Switching proteins on and off: shedding new light on the plant cell
To track a protein inside a living cell, biologists attach a luminous tag to it, most often GFP (Green Fluorescent Protein), a jellyfish protein that fluoresces green when illuminated. Researchers at BIAM and their partners show that an alternative, called FAST, works in plants.
Smaller, brighter and more robust, it has a rare advantage: its light can be switched on and off on demand. This work is published in The Plant Journal.
GFP, an imperfect star
GFP earned the scientists who discovered and developed it the 2008 Nobel Prize in Chemistry. It enabled giant leaps in cell biology. Yet it has its flaws. Being bulky, it can interfere with the properties of the proteins it is attached to. It requires oxygen and loses intensity in acidic conditions (the conditions used to “fix” samples for preservation). Finally, under the microscope’s excitation light, its signal drops sharply. This process, called photobleaching, limits long observations.
Why make proteins glow?
Proteins are the workers of the cell, but they are too small to be seen under a microscope. Attaching a fluorescent tag to them is like fitting them with a beacon: it makes it possible to locate them in the cell and even to reveal their interactions with other proteins. In the past, histological staining techniques required killing the cell. Fluorescent tags make it possible to observe it alive and to follow how it changes over time: it’s the difference between a photo and a film.
A tag in two parts
FAST proteins were developed by Arnaud Gautier’s team (Laboratoire des biomolécules, Paris) from a bacterial protein. They work as a pair: a small protein, half the size of GFP, attached to the protein of interest, and a molecule supplied from outside, the fluorogen. On its own, the fluorogen barely glows. Bound to FAST, it lights up. Adding the fluorogen to the sample switches FAST on, while rinsing removes the signal.
Proven in bacteria, yeast and animals, the system had yet to be tested in plants.
It works in thale cress
The researchers created thale cress plants (Arabidopsis thaliana), the laboratory model plant, producing a green (Green-FAST) or red (Red-FAST) version of the protein. When bathed in fluorogen, the roots light up within seconds, right down to their innermost cells. Stems and leaves need a longer incubation (half an hour), probably because the fluorogen must travel from the roots to the aerial parts. The fluorogen does not affect growth, and a 5- to 15-minute rinse switches the signal off.
Brighter and longer-lasting
Under the same conditions, Green-FAST shines about 2.4 times brighter than GFP, and Red-FAST 1.4 times brighter. Under the microscope, Green-FAST takes four times longer than GFP to lose half of its brightness. Red-FAST also clearly outperforms mCherry, the reference red marker. After fixation or in acidic conditions, FAST proteins remain visible where GFP fades.
A beacon for every destination in the cell
The team attached various FAST tags to plant proteins known to be targeted to distinct cell compartments, in thale cress and in tobacco leaves. This made it possible to label compartments as diverse as the cell membrane, with a phosphate transporter; the nucleus, with two gene regulators; as well as mitochondria (the cell’s power plants), peroxisomes and chloroplasts (where photosynthesis takes place). FAST proteins and their fluorogens prove to be reliable markers.
Visualising interactions between proteins
A variant, Split-FAST, uses a FAST protein cut into two pieces, each fused to a different protein. If the two proteins associate, FAST is reconstituted and, in the presence of the fluorogen, a fluorescent signal appears. This was tested on a known pair: a calcium-sensing protein and an enzyme bound for the chloroplast. Result: a clear light where the pair forms. With a modified enzyme, unable to bind: nothing. The icing on the cake: this FAST protein accepts different fluorogens, giving a green, yellow or red signal.
Why it matters
Plant biologists now have a tool that complements GFP: less bulky, brighter, longer-lasting and usable on fixed samples. The on/off switch separates the true signal from background noise, by comparing the cell before and after adding the fluorogen. The colour can also be changed when FAST has to be combined with other markers.
What next?
Unlike its GFP-based equivalent, which stays locked together, Split-FAST can come apart. It could therefore be used to film proteins as they associate and then separate, revealing their dynamics: this is the next step. FAST also remains to be tested in highly acidic compartments, such as the vacuole.
Reference: David P., Michaud M., Moyet L., Pullara S. et al. (2026). The fluorescence-activating and absorption-shifting tag (FAST), a versatile protein marker for live plant cell imaging. The Plant Journal, 126, e70966. doi:10.1111/tpj.70966
Collaborations: BIAM (EBMP team, Cadarache), LPCV Grenoble, IPSiM Montpellier, LBM Paris, IGH Montpellier, The Twinkle Factory.
Contact: laurent.nussaume@cea.fr