1 Running head: Live Imaging of Inorganic Phosphate 2 3 · 1 Live Imaging of Inorganic Phosphate in...
Transcript of 1 Running head: Live Imaging of Inorganic Phosphate 2 3 · 1 Live Imaging of Inorganic Phosphate in...
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Running head: Live Imaging of Inorganic Phosphate 1
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Corresponding author: 3
Wayne K. Versaw 4
Department of Biology 5
Texas A&M University 6
College Station, Texas, 77843 USA 7
Tel. 979-847-8587 8
Fax: 979-845-2891 9
E-mail [email protected] 10
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Research area: Cell Biology 12
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Plant Physiology Preview. Published on January 26, 2015, as DOI:10.1104/pp.114.254003
Copyright 2015 by the American Society of Plant Biologists
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Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular 1
Resolution 2
Pallavi Mukherjee, Swayoma Banerjee, Amanda Wheeler, Lyndsay A. Ratliff, Sonia 3
Irigoyen, L. Rene Garcia, Steve W. Lockless, Wayne K. Versaw* 4
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Department of Biology, Texas A&M University, College Station, Texas, USA 6
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One-sentence summary: 9
Genetically encoded sensors enable dynamic monitoring of phosphate concentrations in 10
different cells and cell compartments of live plants. 11
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FOOTNOTES 1 1This work was supported by funds from the National Science Foundation (IOS-2
0956486) to W.K.V., the Welch Foundation (A-1742) to S.W.L., and the Howard 3
Hughes Medical Institute to L.R.G. 4
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*Corresponding author: [email protected] 6
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ABSTRACT 1
Despite variable and often scarce supplies of inorganic phosphate (Pi) from 2
soils, plants must distribute appropriate amounts of Pi to each cell and subcellular 3
compartment to sustain essential metabolic activities. The ability to monitor Pi dynamics 4
with subcellular resolution in live plants is therefore critical for understanding how this 5
essential nutrient is acquired, mobilized, recycled and stored. Fluorescence indicator for 6
Pi (FLIPPi) sensors are genetically encoded fluorescence resonance energy transfer 7
(FRET)-based sensors that have been used to monitor Pi dynamics in cultured animal 8
cells. Here we present a series of Pi sensors optimized for use in plants. Substitution of 9
the eYFP component of a FLIPPi sensor with a circularly permuted version of Venus, 10
cpVenus, enhanced sensor dynamic range nearly 2.5-fold. The resulting cpFLIPPi sensor 11
was subjected to a high-efficiency mutagenesis strategy that relied on statistical coupling 12
analysis to identify regions of the protein likely to influence Pi affinity. A series of 13
affinity mutants was selected with Kd values of 0.08-11 mM, which spans the range for 14
most plant cell compartments. The sensors were expressed in Arabidopsis thaliana, and 15
ratiometric imaging was used to monitor cytosolic Pi dynamics in root cells in response 16
to Pi deprivation and resupply. Moreover, plastid-targeted versions of the sensors 17
expressed in wild type and a mutant lacking the PHT4;2 plastidic Pi transporter 18
confirmed a physiological role for this transporter in Pi export from root plastids. These 19
cpFLIPPi sensors therefore enable detailed analysis of Pi dynamics with subcellular 20
resolution in live plants. 21
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INTRODUCTION 1
Phosphorus is an essential element that plants acquire and assimilate in the form 2
of inorganic phosphate (Pi). This macronutrient is a component of numerous metabolites 3
and macromolecules, including ATP, nucleic acids and phospholipids, and serves key 4
roles in energy transfer reactions, signal transduction processes and the regulation of 5
enzyme activities. Of fundamental importance to plants, Pi also serves critical roles in 6
photosynthesis, both as a substrate for ATP synthesis via photophosphorylation and as a 7
regulator in the partitioning of fixed carbon between the starch and sucrose biosynthetic 8
pathways. 9
In many soils, particularly those used for low-input agriculture, the amounts of Pi 10
available to plants are limiting for growth and productivity (Vance et al., 2003). Most of 11
the Pi in soils is unavailable because it is immobilized through formation of insoluble 12
complexes or exists in organic forms such as phytate that plants cannot use directly 13
(Schachtman et al., 1998). As a result, concentrations of free Pi in soil solution range 14
from 1 to 10 μM, whereas cells require Pi in the millimolar range (Bieleski, 1973). 15
To acclimate to Pi limitation, plants have evolved mechanisms to enhance Pi 16
acquisition and also to mobilize, recycle and conserve internal stores. These mechanisms 17
include secretion of organic acids and phosphatases (Vance et al., 2003), increased 18
growth of lateral roots and root hairs (Bates and Lynch, 2000; Peret et al., 2011), 19
production of high-affinity Pi transporters at the root-soil interface (Misson et al., 2004; 20
Shin et al., 2004), formation of symbiotic association with mycorrhizal fungi, which 21
enhance Pi scavenging capabilities (Javot et al., 2007), modification of metabolic 22
pathways (Plaxton and Tran, 2011), and altered patterns of Pi translocation between 23
organs and transport between subcellular compartments (Walker and Sivak, 1986; 24
Mimura, 1999; Raghothama, 1999). Substantial insights have been gained into the 25
underlying biochemical identities and regulatory strategies for such adaptive responses, 26
including those related to sensing and signaling of Pi status (Rouached et al., 2010; 27
Chiou and Lin, 2011; Plaxton and Tran, 2011; Jain et al., 2012; Liu et al., 2014; Zhang et 28
al., 2014). However, a thorough understanding of their respective mechanisms and how 29
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these are integrated is limited by the inability to assess intracellular Pi concentrations 1
with high spatial and temporal resolution. 2
Genetically encoded fluorescent sensors, or biosensors, have proven to be 3
powerful tools for monitoring metabolites and ions in vivo because their expression and 4
subcellular targeting can be manipulated and fluorescence imaging is nondestructive 5
(Lalonde et al., 2005; Okumoto et al., 2012). Sensor proteins are fusions of a ligand-6
binding domain or protein with one or two fluorescent proteins, e.g., GFP and related 7
variants. Sensors with a single fluorescent protein report ligand-dependent changes in 8
conformation as changes in fluorescence intensity, whereas sensors with two fluorescent 9
proteins can yield changes in Förster (or fluorescence) resonance energy transfer 10
(FRET), which can be quantified through ratiometric imaging. FRET-based sensors have 11
been used in live plants to assess a variety of analytes including glucose, maltose, 12
sucrose, glutamine, calcium, zinc and pH (Deuschle et al., 2006; Chaudhuri et al., 2008; 13
Kaper et al., 2008; Rincon-Zachary et al., 2010; Chaudhuri et al., 2011; Adams et al., 14
2012; Gjetting et al., 2012; Krebs et al., 2012; Gjetting et al., 2013). 15
Gu et al. (Gu et al., 2006) engineered FRET-based Pi sensors named 16
fluorescence indicator protein for Pi (FLIPPi) that consist of a cyanobacterial Pi binding 17
protein fused to eCFP and eYFP, and demonstrated the utility of one of these sensors for 18
monitoring cytosolic Pi in cultured animal cells. In this study, we generated a series of 19
second-generation FLIPPi sensors that were modified and optimized for use in live 20
plants. Substitution of eYFP with a circularly permuted form of the fluorescent protein 21
Venus (cpVenus) (Nagai et al., 2002; Nagai et al., 2004) greatly increased the magnitude 22
