From jellyfish to biosensors: the use of fluorescent proteins in plants

Abstract

The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors.

Rule-Based Design of Plant Expression Vectors Using GenoCAD

“…The availability of FTG markers has increased noticeably over the last two decades. The large selection of FTG markers available in plant molecular biology together with the development of sophisticated image acquisition and analysis software contributed to the rapidly and increased adoption of this technology by plant molecular biologists [ 14 ]. Therefore, we implemented a grammar to design constructs for protein localization studies based on fused FTG.…”

“…The use of FTGs in plant biology is especially challenging because of the autofluorescence displayed by several components of plant tissues (chlorophyll, lignified secondary cell walls, and vacuolar contents) overlap with the emission wavelength of green fluorescent protein (GFP) [ 14 ]. The FTGs currently added in the GenoCAD parts library have all been previously tested in Nicotiana benthamiana and Solanum tuberosum leaves, and they include enhanced GFP (eGFP), yellow fluorescent protein (eYFP), cyan fluorescent protein (eCFP) and mCherry.…”

A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase

“…This performance is a thousand-fold improvement over previous cytokinin biosensors. By being able to resolve signals in the low nanomolar, the CKX biosensor is also more sensitive than previously described purine biosensors for adenosine and ATP based on oxidases [32], [37] and such nanomolar sensitivity is uncommon [38], [39]. The excellent specificity of free Zm CKX1 was also retained and the microbiosensor detected a range of cytokinins, including N 6 -(Δ 2 -isopentenyl) adenine, N 6 -(Δ 2 -isopentenyl) adenosine, cis -zeatin, trans -zeatin and trans -zeatin riboside.…”

Moving Toward a Comprehensive Map of Central Plant Metabolism

“…These techniques use either fluorescent tagging (142) or immunolocalization (88), neither of which, unfortunately, is amenable to high-throughput application. In addition to providing information about subcellular localization, these techniques provide information about possible interactions between proteins, or about the binding of proteins to membrane and organelle surfaces and subsequent modification of their activities and/or efficiency of action (56).…”