be used for expression of the fluorescent biosensors. Toyota et al.
used a GCaMP3 sensor driven by the phloem-specific pSUC2 promoter to demonstrate that the wound-evoked Ca
2+ wave is transmitted via the phloem [47].
1.2.3 The Use of Affinity
Variants of the Sensors
Before preliminary experiments are done, the concentration of a
particular analyte of interest within a particular compartment of a
plant cell is not known. Since sensors have different detection
ranges, it is important to test sensors with different affinities for
each target analyte. Moreover, as outlined above, the intrinsic
sensitivity of the FPs can lead to artifacts. Affinity variants are
ideal controls. For example, if two variants that differ in K d by a
factor of 5 give the same in vivo response, the sensor likely reports a
change in another factor, e.g., pH. Note that if pH changes are
known to occur, it is valuable to predict the expected effect on the
FPs used in this sensor, including the direction of change as one
means of isolating the possible cause of the artifact. Note that also
the sensory domain and the linkers can affect the pH sensitivity of
the overall sensor [67].
1.2.4 Potential of
Biosensors to Act as
Buffers or as Components
of Signaling Networks
For quantitative imaging of fluorescent biosensors, it is necessary to
use transgenic plants that allow bright fluorescence. At the same
time, high levels of the sensor can have negative impacts, such as the
sensor itself interfering with cellular processes. If the transgene is
expressed at too high a level, the binding domain of the sensor can
create a large buffer for the metabolite or signal, resulting in a lower
ratio of response signal to the control signal. If the analyte is
scavenged by the sensor, a signal may not be detected. Thus, for
each case, it is necessary to optimize the sensor level and to test for
possible artifacts caused by sensor expression in the plant. These
disruptive activities of biosensors are a process of particular relevance when using endogenous peptides, such as ABA or GA receptors, for sensor construction. Expression of ABACUS1 caused ABA
hypersensitivity [29], while one line expressing ABAleon was resistant to ABA [28], demonstrating that ABA biosensors affect ABA
signaling. Waadt et al. described that expression of GECIs inhibited
plant growth, possibly through interference of endogenous calcium
signaling [59]. The ATP biosensor, ATeam1.03-nD/nA, also
caused plant growth inhibition when it was fused to a mitochondrial targeting sequence [27]. Taken together, biosensors can affect
the physiology of the cells, requiring careful observations and
control experiments. Since the perturbation by the biosensors
might be correlated to their expression levels, it is necessary to
carefully select biosensors which have less physiological perturbation and possibly use weaker promoters to prevent scavenging.
The Use of Genetically Encoded Fluorescent Biosensors
311
used a GCaMP3 sensor driven by the phloem-specific pSUC2 promoter to demonstrate that the wound-evoked Ca
2+ wave is transmitted via the phloem [47].
1.2.3 The Use of Affinity
Variants of the Sensors
Before preliminary experiments are done, the concentration of a
particular analyte of interest within a particular compartment of a
plant cell is not known. Since sensors have different detection
ranges, it is important to test sensors with different affinities for
each target analyte. Moreover, as outlined above, the intrinsic
sensitivity of the FPs can lead to artifacts. Affinity variants are
ideal controls. For example, if two variants that differ in K d by a
factor of 5 give the same in vivo response, the sensor likely reports a
change in another factor, e.g., pH. Note that if pH changes are
known to occur, it is valuable to predict the expected effect on the
FPs used in this sensor, including the direction of change as one
means of isolating the possible cause of the artifact. Note that also
the sensory domain and the linkers can affect the pH sensitivity of
the overall sensor [67].
1.2.4 Potential of
Biosensors to Act as
Buffers or as Components
of Signaling Networks
For quantitative imaging of fluorescent biosensors, it is necessary to
use transgenic plants that allow bright fluorescence. At the same
time, high levels of the sensor can have negative impacts, such as the
sensor itself interfering with cellular processes. If the transgene is
expressed at too high a level, the binding domain of the sensor can
create a large buffer for the metabolite or signal, resulting in a lower
ratio of response signal to the control signal. If the analyte is
scavenged by the sensor, a signal may not be detected. Thus, for
each case, it is necessary to optimize the sensor level and to test for
possible artifacts caused by sensor expression in the plant. These
disruptive activities of biosensors are a process of particular relevance when using endogenous peptides, such as ABA or GA receptors, for sensor construction. Expression of ABACUS1 caused ABA
hypersensitivity [29], while one line expressing ABAleon was resistant to ABA [28], demonstrating that ABA biosensors affect ABA
signaling. Waadt et al. described that expression of GECIs inhibited
plant growth, possibly through interference of endogenous calcium
signaling [59]. The ATP biosensor, ATeam1.03-nD/nA, also
caused plant growth inhibition when it was fused to a mitochondrial targeting sequence [27]. Taken together, biosensors can affect
the physiology of the cells, requiring careful observations and
control experiments. Since the perturbation by the biosensors
might be correlated to their expression levels, it is necessary to
carefully select biosensors which have less physiological perturbation and possibly use weaker promoters to prevent scavenging.
The Use of Genetically Encoded Fluorescent Biosensors
311