of Pi-dependent FRET responses. In keeping with the initial nomenclature, Pi sensors 23
constructed with cpVenus were designated cpFLIPPi. We also used a targeted 24
mutagenesis approach to obtain cpFLIPPi sensors with Pi binding affinities that spanned 25
the physiological range of most cell compartments and expressed these in Arabidopsis. 26
Confocal microscopy coupled with ratiometric analysis or acceptor photobleaching 27
detected changes in cytosolic Pi levels in root epidermal cells in response to Pi 28
starvation, and these changes were fully reversed by Pi replenishment. Plastid-localized 29
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versions of the same sensors expressed in wild-type plants and in mutants lacking the 1
PHT4;2 plastidic Pi transporter (Irigoyen et al., 2011) were used to confirm a role for 2
this transporter in the export of Pi from root plastids. These results demonstrate the 3
utility of cpFLIPPi sensors for monitoring Pi distributions with both cellular and 4
subcellular resolution in live plants. 5
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RESULTS 7
Efficacy of FLIPPi Sensors in Plants 8
In an initial attempt to monitor Pi distributions in live Arabidopsis plants we 9
generated stable transgenic lines for two of the previously described Pi sensors, FLIPPi-10
200μ and FLIPPi-30m (Gu et al., 2006). When assayed in vitro, these sensors yielded 11
robust Pi-dependent FRET responses with Kd values for Pi binding of 200 μM and 30 12
mM, respectively. The FLIPPi-30m sensor was also used to report changes in cytosolic 13
Pi concentrations in cultured animal cells (Gu et al., 2006). It should be noted that these 14
sensors exhibit a Pi-dependent decrease in FRET (Fig. 1A). 15
Constitutive expression of the sensor genes was directed from the 35S promoter, 16
and because no organellar targeting sequences were incorporated in the constructs the 17
proteins were restricted to the cytosol. Independent transgenic lines for both sensors 18
exhibited weak or highly variable fluorescence that diminished rapidly with plant age, 19
suggestive of transgene silencing (Deuschle et al., 2006). Although signal intensity and 20
stability improved when the sensors were expressed in an siRNA-defective mutant host, 21
suppressor of gene silencing 3-13 (sgs3-13) (Kumakura et al., 2009), no changes in 22
FRET were detected when plants were starved for Pi. 23
We hypothesized that one or more attributes of the FLIPPi sensors limited their 24
efficacy when used in plants. For example, if the magnitudes of Pi-dependent responses 25
were small, then subtle changes in cytosolic Pi concentrations would not be detected. 26
Similarly, no change in FRET would be expected if binding affinities for Pi were too far 27
above or below the cytosolic concentrations. To explore these possibilities we first 28
confirmed that purified FLIPPi-200μ and FLIPPi-30m sensor proteins yielded Pi-29
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dependent FRET responses equivalent to those reported previously (Gu et al., 2006). We 1
then examined FRET responses for the purified proteins in a pseudocytosol medium 2
(Messerli and Robinson, 1998; Feijo et al., 1999; Cardenas et al., 2008) to simulate in 3
vivo conditions more closely than the buffer solution used previously (Gu et al., 2006). 4
In this case, the FLIPPi-30m sensor yielded erratic FRET signals regardless of Pi 5
concentration, which may explain the absence of Pi-dependent FRET responses in the 6
corresponding transgenic plants. In contrast, FLIPPi-200μ exhibited the expected Pi-7
dependent reduction in FRET, and the 190 μM Kd was nearly identical to that measured 8
in buffer (Fig. 1B). However, the dynamic range, i.e., the maximum change in FRET 9
ratio (∆Rmax), was relatively small (Fig. 1B), and the change was very small over the 2.5 10
to 10 mM concentration range that encompasses most estimates for cytosolic Pi in plants 11
grown in Pi-replete conditions. (Rebeille et al., 1984; Stitt et al., 1988; Lee and Ratcliffe, 12
1993; Copeland and Zammit, 1994; Gout et al., 2011). Based on these results, we chose 13
to optimize FLIPPi-200μ. 14
15
Expanded Dynamic Range of a Pi sensor with a Circularly Permuted Venus 16
Substitution of the YFP acceptor component of a FRET-based sensor for Ca2+ 17
with a circularly permuted version of Venus (cpVenus) improved dynamic range and 18
stability and reduced sensitivity to acidic pH (Nagai et al., 2004). This modified sensor 19
has been effective for monitoring Ca2+ dynamics in plants (Rincon-Zachary et al., 2010; 20
Krebs et al., 2012). To test whether this modification would also increase the dynamic 21
range of a Pi sensor we replaced the eYFP portion of FLIPPi-200μ with cp173Venus, 22
the same cpVenus variant used to improve the YC3.60 Ca2+ sensor (Nagai et al., 2004). 23
As shown in Fig. 1, the modified Pi sensor, cpFLIPPi-200μ, exhibited greater FRET 24
ratios in the absence of Pi and at all concentrations tested. More importantly, the 25
dynamic range of the modified sensor (∆Rmax -1.08) was nearly 2.5-fold greater than that 26
of the original sensor (∆Rmax -0.44). The emission spectra for cpFLIPPi-200μ (with 27
eCFP excitation) indicated that the Pi-dependent response of this sensor was due to a 28
change in FRET (increase in eCFP emission coupled to a decrease in cpVenus emission) 29
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rather than unrelated changes in the fluorescence intensities of either eCFP or cpVenus 1
alone (Suppl. Fig. 1). 2
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Mutant cpFLIPPi Sensor Variants with Altered Affinities for Pi 4
Although the use of cpVenus increased sensor dynamic range it had no 5
significant effect on affinity for Pi. Consequently, the magnitude of the response change 6
over the target concentration range of 2.5 to 10 mM remained small for cpFLIPPi-200μ 7
(Fig. 1). This situation necessitated a mutant screen to obtain cpFLIPPi variants with 8
altered binding affinities. 9
To limit the scope of mutagenesis we used statistical coupling analysis (SCA), a 10
sequence-based method for identifying protein residues or regions responsible for ligand 11
binding affinity and potential allosteric sites (Lockless and Ranganathan, 1999; Socolich 12
et al., 2005). SCA was conducted with an alignment of the Synechococcus Pi binding 13
protein (PiBP) sequence with 1,020 of the most closely related protein sequences (PSI-14
BLAST e-score <1e-10) available from GenBank. We found that the region spanning 15
amino acids 18 to 23 contained multiple coupled sites that map near the predicted Pi 16
binding site (Suppl. Figs. 2 and 3). We predicted that changes in this region would affect 17
the Pi binding affinity of the sensor protein. 18
We used PCR to introduce random mutations in the corresponding region of the 19
cpFLIPPi-200μ plasmid clone. The mutant library was transformed into E. coli, and 20
lysates of individual clones were screened for Pi-dependent FRET. The mutagenesis 21
strategy proved to be remarkably effective. Of the nearly 600 mutants screened, less than 22
1% resembled the parent sensor and 80% of the mutants exhibited Pi-dependent FRET 23
with altered affinity. The remaining 20% of the mutant population were either 24
unresponsive to Pi or had ratiometric signals that could not be accurately fit to a binding 25
isotherm. Eight candidate sensor mutants were selected, and their Pi-dependent FRET 26
responses were verified using the purified proteins (Table I). The Kd values ranged from 27
0.08 to 11 mM, which spans the predicted physiological range for the cytosol and other 28
cell compartments (Rebeille et al., 1984; Stitt et al., 1988; Sharkey and Vanderveer, 29
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1989; Lee and Ratcliffe, 1993; Copeland and Zammit, 1994; Gout et al., 2011; Tiessen 1
et al., 2012). 2
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Specificity and pH Sensitivity of cpFLIPPi Sensors 4
