and a FRET pair of CFP and circularly permutated Venus (cpVenus). The FRET efficiency of cpFLIPPi negatively correlates within
the concentration of intracellular inorganic phosphate
[16]. cpFLIPPi-6.4 m, one of the variants of cpFLIPPi with a K d
for phosphate, of 6.4 mM, was used to measure phosphate levels in
the cytosol and in plastids of Arabidopsis plants [16]. eCALWY,
which utilizes two cysteine-containing metal-binding domains
(ATOX1 and WD4) as the sensor and a FRET pair of cerulean
and citrine [17], was used to analyze and visualize zinc concentrations in Arabidopsis roots and organelles [18].
Soluble sugar levels in plant cells have been measured with the
help of the glucose and sucrose biosensors FLIPglu and FLIPsuc,
respectively [19, 20]. FLIPglu consists of an E. coli periplasmic
glucose/galactose-binding protein and the FRET pair CFP and
YFP [19]. FLIPsuc consists of a sucrose-binding protein, the Agrobacterium tumefaciens ThuE, and the same FRET pair [19]. Use of
these fluorescent sugar biosensors provided insights into sugar
translocation and metabolism in living plant cells. These sensors
were also used to identify a new class of sugar transporters, the
SWEETs [21, 22]. Similarly, the bacterial periplasmic-binding protein was used to create series of FRET sensors for glutamate
[23, 24], which would be very useful in deciphering the plant
glutamate signaling pathway.
To engineer a biosensor for ATP, the ε-subunit of Bacillus
subtilis ATP synthase was chosen as the sensory domain in the
FRET-based biosensor ATeam1.03-nD/nA. When the ε-subunit
binds MgATP
2À , the conformational changes in the sensor enhance
the FRET between monomeric super-enhanced CFP (mseCFP)
and cpVenus (cp173-Venus) located at the sensor’s N- and
C-terminus, respectively [25]. ATeam1.03-nD/nA facilitated the
visualization of intercellular ATP levels during hypersensitive cell
death in Arabidopsis leaves and the measurement of MgATP
2À
concentrations both between plant tissues and within individual
cell such as root hairs [26, 27].
FRET-based phytohormone biosensors is one of the newest
areas of research. Jones et al. and Waadt et al. independently developed FRET-based abscisic acid (ABA) biosensors, ABACUS and
ABAleon, respectively, in which the ABA receptor PYL1 or PYR1
and the ABA co-receptor ABI1 act as the sensory domain
[28, 29]. ABACUS was used to reveal the effects of exogenous
ABA on ABA homeostasis in Arabidopsis, and ABAleon was used to
track ABA transport from hypocotyl to the shoot and root
[28, 29]. Rizza et al. developed the FRET-based gibberellin
(GA) biosensor, GPS1 [30], in which the GA receptor AtGID1C
and the coreceptor AtGAI were utilized in the sensory domain and
edCerulean and edAphrodite were used as the FRET reporter pair
[30]. GPS1 showed an increase in the yellow-to-cyan emission ratio
The Use of Genetically Encoded Fluorescent Biosensors
307
the concentration of intracellular inorganic phosphate
[16]. cpFLIPPi-6.4 m, one of the variants of cpFLIPPi with a K d
for phosphate, of 6.4 mM, was used to measure phosphate levels in
the cytosol and in plastids of Arabidopsis plants [16]. eCALWY,
which utilizes two cysteine-containing metal-binding domains
(ATOX1 and WD4) as the sensor and a FRET pair of cerulean
and citrine [17], was used to analyze and visualize zinc concentrations in Arabidopsis roots and organelles [18].
Soluble sugar levels in plant cells have been measured with the
help of the glucose and sucrose biosensors FLIPglu and FLIPsuc,
respectively [19, 20]. FLIPglu consists of an E. coli periplasmic
glucose/galactose-binding protein and the FRET pair CFP and
YFP [19]. FLIPsuc consists of a sucrose-binding protein, the Agrobacterium tumefaciens ThuE, and the same FRET pair [19]. Use of
these fluorescent sugar biosensors provided insights into sugar
translocation and metabolism in living plant cells. These sensors
were also used to identify a new class of sugar transporters, the
SWEETs [21, 22]. Similarly, the bacterial periplasmic-binding protein was used to create series of FRET sensors for glutamate
[23, 24], which would be very useful in deciphering the plant
glutamate signaling pathway.
To engineer a biosensor for ATP, the ε-subunit of Bacillus
subtilis ATP synthase was chosen as the sensory domain in the
FRET-based biosensor ATeam1.03-nD/nA. When the ε-subunit
binds MgATP
2À , the conformational changes in the sensor enhance
the FRET between monomeric super-enhanced CFP (mseCFP)
and cpVenus (cp173-Venus) located at the sensor’s N- and
C-terminus, respectively [25]. ATeam1.03-nD/nA facilitated the
visualization of intercellular ATP levels during hypersensitive cell
death in Arabidopsis leaves and the measurement of MgATP
2À
concentrations both between plant tissues and within individual
cell such as root hairs [26, 27].
FRET-based phytohormone biosensors is one of the newest
areas of research. Jones et al. and Waadt et al. independently developed FRET-based abscisic acid (ABA) biosensors, ABACUS and
ABAleon, respectively, in which the ABA receptor PYL1 or PYR1
and the ABA co-receptor ABI1 act as the sensory domain
[28, 29]. ABACUS was used to reveal the effects of exogenous
ABA on ABA homeostasis in Arabidopsis, and ABAleon was used to
track ABA transport from hypocotyl to the shoot and root
[28, 29]. Rizza et al. developed the FRET-based gibberellin
(GA) biosensor, GPS1 [30], in which the GA receptor AtGID1C
and the coreceptor AtGAI were utilized in the sensory domain and
edCerulean and edAphrodite were used as the FRET reporter pair
[30]. GPS1 showed an increase in the yellow-to-cyan emission ratio
The Use of Genetically Encoded Fluorescent Biosensors
307