in response to addition of GA 1 , GA 3 , or GA 4, with different affinities for the three analogs. A nuclear-targeted derivative, nlsGPS1,
was used to investigate GA distribution in the nuclei of elongating
root and shoot tissues [30].
1.1.4 Intrinsic Biosensors
Fluorescent proteins are generally sensitive to acidic pH and to
halides [31, 32]. Different FPs have different pK a values and therefore different ranges of pH sensitivity. The properties of FPs have
been utilized for engineering intrinsic, single-FP biosensors for pH,
reactive oxygen species (ROS), and the redox state. The pHluorin
pH biosensors are derivatives of GFP with amino acid substitutions
that increase its pH sensitivity. Both ratiometric and elliptic
pHluorin biosensors have been used in plant research [33–
35]. The pH-sensitive GFP(H148D) was used to visualize local
pH changes in response to mechanical stimulation at the cell surface
[36]. Gjetting et al. took advantage of differences in pH sensitivity
of two FPs, Enhanced GFP (EGFP) and monomeric Red Fluorescent Protein 1 (mRFP1), and developed a ratiometric pH biosensor, pHusion, consisting of EGFP and mRFP1 fused via a short
amino acid linker [37]. The fluorescence intensity of the EGFP in
pHusion reversibly decreases by acidification, while that of the
mRFP1 is relatively stable through different pH values. By fusing
different localization signal peptides or organelle-specific proteins
to ratiometric pHluorin and pHusion sensors, organellar pH has
been measured in Arabidopsis cells [34, 35, 37].
Redox-sensitive FP-based biosensors (roGFPs) are GFP derivatives in which some surface-exposed amino acid residues are
substituted with cysteine residues, potentially forming disulfide
bonds. roGFPs have two excitation maxima that correspond to
the cellular redox potential, which function as ratiometric sensors
[38]. roGFP2 has been used for investigating intracellular redox
states in plants [39–41]. Schwarzl€ ander et al. used roGFP2 targeted
to the mitochondria to reveal that abiotic stresses, including temperature, salt, and heavy metal stresses, affect the mitochondrial
redox state [40].
1.1.5 Extrinsic
Intensiometric Biosensors
Intensiometric biosensors have one excitation and one emission
maximum. Most extrinsic intensiometric biosensors consist of one
cpFP and a sensory domain for an analyte. Generally, the sensory
domain is based on a whole or partial region of a protein that binds
a ligand. Binding of ligands to the sensory domain triggers conformational changes, leading to an increase or decrease of fluorescence
from the cpFP [42].
GCaMP is a widely used single FP-based GECI, consisting of
the CaM–M13 sensory domain inserted into cpGFP [43] and has
been employed in many plant studies [44–48]. To increase the
range of fluorophores available for GECI, Zhao et al. engineered
308
Akira Yoshinari et al.
was used to investigate GA distribution in the nuclei of elongating
root and shoot tissues [30].
1.1.4 Intrinsic Biosensors
Fluorescent proteins are generally sensitive to acidic pH and to
halides [31, 32]. Different FPs have different pK a values and therefore different ranges of pH sensitivity. The properties of FPs have
been utilized for engineering intrinsic, single-FP biosensors for pH,
reactive oxygen species (ROS), and the redox state. The pHluorin
pH biosensors are derivatives of GFP with amino acid substitutions
that increase its pH sensitivity. Both ratiometric and elliptic
pHluorin biosensors have been used in plant research [33–
35]. The pH-sensitive GFP(H148D) was used to visualize local
pH changes in response to mechanical stimulation at the cell surface
[36]. Gjetting et al. took advantage of differences in pH sensitivity
of two FPs, Enhanced GFP (EGFP) and monomeric Red Fluorescent Protein 1 (mRFP1), and developed a ratiometric pH biosensor, pHusion, consisting of EGFP and mRFP1 fused via a short
amino acid linker [37]. The fluorescence intensity of the EGFP in
pHusion reversibly decreases by acidification, while that of the
mRFP1 is relatively stable through different pH values. By fusing
different localization signal peptides or organelle-specific proteins
to ratiometric pHluorin and pHusion sensors, organellar pH has
been measured in Arabidopsis cells [34, 35, 37].
Redox-sensitive FP-based biosensors (roGFPs) are GFP derivatives in which some surface-exposed amino acid residues are
substituted with cysteine residues, potentially forming disulfide
bonds. roGFPs have two excitation maxima that correspond to
the cellular redox potential, which function as ratiometric sensors
[38]. roGFP2 has been used for investigating intracellular redox
states in plants [39–41]. Schwarzl€ ander et al. used roGFP2 targeted
to the mitochondria to reveal that abiotic stresses, including temperature, salt, and heavy metal stresses, affect the mitochondrial
redox state [40].
1.1.5 Extrinsic
Intensiometric Biosensors
Intensiometric biosensors have one excitation and one emission
maximum. Most extrinsic intensiometric biosensors consist of one
cpFP and a sensory domain for an analyte. Generally, the sensory
domain is based on a whole or partial region of a protein that binds
a ligand. Binding of ligands to the sensory domain triggers conformational changes, leading to an increase or decrease of fluorescence
from the cpFP [42].
GCaMP is a widely used single FP-based GECI, consisting of
the CaM–M13 sensory domain inserted into cpGFP [43] and has
been employed in many plant studies [44–48]. To increase the
range of fluorophores available for GECI, Zhao et al. engineered
308
Akira Yoshinari et al.