To determine whether cpFLIPPi mutations that altered affinity for Pi also 5
affected specificity we examined the effect of other oxyanions, including 6
organophosphates, on FRET responses. None of the sensors showed responses indicative 7
of binding to sulfate or nitrate, and results for one sensor, cpFLIPPi-6.4m, are shown in 8
Supp. Fig. S4A. Similarly, treatment with glycerol-2-phosphate and pyrophosphate, a 9
phosphate ester and anhydride, respectively, yielded little or no FRET responses (Supp. 10
Fig. S4B). These results suggest that the cpFLIPPi sensors have retained high specificity 11
for Pi. 12
Our in vitro calibration assays and related tests for specificity were conducted at 13
pH 7.5 to simulate the cytosol (Pratt et al., 2009; Gout et al., 2011). Although the cytosol 14
is tightly buffered at this pH, the chloroplast stroma can vary from pH 7 to 8 in response 15
to light (Demmig and Gimmler, 1983). Because one of our goals was to monitor 16
plastidic Pi concentrations, we examined Pi-dependent FRET responses over this pH 17
range for each of the cpFLIPPi sensors. We observed a consistent trend in which affinity 18
decreased with increasing pH and results for one representative sensor, cpFLIPPi-6.4m, 19
are shown in Fig. 2. The apparent differences in affinity over this pH range can be 20
largely attributed to the effect of pH on ionization of Pi with specificity for the 21
monobasic form. Unfortunately, these sensors had very low dynamic ranges when 22
assayed at pH 5.5, which would limit their utility in acidic compartments like the 23
vacuole and apoplast. 24
25
Subcellular Targeting of cpFLIPPi Sensors in Arabidopsis 26
To test the fidelity of targeting cpFLIPPi sensors to different compartments of 27
plant cells we constructed expression clones for each of the eight different cpFLIPPi 28
sensors to yield proteins with and without an N-terminal RbcS chloroplast transit peptide 29
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(Lee et al., 2006). To avoid potential complications associated with the 35S promoter, 1
the Arabidopsis UBQ10 promoter was used to drive constitutive expression at moderate 2
levels (Norris et al., 1993; Geldner et al., 2009; Grefen et al., 2010). Constructs were 3
introduced into the siRNA mutant sgs3-13 (Kumakura et al., 2009) to minimize potential 4
loss of fluorescent signals due to post-transcriptional gene silencing (Deuschle et al., 5
2006). Protoplasts were prepared from leaves of 5-week-old plants and fluorescent 6
signals were viewed with confocal microscopy. Co-localization with chlorophyll 7
autofluorescence shown in Fig. 3 confirmed that addition of the RbcS chloroplast transit 8
peptide was sufficient to target sensors to plastids, whereas unmodified sensors were 9
excluded from plastids and appeared to be restricted to the cytosol. FRET emission was 10
detected with the same localization patterns in palisade mesophyll cells of intact leaves 11
(Supp. Fig. S5), suggesting that live Pi imaging could be conducted in aerial tissues. 12
13
Live Imaging of Cytosolic Pi in Root Epidermal Cells 14
Live cell imaging using a spinning disk confocal microscope equipped with a 15
beam splitter for simultaneous dual emission fluorescence was used to test the 16
functionality of cpFLIPPi sensors for reporting changes in cytosolic Pi concentrations. 17
Untransformed plants and transgenic plants expressing eCFP and cpVenus separately 18
were imaged each day to correct for fluorescence background, spectral bleed-through 19
and cross excitation. T2 generation transgenic seedlings were grown for 5 days in 20
hydroponic medium containing 0.5 mM Pi then placed in an imaging chamber 21
containing the same medium. 22
Images for multiple epidermal cells within the root differentiation zone were 23
captured from at least six independent seedlings of each genotype to establish the steady-24
state baseline for the FRET ratio under this Pi-sufficient growth condition. The same 25
seedlings were then transferred to medium that lacked Pi and imaged again after 24 26
hours of Pi starvation. This duration of Pi starvation was chosen to avoid morphological 27
changes or cell death associated with more severe Pi deprivation (Sanchez-Calderon et 28
al., 2005). Specifically, cell viability tests with SYTOX orange (Truenit and Haseloff, 29
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2008) consistently revealed a substantial number of non-viable cells when plants were Pi 1
starved for 72 hours (Supp. Fig. S6), but none or very few were detected when plants 2
were held in Pi-replete conditions or starved for 24 hours. Sensitivity to prolonged Pi 3
deprivation is undoubtedly a function of growth conditions and the age of the plant when 4
deprivation is initiated. 5
Separate experiments demonstrated that our Pi starvation regime was sufficient 6
to reduce the spatially-undefined Pi contents of root extracts by 19% (from 5.3 ± 0.2 7
nmol Pi/ “fed” seedling to 4.3 ± 0.2 nmol Pi/ starved seedling, mean ± SE, n=3), and was 8
also sufficient to elicit a 20% increase in secreted acid phosphatase activity (from 60 ± 4 9
mUnits/ “fed” seedling to 72 ± 5 mUnits/ starved seedling, mean ± SE, n=12), which is 10
indicative of Pi starvation (Robinson et al., 2012). Both of these changes were 11
statistically significant as determined by Student’s t-test, p<0.05). Moreover, secreted 12
acid phosphatase activities were identical in wild type and sgs3-13 plants, indicating that 13
this mutation has no obvious effect on normal responses to Pi starvation. Consequently, 14
sgs3-13 was used as the “wild-type” background for all subsequent experiments. 15
We screened transgenic lines for cpFLIPPi-80μ, -200μ, -1.6m, -4.8m, -5.3m, and 16
-6.4m under Pi-replete and 24 hr Pi starvation conditions and found that only those with 17
Kd values of 4.8 mM and greater showed a significant change in FRET ratio and in each 18
of these cases the ratio increased in response to Pi starvation, consistent with decreased 19
cytosolic Pi concentrations. Transgenic lines expressing the cpFLIPPi-6.4m sensor 20
showed the largest response and representative FRET ratio images and quantitation 21
derived from six independent seedlings are shown in Fig. 4. We attributed this response 22
to altered Pi concentrations rather than unrelated changes in the emission of one or both 23
fluorophores because no differences in FRET ratios were detected in seedlings that 24
express a high-affinity sensor, i.e., cpFLIPPi-80μ (Supp. Fig. S7A). 25
Comparisons of fluorescence intensities in cpFLIPPi-6.4m seedlings to those of 26
purified sensor protein standards imaged under identical conditions indicated that the 27
cytosolic concentrations of this sensor was only ~1 μM (Supp. Fig. S8). Consequently, 28
we suggest that the fraction of cytosolic Pi sequestered by this sensor is negligible. 29
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To test whether FRET ratios in Pi-starved plants could be reversed by 1
replenishment with Pi we added 0.5 mM Pi to 24 hour-starved cpFLIPPi-6.4 lines and 2
monitored FRET over time (Fig. 5). No changes were detected when FRET was 3
monitored for the first few minutes. However, changes in FRET ratios indicative of 4
replenishment were detected after 1 hour, and FRET signals were nearly equivalent to 5
the initial “fed” state within 8 hours (Fig. 5). These results strongly indicate that the in 6
vivo FRET responses are specific for Pi. 7
It was possible that, within limits, the steady state concentration of Pi in the 8
cytosol is a variable continuum dependent on the magnitude of Pi supply. Alternatively, 9
if other cellular pools buffer cytosolic Pi (Mimura, 1999) then the concentration would 10
be relatively constant until buffering capacity is exceeded. To distinguish these 11
possibilities we grew transgenic cpFLIPPi-6.4 lines in hydroponic medium containing 12
