G-GECO (green fluorescence), R-GECO (red fluorescence), and
B-GECO (blue fluorescence), based on GCaMP3 [49]. Keinath
et al. used R-GECO1 to demonstrate the involvement of calcium
signaling in biotic responses in Arabidopsis thaliana [50].
HyPer and HyPer2 are fluorescent biosensors consisting of
cp-Yellow Fluorescent Protein (cpYFP) and the prokaryotic
H 2 O 2 -sensing protein OxyR and are used for detecting the ROS
hydrogen peroxide (H 2 O 2 ) [51, 52]. The HyPer biosensors
enabled observation of dynamic changes in H 2 O 2 levels in plants
responding to environmental stimuli such as exposure to high-light
[52–55].
As a modification of the FRET glutamate sensor described
above, iGluSnFR was developed as a single FP-based biosensor
[56] and used in plant studies [47]. iGluSnFR is built from
cpGFP fused with the E. coli glutamate/aspartate periplasmicbinding protein, GltI [56]. In Arabidopsis thaliana, glutamate is
involved in long-distance signal transduction through activation of
cation channels belonging to Glutamate Receptor Likes (GLRs)
[47, 48, 57]. Toyota et al. revealed that wound stimulation triggers
rapid increase of glutamate concentration in the apoplasm by using
cell wall-targeted iGluSnFR [47].
1.1.6 Improvement of
Intensiometric Biosensors
Many intensiometric biosensors described above are characterized
by having a higher dynamic range compared to FRET-based biosensors [58]. However, these single FP sensors are not ratiometric
and are thus subject to artifacts. For instance, changes in sensor
level will lead to a change in the output. Therefore, single FP-based
biosensors must be improved by introducing an internal reference
FP. R-GECO1-mTurquoise carries a blue fluorescent reference FP,
mTurquoise [59]. In parallel, Ast et al. engineered a ratiometric
GECI named MatryoshCaMP6s based on GCaMP6s, containing a
nested LSSmOrange in cpGFP (Fig. 2) [60]. These novel ratiometric GECIs are able to accurately map dynamic changes in Ca
2+
concentration in Arabidopsis roots.
1.1.7 Activity Sensors
While it is important to gain insights into the distribution and
dynamics of small molecules, it is also important to be able to
monitor the activity of enzymes and transporters in vivo. FRETbased, single FP, and Matryoshka strategies have successfully been
used to engineer activity sensors for nitrate, oligopeptides, and
ammonium transporters [60, 61, 63]. These sensors can be used
to study metabolite sensing and signaling pathways in plants.
The Use of Genetically Encoded Fluorescent Biosensors
309
B-GECO (blue fluorescence), based on GCaMP3 [49]. Keinath
et al. used R-GECO1 to demonstrate the involvement of calcium
signaling in biotic responses in Arabidopsis thaliana [50].
HyPer and HyPer2 are fluorescent biosensors consisting of
cp-Yellow Fluorescent Protein (cpYFP) and the prokaryotic
H 2 O 2 -sensing protein OxyR and are used for detecting the ROS
hydrogen peroxide (H 2 O 2 ) [51, 52]. The HyPer biosensors
enabled observation of dynamic changes in H 2 O 2 levels in plants
responding to environmental stimuli such as exposure to high-light
[52–55].
As a modification of the FRET glutamate sensor described
above, iGluSnFR was developed as a single FP-based biosensor
[56] and used in plant studies [47]. iGluSnFR is built from
cpGFP fused with the E. coli glutamate/aspartate periplasmicbinding protein, GltI [56]. In Arabidopsis thaliana, glutamate is
involved in long-distance signal transduction through activation of
cation channels belonging to Glutamate Receptor Likes (GLRs)
[47, 48, 57]. Toyota et al. revealed that wound stimulation triggers
rapid increase of glutamate concentration in the apoplasm by using
cell wall-targeted iGluSnFR [47].
1.1.6 Improvement of
Intensiometric Biosensors
Many intensiometric biosensors described above are characterized
by having a higher dynamic range compared to FRET-based biosensors [58]. However, these single FP sensors are not ratiometric
and are thus subject to artifacts. For instance, changes in sensor
level will lead to a change in the output. Therefore, single FP-based
biosensors must be improved by introducing an internal reference
FP. R-GECO1-mTurquoise carries a blue fluorescent reference FP,
mTurquoise [59]. In parallel, Ast et al. engineered a ratiometric
GECI named MatryoshCaMP6s based on GCaMP6s, containing a
nested LSSmOrange in cpGFP (Fig. 2) [60]. These novel ratiometric GECIs are able to accurately map dynamic changes in Ca
2+
concentration in Arabidopsis roots.
1.1.7 Activity Sensors
While it is important to gain insights into the distribution and
dynamics of small molecules, it is also important to be able to
monitor the activity of enzymes and transporters in vivo. FRETbased, single FP, and Matryoshka strategies have successfully been
used to engineer activity sensors for nitrate, oligopeptides, and
ammonium transporters [60, 61, 63]. These sensors can be used
to study metabolite sensing and signaling pathways in plants.
The Use of Genetically Encoded Fluorescent Biosensors
309