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Plant Physiology 135:1879-1887 (2004)
© 2004 American Society of Plant Biologists

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BREAKTHROUGH TECHNOLOGIES

Use of the Fluorescent Timer DsRED-E5 as Reporter to Monitor Dynamics of Gene Activity in Plants1

Rossana Mirabella2, Carolien Franken, Gerard N.M. van der Krogt3, Ton Bisseling* and René Geurts

Wageningen University, Department of Plant Sciences, Laboratory of Molecular Biology, 6703 HA, Wageningen, The Netherlands

Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this problem. DsRED-E5 changes its emission spectrum over time from green to red in a concentration independent manner. Therefore, the green to red fluorescence ratio indicates the age of the protein and can be used as a fluorescent timer to monitor dynamics of gene expression. Here, we analyzed the potential of DsRED-E5 as reporter in plant cells. We showed that in cowpea (Vigna unguiculata) mesophyll protoplasts, DsRED-E5 changes its fluorescence in a way similar to animal cells. Moreover, the timing of this shift is suitable to study developmental processes in plants. To test whether DsRed-E5 can be used to monitor gene regulation in plant organs, we placed DsRED-E5 under the control of promoters that are either up- or down-regulated (MtACT4 and LeEXT1 promoters) or constitutively expressed (MtACT2 promoter) during root hair development in Medicago truncatula. Analysis of the fluorescence ratios clearly provided more accurate insight into the timing of promoter activity.

1 This work was supported by the European Community Training and Mobility of Researchers program FMRX CT 980239 (grant to R.M.).

2 Present address: Department of Plant Physiology, Swammerdam Institute for Life Science, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.

3 Present address: Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

www.plantphysiol.org/cgi/doi/10.1104/pp.103.038539.

* Corresponding author; e-mail ton.bisseling{at}wur.nl; fax 31–317–483584.

Received January 8, 2004; returned for revision May 6, 2004; accepted May 10, 2004.


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