0.5 mM Pi for 5 days then replaced the medium with fresh media containing the same or 13
reduced concentrations of Pi. Plants were grown in the new condition for another 24 14
hours then imaged in the same final growth medium. As shown in Fig. 6, FRET ratios 15
increased when supplied with 25 μM or less Pi, but remained nearly constant when 16
plants were supplied with Pi concentrations greater than 25 μM. FRET ratios reached a 17
maximum when plants were supplied with 10 μM Pi, but the values were not 18
significantly different from those measured with plants completely deprived of Pi. These 19
results support the idea that cytosolic Pi is buffered over a broad range of external Pi 20
concentrations, and that this buffering capacity is overwhelmed when Pi supply drops 21
below a critical threshold. 22
It was formally possible that the Pi-dependent changes in FRET ratios we 23
detected were caused by a mechanism other than FRET. Because it was not possible to 24
address this issue through analysis of the emission spectrum in vivo as was done for our 25
in vitro studies, we used partial acceptor photobleaching as an alternative approach 26
(Roszik et al., 2008). Evidence for FRET would be revealed by this method if 27
photodestruction of the acceptor cpVenus resulted in an increase in donor eCFP 28
emission due to dequenching of the donor. Moreover, the increase in eCFP emission 29
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would be proportional to FRET efficiency, which would be dependent on Pi 1
concentration. No consistent change in eCFP emission would be expected if sensor 2
responses were unrelated to FRET. 3
We conducted partial acceptor photobleaching with each of the same plants used 4
to evaluate the effect of varied Pi supply on its accumulation in the cytosol (Fig. 6). In 5
each case, eCFP emission clearly increased as cpVenus emission diminished, indicating 6
a FRET response (Suppl. Fig. S9A). FRET efficiencies were calculated from the 7
photobleach responses as described previously (Roszik et al., 2008). The FRET 8
efficiency values calculated for each concentration of supplied Pi correlated with the 9
corresponding FRET ratios (Suppl. Fig. S9B) and thus further validated the efficacy of 10
the cpFLIPPi-6.4m sensor for monitoring cytosolic Pi. 11
12
Live imaging of Pi in Root Plastids of Wild Type and a Plastidic Pi Transport 13
Mutant 14
Nonphotosynthetic plastids import ATP from the cytosol in stoichiometric 15
exchange for stromal ADP (Reiser et al., 2004; Reinhold et al., 2007). A consequence 16
of the unbalanced phosphate moieties associated with this exchange is that Pi would 17
accumulate to deleterious levels if not balanced by export. We proposed previously 18
that the plastidic Pi transporter PHT4;2 confers this export activity in root plastids 19
(Irigoyen et al., 2011). Transport activities measured with root plastids isolated from 20
wild type and a pht4;2 mutant support this idea. However, the mutation also reduced 21
Pi import. It was possible that the import activity simply reflected the reversibility of 22
the transport process since maintenance of relevant electrochemical gradients cannot 23
be assured with isolated organelles. Alternatively, PHT4;2 may have an undefined 24
physiological role in Pi import. 25
To directly assess the role of PHT4;2 in plastidic Pi transport within the context 26
of an intact plant we introduced the plastid-targeted version of the cpFLIPPi-6.4m sensor 27
into the pht4;2 background then compared FRET ratios in this line to those of the same 28
sensor in wild type. Plants were grown for 5 days in medium containing 0.5 mM Pi to 29
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establish uniform growth then imaged in the same medium. FRET ratios were 1
significantly lower in mutant plastids, which is consistent with increased accumulation 2
of Pi. Representative ratio images and quantitation from six independent seedlings of 3
each genotype are shown in Fig. 7. The FRET ratio images were merged with DIC 4
images to distinguish individual cells, and this revealed that there is little variation in the 5
amounts of Pi accumulated in plastids within the same cell or in different epidermal cells 6
(Fig. 7A). The differences in FRET ratios can be attributed to altered Pi concentrations 7
rather than unrelated effects of the pht4;2 mutation because no differences in FRET 8
ratios were detected when the high-affinity plastid cpFLIPPi-80μ sensor was used in 9
these same genotypes (Supp. Fig. S7B). These results strongly support the hypothesis 10
that the physiologically-relevant role of PHT4;2 is Pi export from root plastids. 11
12
DISCUSSION 13
Despite variable and often scarce supplies of Pi from soils, plants must distribute 14
appropriate amounts of Pi to each cell and subcellular compartment to sustain essential 15
metabolic activities. Because its concentrations in different locations can vary widely 16
and in some cases may also serve as a signal of Pi sufficiency (Carswell et al., 1996; 17
Ticconi et al., 2001; Varadarajan et al., 2002; Kanno et al., 2012), dynamic assessment 18
of these concentrations in live plants is critical for understanding the complex 19
mechanisms underlying Pi homeostasis. Nonaqueous fractionation and 31P-NMR have 20
been used to probe Pi contents of some cell compartments, but these methods lack 21
cellular resolution and have no or limited temporal resolution (Rebeille et al., 1984; 22
Sharkey and Vanderveer, 1989; Stitt et al., 1989; Pratt et al., 2009; Gout et al., 2011). 23
Therefore, to achieve the necessary spatial and temporal resolution we chose to adapt for 24
use in plants a genetically encoded Pi sensor (FLIPPi) that had been used previously to 25
track Pi in cultured animal cells (Gu et al., 2006). 26
Our efforts to adapt FLIPPi sensors for use in plants included modification of the 27
sensor proteins first to enhance dynamic range and then to optimize affinity. To enhance 28
dynamic range, i.e., the maximum change in Pi-dependent FRET, we used a strategy that 29
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16
had proven successful for improvement of yellow cameleon Ca2+ sensors (Nagai et al., 1
2004). Specifically, we substituted the eYFP component of a FLIPPi sensor with a 2
circularly permuted form of Venus (cpVenus). The resulting circularly permuted FLIPPi 3
(cpFLIPPi) sensor exhibited increased FRET even in the absence of Pi. More 4
importantly, its dynamic range was more than twice that of the parent sensor. This 5
enhancement is presumably a function of the relative orientation of the two 6
chromophores in the sensor rather than their distance because the chromophores remain 7
equally separated by the Pi binding protein (Nagai et al., 2004). Regardless, it is unlikely 8
that cpVenus and eYFP interact differently with the Pi binding protein component of the 9
sensor because binding affinity was unaffected. 10
Most estimates of cellular Pi concentrations in Pi-replete plants range from 2.5 to 11
10 mM and are derived from enzyme kinetic parameters, nonaqueous fractionation and 12 31P-NMR (Rebeille et al., 1984; Stitt et al., 1988; Sharkey and Vanderveer, 1989; Lee 13
and Ratcliffe, 1993; Copeland and Zammit, 1994; Gout et al., 2011; Tiessen et al., 14
2012), although cytosolic concentrations as low as 60 to 80 μM have also been reported 15
(Pratt et al., 2009). We used these estimates as a guide for selecting cpFLIPPi affinity 16
mutants with a goal of encompassing the entire range. Our mutagenesis strategy 17
involved a novel use of statistical coupling analysis (SCA) (Lockless and Ranganathan, 18
1999; Socolich et al., 2005) as a means to enhance the efficiency of the screen. The 19
premise of SCA is that coupling represents an evolutionary constraint for structural or 20
functional properties, e.g., ligand binding, protein-protein interactions and allosteric 21
sites. We conducted SCA with a set of 1,020 conserved Pi binding proteins and 22
