biosensor consisting of the DII degron domain of the Aux/IAA
protein fused with Venus, a rapid-maturating FP [5]. DII-VENUS
is rapidly degraded by the proteasome after auxin-initiated ubiquitination through TIR1/AFB co-receptors. Thus, DII-VENUS
fluorescence was negatively correlated with intracellular auxin
levels. DII-VENUS has been used to monitor rapid changes in
auxin distribution during root gravitropism [5]. A similar strategy
has been applied to engineer biosensors for gibberellin (GFP-RGA)
[6, 7], jasmonic acid (Jas9-Venus) [8], and strigolactone
(StrigoQuant) [9].
A Venus-based biosensor has been more recently used to study
the boric acid transport in Arabidopsis thaliana. The mRNA level
of NIP5;1 is downregulated by intracellular boric acid through
NIP5;1 mRNA decay [10]. The 5
0 -UTR of NIP5;1 is required for
the boron-induced ribosomal stalling and subsequent mRNA decay
[10, 11]. Fukuda et al. engineered a fluorescent biosensor to monitor boric acid levels, uNIP5;1-Venus, in which the 5
0 -UTR of
NIP5;1 was fused to the gene for Venus. The Venus signal would
decrease as boric acid levels increased due to increased RNA decay.
uNIP5;1-Venus responded over a range of 30–500 μM boric acid
and enabled quantification of boric acid distribution in the root at
the single-cell level [12]. This still represents an additional step for
RNA to protein, but any delay in sensing is less than a sensor that
requires a transcriptional response.
1.1.3 FRET-Based
Biosensors
Biosensors based on Fo ¨rster resonance energy transfer (FRET)
generally have a sensory domain for an analyte sandwiched between
two different FPs (a donor and an acceptor) at each protein terminus. Distance and/or orientation between the FPs is altered by
conformational changes of the sensory domain, resulting in a
change in the relative fluorescence through alteration of the
FRET efficiency. FRET-based biosensors have been used to visualize the concentrations of inorganic ions involved in both plant
nutrition and signaling.
Calcium ion (Ca
2+ ) is an important cellular signaling molecule.
For over two decades, genetically encoded Ca
2+ indicators (GECIs)
have been used to measure intracellular Ca
2+ concentration in vivo.
The first GECI, Yellow Cameleon (YC), was engineered as a fourprotein fusion of calmodulin and M13, a calmodulin-binding
domain of the myosin light chain kinase, to form a
two-component sensory domain for Ca
2+
, and cyan fluorescent
protein (CFP) and yellow FP (YFP) as a FRET pair at the N- and
C-termini, respectively [13]. YC2.1, one of the YC variants, was
used for calcium imaging in guard cells of Arabidopsis thaliana
[14, 15].
cpFLIPPi is an inorganic phosphate biosensor consisting of the
phosphate-binding protein (PiBP) cloned from Synechococcus spp.
306
Akira Yoshinari et al.
protein fused with Venus, a rapid-maturating FP [5]. DII-VENUS
is rapidly degraded by the proteasome after auxin-initiated ubiquitination through TIR1/AFB co-receptors. Thus, DII-VENUS
fluorescence was negatively correlated with intracellular auxin
levels. DII-VENUS has been used to monitor rapid changes in
auxin distribution during root gravitropism [5]. A similar strategy
has been applied to engineer biosensors for gibberellin (GFP-RGA)
[6, 7], jasmonic acid (Jas9-Venus) [8], and strigolactone
(StrigoQuant) [9].
A Venus-based biosensor has been more recently used to study
the boric acid transport in Arabidopsis thaliana. The mRNA level
of NIP5;1 is downregulated by intracellular boric acid through
NIP5;1 mRNA decay [10]. The 5
0 -UTR of NIP5;1 is required for
the boron-induced ribosomal stalling and subsequent mRNA decay
[10, 11]. Fukuda et al. engineered a fluorescent biosensor to monitor boric acid levels, uNIP5;1-Venus, in which the 5
0 -UTR of
NIP5;1 was fused to the gene for Venus. The Venus signal would
decrease as boric acid levels increased due to increased RNA decay.
uNIP5;1-Venus responded over a range of 30–500 μM boric acid
and enabled quantification of boric acid distribution in the root at
the single-cell level [12]. This still represents an additional step for
RNA to protein, but any delay in sensing is less than a sensor that
requires a transcriptional response.
1.1.3 FRET-Based
Biosensors
Biosensors based on Fo ¨rster resonance energy transfer (FRET)
generally have a sensory domain for an analyte sandwiched between
two different FPs (a donor and an acceptor) at each protein terminus. Distance and/or orientation between the FPs is altered by
conformational changes of the sensory domain, resulting in a
change in the relative fluorescence through alteration of the
FRET efficiency. FRET-based biosensors have been used to visualize the concentrations of inorganic ions involved in both plant
nutrition and signaling.
Calcium ion (Ca
2+ ) is an important cellular signaling molecule.
For over two decades, genetically encoded Ca
2+ indicators (GECIs)
have been used to measure intracellular Ca
2+ concentration in vivo.
The first GECI, Yellow Cameleon (YC), was engineered as a fourprotein fusion of calmodulin and M13, a calmodulin-binding
domain of the myosin light chain kinase, to form a
two-component sensory domain for Ca
2+
, and cyan fluorescent
protein (CFP) and yellow FP (YFP) as a FRET pair at the N- and
C-termini, respectively [13]. YC2.1, one of the YC variants, was
used for calcium imaging in guard cells of Arabidopsis thaliana
[14, 15].
cpFLIPPi is an inorganic phosphate biosensor consisting of the
phosphate-binding protein (PiBP) cloned from Synechococcus spp.
306
Akira Yoshinari et al.