverse plants. The technique enables the monitoring of gene expression and protein localization in diverse organisms with greater flexibility and efficiency.
“One advantage of eYGFPuv as a reporter is that UV‐excitable eYGFPuv can be easily imaged at a wide range of scales from the sub‐meter level seedlings to whole plants without the need for emission filters. Another advantage is that the eYGFPuv-expressing tissue or whole plants can be directly visualized in a petri dish or tissue culture vessel without removing the lid, and eYGFPuv signals can be seen in dim light or dark conditions.” said the team of researchers from the Biosciences Division and the Center for Bioenergy Innovation, both at Oak Ridge National Laboratory. These features suggest that 1× eYGFPuv has a wide range of applications in plant science research.
The researchers described a number of potential applications of 1× eYGFPuv in plant science research. Its use during in planta transformation could eliminate the need for substrates and luciferase imaging systems, enabling the identification of transgenic shoots in real time. 1× eYGFPuv could be used directly for seed selection after floral dip transformation or as a reporter gene for antibiotic-free in vitro transformation. It could be linked to a target gene in order to track gene expression. 1× eYGFPuv could also be used for promoter characterization, including studies of tissue specificity and hormonal regulation, screening of synthetic promoters, and assessing the tightness of inducible promoters. It could be used to reveal pathogen invasion or drought stress during plant growth and to monitor the transport of targets such as proteins from roots to shoots. Finally, 1× eYGFPuv could also be used in long-term studies of transgene stability.
More information can be found in the article ‘Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants’ published in the journal Horticulture Research.
Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants
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