identified a six amino acid region that contained coupled residues that also map near the 23
predicted Pi binding site. By limiting our mutagenesis to alter only this six amino acid 24
region rather than exploring the entire 340 amino acids of the Pi binding protein 25
included in the cpFLIPPi sensor, we reduced the scope of mutagenesis numerically and 26
also greatly biased our screen for those likely to alter Pi binding properties. 27
The mutant screen was highly efficient with nearly 80% of the individuals 28
screened showing altered affinity. Eight of these mutants were selected for further 29
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17
analysis. Each of these were highly specific for Pi and had Kd values for Pi binding that 1
ranged from 0.08 to 11 mM, which spanned the entire target range. Although the in vitro 2
binding assays were conducted in a medium that approximated cellular viscosity and 3
primary ionic composition (Messerli and Robinson, 1998; Feijo et al., 1999), we 4
recognize that the binding affinities may differ in vivo. Nevertheless, the large (>100-5
fold) concentration range collectively probed by the cpFLIPPi sensors was more than 6
sufficient to monitor relative changes in the Pi contents of cytosol and plastid stroma of 7
Arabidopsis root epidermal cells and are likely to also be useful for monitoring Pi 8
contents in other cells and other cell compartments with near-neutral pH. 9
The cpFLIPPi sensors yielded weak or unstable fluorescent signals when 10
expressed in wild-type plants suggesting that they were subject to transgene silencing. 11
Silencing was also reported for related FRET-based sensors for glucose and glutamine, 12
and in these cases silencing was eliminated when the sensors were expressed in siRNA-13
deficient mutants sgs3 and rdr6 (Deuschle et al., 2006; Chaudhuri et al., 2008; Yang et 14
al., 2010). It is possible that some aspect of the ligand-binding portions of these sensors 15
promotes their silencing because a related Ca2+ sensor that contains the same 16
fluorophores as cpFLIPPi and is expressed from the same Arabidopsis UBQ10 promoter 17
is not subject to silencing (Krebs et al., 2012). Regardless, we attained strong, stable 18
fluorescence when cpFLIPPi sensors were expressed in the sgs3-13 background. It 19
should be noted that although rdr6 mutations are more effective at eliminating transgene 20
silencing than sgs3 mutations (Deuschle et al., 2006), we chose to use sgs3-13 for our 21
studies because, unlike rdr6 mutations, it had little effect on plant growth and 22
morphology. Moreover, secreted acid phosphatase activities measured in Pi-sufficient 23
and Pi-starved sgs3-13 seedlings were equivalent to those of wild-type seedlings grown 24
under the same conditions suggesting that this mutation has no significant effect on 25
normal responses to Pi starvation. 26
Analysis of root extracts revealed that depriving 5-day old seedlings of Pi for 24 27
hours was sufficient to reduce free, cellular- and subcellular-undefined Pi contents by 28
~19%. Live imaging of cpFLIPPi sensors allowed us to unambiguously monitor the 29
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18
fraction attributed to cytosolic Pi contents of epidermal cells. A significant change in 1
FRET ratio was detected only for sensors with Kd values of 4.8 mM or greater, which 2
suggests that sensors with higher affinity remained effectively saturated under this 3
relatively mild starvation regime. Transgenic lines with the cpFLIPPi-6.4m sensor 4
yielded the largest FRET response upon Pi deprivation, which may reflect an optimal 5
pairing of the binding affinity of this sensor with cytosolic Pi concentrations. 6
Multiple lines of evidence indicate that the changes in FRET ratios we measured 7
in epidermal cells represent genuine Pi-dependent responses. First, FRET responses 8
elicited by Pi deprivation were completely reversed by Pi replenishment. Second, high-9
affinity sensors exhibited no detectable FRET responses suggesting little or no changes 10
in Pi-independent factors that could influence sensor properties. And third, Pi-dependent 11
FRET efficiencies determined from acceptor photobleaching experiments directly 12
correlated with the corresponding FRET ratios. Consequently, we conclude that the 13
cpFLIPPi-6.4m sensor reports changes in cytosolic Pi concentrations with high 14
specificity. Our results also suggest that the concentration of Pi in the cytosol is 15
relatively constant despite large differences in the amount of Pi supplied in the growth 16
medium, presumably due to buffering by vacuolar pools (Mimura, 1999). This buffering, 17
however, can be overwhelmed when Pi supply falls below a critical threshold. For our 18
growth and assay conditions, this threshold was ~25 μM. 19
In addition to cytosolic Pi, the cpFLIPPi-6.4 sensor also enabled monitoring of Pi 20
at the subcellular level. We proposed previously that the physiologically relevant role of 21
PHT4;2, a plastidic Pi transporter, was export of Pi from root plastids (Irigoyen et al., 22
2011). This hypothesis was based on transport assays conducted with isolated root 23
plastids, but the assays also revealed an import activity, which we viewed as a 24
consequence of transport reversibility under our assay conditions. However, it was 25
formally possible that Pi import was the relevant biological activity. Ratiometric 26
imaging of a plastid-targeted version of cpFLIPPi-6.4m expressed in wild-type and 27
pht4;2 plants distinguished these possibilities in the context of a live plant and thereby 28
confirmed that Pi export is the physiologically relevant role for PHT4;2. 29
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19
1
CONCLUSIONS 2
The current series of cpFLIPPi sensors provide the necessary spatial and 3
temporal resolution to follow critical aspects of Pi metabolism in live plants. One of 4
these, cpFLIPPi-6.4m, was particularly useful because it detected reduced cytosolic Pi 5
concentrations in response to varied external supply and also detected a mutation-6
induced increase in Pi accumulation within an organelle. These sensor proteins are also 7
likely to be suitable for studies in microbial and animal cell systems, and the approaches 8
described here for their optimization may be broadly applicable to other genetically 9
encoded sensors. Future efforts to target cpFLIPPi sensors to additional subcellular 10
compartments and further engineering to enhance signal responses in acidic 11
environments will provide valuable insight into the mechanisms underlying Pi 12
metabolism as well as the sensing and signaling of Pi status. 13
14
15
MATERIALS AND METHODS 16
Plasmids 17
We obtained FLIPPi plasmid clones pRSET FLIPPi-200μ and pRSET FLIPPi-18
30m (Gu et al., 2006) from Addgene (www.Addgene.com). To replace the eYFP coding 19
region of pRSET FLIPPi-200μ with a circularly permuted version of Venus, we first 20
amplified the cp173 coding region from the YC3.60 Ca2+ sensor (Nagai et al., 2004). We 21
then used PCR and the In-Fusion (Clontech) cloning kit to introduce this sequence into 22
the same region occupied by the eYFP coding sequence in the pRSET FLIPPi-200μ 23
plasmid to generate pRSET cpFLIPPi-200μ. 24
For cpFLIPPi sensor expression in plants, a 2455 bp Hind III-BamHI segment of 25
the desired pRSET cpFLIPPi clone was ligated into pCN-UBQ10, a modified 26
pCAMBIA3300 plasmid. The modifications included insertion of the Arabidopsis 27
UBQ10 promoter (Norris et al., 1993) to direct expression of the sensor gene and 28
replacement of the 35S promoter located upstream of the phosphinothricin resistance 29
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20
gene with the Nos promoter of pBI121. A plastid-targeted variant of each clone was 1
constructed by inserting a 237 bp fragment encoding the 79 amino acid RbcS chloroplast 2
transit peptide (Lee et al., 2006) in frame at the 5’ end of the sensor gene. 3
4
Construction of a cpFLIPPi affinity mutant library 5
We used statistical coupling analysis (SCA) (Lockless and Ranganathan, 1999; 6
Socolich et al., 2005) as a guide to generate a sensor mutant library biased for altered 7
affinity. Sequences were collected from the nr database (as of 5/5/2010) using PSI-8
BLAST (Altschul et al., 1997). The Escherichia coli PiBP protein (GI:157829674) was 9
used as the query for five iterations of PSI-BLAST. Sequences with an e-score<1e-10 10
were aligned using MUSCLE (Edgar, 2004). Identical sequences were eliminated and 11
the alignment of the remaining 1020 sequences was used for SCA. SCA was performed 12
as previously described (Lockless and Ranganathan, 1999; Socolich et al., 2005) to 13
identify the set of positions that co-evolve with one another. Two rounds of hierarchical 14
clustering were performed to yield a self-consistent set of positions that statistically co-15
vary with each other. PiBPs have one self-consistent cluster consisting of 43 amino acids 16
(13% of protein), which was mapped onto the structure of the E. coli PiBP using PyMol 17
(Suppl. Fig. S3). 18
A stretch of six amino acids (positions 18-23) within the 43 amino acid cluster 19
was identified as a high-value mutational target based on the following observations: 1) 20
four of the positions (#18, 19, 21 and 23) co-evolve with one another, 2) one position 21
(#20) is highly conserved, 3) they are located near the Pi binding site, and 4) the 22
positions could be simultaneously mutated with one set of oligonucleotides. The 23
mutagenesis oligonucleotides were designed such that each position was mutated to the 24
three most frequently observed amino acids in the alignment that are found in at least 9% 25
of sequences. In practice, this means that more than the desired amino acids are possible 26
at some positions since the nucleotides required to encoded the target amino acids can 27
also encode other amino acids. For example, alanine (GCT codon), aspartate (GAT 28
codon) and serine (TCT codon) are needed at position 19, but the nucleotides required in 29
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21
the oligonucleotide at each position can also encode for tyrosine (TAT codon). 1
The pRSET cpFLIPPi-200μ plasmid was used as template for PCR mutagenesis. 2
The mutagenesis oligonucleotides 5’- 3
ccaaaccgtgcaaatctccRBCKMTgggcKCGDMCDYCgcggctcctttgctgcaacgttg and 5’- 4
gttgcagcaaaggagccgcGRHGKHCGMgccAKMGVYggagatttgcacggtttgggctt introduce 5
ca. 1728 possible amino acid combinations for the six amino acid (positions 18-23) 6
target region. The amplicon population was restriction digested then ligated into pRSET 7
cpFLIPPi-200μreplacing the original sequence. A portion of this mixed ligation product 8
was introduced into E. coli DH5α cells and >10,000 colonies were pooled for bulk 9
plasmid isolation. The isolated plasmids collectively represent the affinity mutant 10
library. 11
12
cpFLIPPi affinity mutant screen 13
Portions (0.1 ng) of the sensor affinity mutant plasmid library were introduced 14
into E. coli BL21 (DE3) cells. The pRSET cpFLIPPi-200μ plasmid served as a positive 15
control and pUC19 served as a negative control for each transformation and subsequent 16
screen. Individual colonies were transferred to a 96-well plate containing 45 μL LB 17
medium in each well and mixed thoroughly. Aliquots of 20 µL were transferred to 18
duplicate 96-well deep well plates containing 1.5 mL LB medium then covered with a 19
gas-permeable seal. Plates were shaken at 300 rpm for 16 hours at 25°C. Glycerol stocks 20
were prepared from 0.1 mL aliquots of each culture in a separate 96-well plate and 21
stored at -80°C. The growth plates were centrifuged for 30 min to pellet cells then the 22
supernatants were removed by vacuum aspiration. Cell pellets were washed twice with 23
20 mM Tris-HCl, pH 7.5 then frozen at -20°C. To lyse cells, 0.35 mL lysis solution (1x 24
BugBuster (Novagen), 20 mM Tris-HCl pH 7.5, 150 mM K-gluconate, 3 μL 25
Lysonase/mL) was added to each well, mixed thoroughly then incubated at room temp 26
for 20 min with mixing every 5 minutes. Plates were then centrifuged for 10 min at 2800 27
rpm, 20°C to remove cell debris. 28
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22
Fluorescence assays were conducted with lysates in 96-well black plates. We 1
transferred 20 μL aliquots of each lysate to eight wells containing 200 μL pseudocytosol 2
medium (100 mM K-gluconate, 30 mM NaCl, 25 mM MES, 25 mM HEPES, 40% 3
sucrose, 1 mg/mL BSA, pH 7.5) (Messerli and Robinson, 1998; Feijo et al., 1999) and 4
varied amounts of Pi (0 to 30 mM). Fluorescence was measured using a microplate 5
reader (Synergy HT) with excitation 420/27 nm and emission 485/20 and 540/25 nm. 6
Fluorescence from direct excitation of cpVenus (500/20 nm) was also monitored with 7
emission at 540/25 nm. The Kd value for each sensor was estimated by fitting data to a 8
single-site binding isotherm as described (Gu et al., 2006). 9
10
In vitro characterization of cpFLIPPi proteins 11
Selected cpFLIPPi mutant clones were re-introduced into E. coli BL21 (DE3) 12
cells, and single colonies were cultured in 100 mL medium then harvested and lysed 13
essentially as described above for the mutant screen. The expressed sensor proteins were 14
purified from cell lysates using His-affinity chromatography with elution in 20 mM Tris-15
HCl pH 7.5, 150 mM K-gluconate, 400 mM imidazole. The eluent was dialyzed 16
overnight at 4°C against 20 mM Tris-HCl pH 7.5 then transferred to a new tube and 17
stored at 4°C. The purified sensors could be stored for at least one month without 18
measurable change in fluorescence or binding properties. Kd values were determined as 19
described above for the mutant screen, but twelve Pi concentrations were used to 20
generate binding curves. Assays for binding affinity, specificity and pH sensitivity were 21
all conducted with at least three independent protein preparations. 22
23
Generation of transgenic Arabidopsis plants 24
Sensor transgenes were introduced into Arabidopsis plants by floral dip 25
transformation (Clough and Bent, 1998). 26
27
Subcellular localization of sensor proteins 28
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23
Protoplasts were prepared as described (Yoo et al., 2007) from leaves of 5-week-1
old transgenic plants expressing cpFLIPPi sensors engineered with and without an N-2
terminal RbcS chloroplast transit peptide. Protoplasts were imaged with a BioRad ES 3
confocal laser scanning microscope equipped with a 63x (numerical aperture, 1.2) water-4
immersion objective (515 nm excitation, 540 nm emission for direct excitation of the 5
cpVenus sensor component, and 488 nm excitation, 680 nm emission for chlorophyll 6
autofluorescence. 7
8
Live imaging of sensors in plants 9
Transgenic plants were grown in individual wells of 96-well plates with 0.3 mL 10
of half-strength MS medium containing 1% sucrose and unless indicated otherwise, 0.5 11
mM Pi. The plates were incubated in a growth chamber (60% relative humidity, 21°C, 12
90 μmol m-2 s-1 light intensity, 16 hour photoperiod). After 5 days the seedlings were 13
either imaged or transferred to fresh medium as indicated. For imaging, seedlings were 14
placed in a custom-made chamber (http://microscopy.tamu.edu/lab-protocols/light-15
microscopy-protocols.html) containing the same growth medium. Seedlings were 16
covered with a small square of nylon mesh then gently immobilized with a glass weight. 17
A similar procedure was described recently (Kruger et al, 2013). Seedlings were 18
analyzed for FRET using an inverted Olympus IX81 laser spinning disk confocal 19
microscope equipped with an Andor Opti-splitter to allow simultaneous dual emission. 20
Root epidermal cells were viewed with a 40x (1.3 n.a.) oil-immersion objective (445 nm 21
excitation for eCFP and 483/32 and 542/27 nm emission wavelengths for eCFP and 22
FRET-derived cpVenus, respectively. Laser intensity and exposure settings were kept 23
constant. For acceptor photobleaching experiments, a 515 nm excitation laser was used 24
with maximum laser intensity. Untransformed seedlings and transgenic seedlings 25
expressing eCFP alone and cpVenus separately were imaged each day to allow 26
correction for background fluorescence, spectral bleedthrough and cross excitation. 27
All image analyses were done using ImageJ software. Mean intensity values for 28
untransformed seedlings were used to establish background fluorescence values, and 29
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24
these were subtracted from all sensor images before conducting further analyses. For 1
analysis of cytosol-localized sensors, mean intensity values for eCFP and cpVenus were 2
measured for selected regions of interests (ROIs), which typically included 3-4 cells for 3
each field of view. For analysis of plastid-localized sensors, a uniform threshold was 4
applied to the eCFP and FRET-cpVenus images, and mean intensity values above the 5
threshold were used to calculate sensitized FRET ratios. FRET efficiencies (E values) 6
were calculated from partial acceptor photobleaching images as described (Roszik et al., 7
2008). Briefly, mean intensity values for eCFP (donor) and directly excited cpVenus 8
(acceptor) for each sample were recorded prior to photobleach and again for at least four 9
photobleach time points. These values were corrected for spectral bleedthrough and 10
cross-excitation then used to calculate mean FRET efficiencies. An ImageJ plugin, 11
AccPbFRET, is available to automate much of this analysis (Roszik et al., 2008). 12
13
Colorimetric Pi assay 14
Plants were grown exactly as for live imaging with and without a final 24 hour 15
Pi deprivation. Roots were harvested and pooled in groups of 15. Pi contents were 16
determined using a phosphomolybdate colorimetric assay as described previously 17
(Ames, 1966; Versaw and Harrison, 2002). 18
19
Secreted acid phosphatase assay 20
Plants were grown exactly as for live imaging with and without a final 24 hour 21
Pi deprivation. Individual seedlings were then transferred to 0.3 mL fresh medium 22
containing no Pi. Secreted acid phosphatase activity in the medium was measured after 23
30 minutes using p-nitrophenol phosphate as described (Robinson et al., 2012) except 24
seedlings were removed from the assay mixture immediately prior to termination with 25
1M sodium carbonate. One unit of phosphatase activity is defined as 1 μmol p-26
nitrophenol liberated per minute. 27
28
Upon request, all novel materials described in this publication will be made 29
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25
available in a timely manner for non-commercial research purposes, subject to the 1
requisite permission from any third-party owners of all or parts of the material. 2
Obtaining any permissions will be the responsibility of the requestor. 3
4
SUPPLEMENTAL MATERIAL 5
Supplemental Figure S1. Emission spectrum of cpFLIPPi-200μ in pseudcytosol 6
medium containing 0 or 10 mM Pi. 7
8
Supplemental Figure S2. Analysis to identify residues that co-evolve in the PiBP 9
protein family. 10
11
Supplemental Figure S3. Mapping of co-evolving and mutated residues onto the PiBP 12
structure (pdb1A40). 13
14
Supplemental Figure S4. Specificity of cpFLIPPi-6.4m. 15
16
Supplemental Figure S5. Differential targeting oof cpFLIPPi-6.4m in palisade 17
mesophyll cells of intact Arabidopsis leaves. 18
19
Supplemental Figure S6. SYTOX orange staining shows non-viable cells in roots after 20
72 hr Pi starvation. 21
22
Supplemental Figure S7. Live imaging of cytosolic and plastidic Pi in root epidermal 23
cells using a high-affinity Pi sensor, cpFLIPPi-80μ. 24
25
Supplemental Figure S8. Estimates of cpFLIPPi-6.4m protein concentrations in the 26
cytosol of root epidermal cells. 27
28
Supplemental Figure S9. Pi-dependent FRET efficiencies determined from partial 29
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26
acceptor photobleaching of cytosolic cpFLIPPi-6.4m correlate with FRET ratio values. 1
2
ACKNOWLEDGEMENTS 3
We thank Shian Liu for assistance with analysis of the mutant library and Tom 4
McKnight for critical reading of this manuscript. 5
6
7
8
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27
LITERATURE CITED 1
Adams JP, Adeli A, Hsu CY, Harkess RL, Page GP, Depamphilis CW, Schultz EB, 2 Yuceer C (2012) Plant-based FRET biosensor discriminates environmental zinc 3 levels. Plant Biotechnol J 10: 207-216 4
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ 5 (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database 6 search programs. Nucleic Acids Res 25: 3389-3402 7
Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases. 8 Methods Enzymol 8: 115-118 9
Bates TR, Lynch JP (2000) The efficiency of Arabidopsis thaliana (Brassicaceae) root 10 hairs in phosphorus acquisition. Am J Bot 87: 964-970 11
Bieleski RL (1973) Phosphate pools, phosphate transport and phosphate availability. 12 Ann Rev Plant Physiol 24: 225-252 13
Cardenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK (2008) Pollen tube growth 14 oscillations and intracellular calcium levels are reversibly modulated by actin 15 polymerization. Plant Physiol 146: 1611-1621 16
Carswell C, Grant BR, Theodorou ME, Harris J, Niere JO, Plaxton WC (1996) The 17 Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica 18 nigra Seedlings. Plant Physiol 110: 105-110 19
Chaudhuri B, Hormann F, Frommer WB (2011) Dynamic imaging of glucose flux 20 impedance using FRET sensors in wild-type Arabidopsis plants. J Exp Bot 62: 21 2411-2417 22
Chaudhuri B, Hormann F, Lalonde S, Brady SM, Orlando DA, Benfey P, Frommer 23 WB (2008) Protonophore- and pH-insensitive glucose and sucrose accumulation 24 detected by FRET nanosensors in Arabidopsis root tips. Plant J 56: 948-962 25
Chiou TJ, Lin SI (2011) Signaling network in sensing phosphate availability in plants. 26 Annu Rev Plant Biol 62: 185-206 27
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-28 mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 29
Copeland L, Zammit A (1994) Kinetic properties of NAD-dependent glyceraldehyde-30 3-phosphate dehydrogenase from the host fraction of soybean root nodules. Arch 31 Biochem Biophys 312: 107-113 32
Demmig B, Gimmler H (1983) Properties of the isolated intact chloroplast at 33 cytoplasmic K concentrations: I. light-induced cation uptake into intact 34 chloroplasts is driven by an electrical potential difference. Plant Physiol 73: 169-35 174 36
Deuschle K, Chaudhuri B, Okumoto S, Lager I, Lalonde S, Frommer WB (2006) 37 Rapid metabolism of glucose detected with FRET glucose nanosensors in 38 epidermal cells and intact roots of Arabidopsis RNA-silencing mutants. Plant 39 Cell 18: 2314-2325 40
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high 41 throughput. Nucleic Acids Res 32: 1792-1797 42
www.plantphysiol.orgon April 17, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
28
Feijo JA, Sainhas J, Hackett GR, Kunkel JG, Hepler PK (1999) Growing pollen 1 tubes possess a constitutive alkaline band in the clear zone and a growth-2 dependent acidic tip. J Cell Biol 144: 483-496 3
Geldner N, Denervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J 4 (2009) Rapid, combinatorial analysis of membrane compartments in intact plants 5 with a multicolor marker set. Plant J 59: 169-178 6
Gjetting KS, Ytting CK, Schulz A, Fuglsang AT (2012) Live imaging of intra- and 7 extracellular pH in plants using pHusion, a novel genetically encoded biosensor. 8 J Exp Bot 63: 3207-3218 9
Gjetting SK, Schulz A, Fuglsang AT (2013) Perspectives for using genetically encoded 10 fluorescent biosensors in plants. Front Plant Sci 4: 234 11
Gout E, Bligny R, Douce R, Boisson AM, Rivasseau C (2011) Early response of plant 12 cell to carbon deprivation: in vivo 31P-NMR spectroscopy shows a quasi-13 instantaneous disruption on cytosolic sugars, phosphorylated intermediates of 14 energy metabolism, phosphate partitioning, and intracellular pHs. New Phytol 15 189: 135-147 16
Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) A 17 ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates 18 temporal stability and native protein distribution in transient and stable 19 expression studies. Plant J 64: 355-365 20
Gu H, Lalonde S, Okumoto S, Looger LL, Scharff-Poulsen AM, Grossman AR, 21 Kossmann J, Jakobsen I, Frommer WB (2006) A novel analytical method for 22 in vivo phosphate tracking. FEBS Lett 580: 5885-5893 23
Irigoyen S, Karlsson PM, Kuruvilla J, Spetea C, Versaw WK (2011) The sink-24 specific plastidic phosphate transporter PHT4;2 influences starch accumulation 25 and leaf size in Arabidopsis. Plant Physiol 157: 1765-1777 26
Jain A, Nagarajan VK, Raghothama KG (2012) Transcriptional regulation of 27 phosphate acquisition by higher plants. Cell Mol Life Sci 69: 3207-3224 28
Javot H, Pumplin N, Harrison MJ (2007) Phosphate in the arbuscular mycorrhizal 29 symbiosis: transport properties and regulatory roles. Plant Cell Environ 30: 310-30 322 31
Kanno S, Yamawaki M, Ishibashi H, Kobayashi NI, Hirose A, Tanoi K, Nussaume 32 L, Nakanishi TM (2012) Development of real-time radioisotope imaging 33 systems for plant nutrient uptake studies. Philos Trans R Soc Lond B Biol Sci 34 367: 1501-1508 35
Kaper T, Lager I, Looger LL, Chermak D, Frommer WB (2008) Fluorescence 36 resonance energy transfer sensors for quantitative monitoring of pentose and 37 disaccharide accumulation in bacteria. Biotechnol Biofuels 1: 11 38
Krebs M, Held K, Binder A, Hashimoto K, Den Herder G, Parniske M, Kudla J, 39 Schumacher K (2012) FRET-based genetically encoded sensors allow high-40 resolution live cell imaging of Ca(2)(+) dynamics. Plant J 69: 181-192 41
Kumakura N, Takeda A, Fujioka Y, Motose H, Takano R, Watanabe Y (2009) 42 SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6-bodies. 43 FEBS Lett 583: 1261-1266 44
www.plantphysiol.orgon April 17, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
29
Lalonde S, Ehrhardt DW, Frommer WB (2005) Shining light on signaling and 1 metabolic networks by genetically encoded biosensors. Curr Opin Plant Biol 8: 2 574-581 3
Lee DW, Lee S, Lee GJ, Lee KH, Kim S, Cheong GW, Hwang I (2006) Functional 4 characterization of sequence motifs in the transit peptide of Arabidopsis small 5 subunit of rubisco. Plant Physiol 140: 466-483 6
Lee RB, Ratcliffe RG (1993) Subcellular distribution of inorganic phosphate, and levels 7 of nucleoside triphosphate, in mature maize roots at low external phosphate 8 concentrations: Measurements with 31P-NMR. J Exp Bot 44: 587-598 9
Liu TY, Lin WY, Huang TK, Chiou TJ (2014) MicroRNA-mediated surveillance of 10 phosphate transporters on the move. Trends Plant Sci 19: 647-655 11
Lockless SW, Ranganathan R (1999) Evolutionarily conserved pathways of energetic 12 connectivity in protein families. Science 286: 295-299 13
Messerli MA, Robinson KR (1998) Cytosplasmic acidificaiton and current influc 14 follow growth pulses of Lilium longiflorum pollen tubes. Plant J 16: 87-91 15
Mimura T (1999) Regulation of phosphate transport and homeostasis in plant cells. Int 16 Rev Cytol 191: 149-200 17
Misson J, Thibaud MC, Bechtold N, Raghothama K, Nussaume L (2004) 18 Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high 19 affinity transporter contributing greatly to phosphate uptake in phosphate 20 deprived plants. Plant Mol Biol 55: 727-741 21
Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant 22 of yellow fluorescent protein with fast and efficient maturation for cell-biological 23 applications. Nat Biotech 20: 87-90 24
Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A (2004) Expanded 25 dynamic range of fluorescent indicators for Ca(2+) by circularly permuted 26 yellow fluorescent proteins. Proc Natl Acad Sci USA 101: 10554-10559 27
Norris SR, Meyer SE, Callis J (1993) The intron of Arabidopsis thaliana polyubiquitin 28 genes is conserved in location and is a quantitative determinant of chimeric gene 29 expression. Plant Mol Biol 21: 895-906 30
Okumoto S, Jones A, Frommer WB (2012) Quantitative imaging with fluorescent 31 biosensors. Annu Rev Plant Biol 63: 663-706 32
Peret B, Clement M, Nussaume L, Desnos T (2011) Root developmental adaptation to 33 phosphate starvation: better safe than sorry. Trends Plant Sci 16: 442-450 34
Plaxton WC, Tran HT (2011) Metabolic adaptations of phosphate-starved plants. Plant 35 Physiol 156: 1006-1015 36
Pratt J, Boisson AM, Gout E, Bligny R, Douce R, Aubert S (2009) Phosphate (Pi) 37 starvation effect on the cytosolic Pi concentration and Pi exchanges across the 38 tonoplast in plant cells: an in vivo 31P-nuclear magnetic resonance study using 39 methylphosphonate as a Pi analog. Plant Physiol 151: 1646-1657 40
Raghothama K (1999) Phosphate acquisition. Annu Rev Plant Physiol Plant Mol Biol 41 50: 665-693 42
Rebeille F, Bligny R, Douce R (1984) Is the cytosolic Pi concentration a limiting factor 43 for plant cell respiration? Plant Physiol 74: 355-359 44
www.plantphysiol.orgon April 17, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
30
Reinhold T, Alawady A, Grimm B, Beran KC, Jahns P, Conrath U, Bauer J, Reiser 1 J, Melzer M, Jeblick W, Neuhaus HE (2007) Limitation of nocturnal import of 2 ATP into Arabidopsis chloroplasts leads to photooxidative damage. Plant J 50: 3 293-304 4
Reiser J, Linka N, Lemke L, Jeblick W, Neuhaus HE (2004) Molecular physiological 5 analysis of the two plastidic ATP/ADP transporters from Arabidopsis. Plant 6 Physiol 136: 3524-3536 7
Rincon-Zachary M, Teaster ND, Sparks JA, Valster AH, Motes CM, Blancaflor EB 8 (2010) Fluorescence resonance energy transfer-sensitized emission of yellow 9 cameleon 3.60 reveals root zone-specific calcium signatures in Arabidopsis in 10 response to aluminum and other trivalent cations. Plant Physiol 152: 1442-1458 11
Robinson WD, Park J, Tran HT, Del Vecchio HA, Ying S, Zins JL, Patel K, 12 McKnight TD, Plaxton WC (2012) The secreted purple acid phosphatase 13 isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-14 scavenging by Arabidopsis thaliana. J Exp Bot 63: 6531-6542 15
Roszik J, Szollosi J, Vereb G (2008) AccPbFRET: an ImageJ plugin for semi-16 automatic, fully corrected analysis of acceptor photobleaching FRET images. 17 BMC Bioinformatics 9: 346 18
Rouached H, Arpat AB, Poirier Y (2010) Regulation of phosphate starvation 19 responses in plants: signaling players and cross-talks. Mol Plant 3: 288-299 20
Sanchez-Calderon L, Lopez-Bucio J, Chacon-Lopez A, Cruz-Ramirez A, Nieto-21 Jacobo F, Dubrovsky JG, Herrera-Estrella L (2005) Phosphate starvation 22 induces a determinate developmental program in the roots of Arabidopsis 23 thaliana. Plant Cell Physiol 46: 174-184 24
Schachtman DP, Reid RJ, Ayling SM (1998) Phosphorus uptake by plants: from soil 25 to cell. Plant Physiol 116: 447-453 26
Sharkey TD, Vanderveer PJ (1989) Stromal phosphate concentration is low during 27 feedback limited photosynthesis. Plant Physiol 91: 679-684 28
Shin H, Shin HS, Dewbre GR, Harrison MJ (2004) Phosphate transport in 29 Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from 30 both low- and high-phosphate environments. Plant J 39: 629-642 31
Socolich M, Lockless SW, Russ WP, Lee H, Gardner KH, Ranganathan R (2005) 32 Evolutionary information for specifying a protein fold. Nature 437: 512-518 33
Stitt M, McLilley R, Gerhardt R, Heldt HW (1989) Metabolite levels in specific cells 34 and subcellular compartments of plant leaves. Methods Enzymol 174: 518-552 35
Stitt M, Wilke I, Feil R, Heldt HW (1988) Coarse control of sucrose-phosphate 36 synthase in leaves: Alterations of the kinetic properties in response to the rate of 37 photosynthesis and the accumulation of sucrose. Planta 174: 217-230 38
Ticconi CA, Delatorre CA, Abel S (2001) Attenuation of phosphate starvation 39 responses by phosphite in Arabidopsis. Plant Physiol 127: 963-972 40
Tiessen A, Nerlich A, Faix B, Hummer C, Fox S, Trafford K, Weber H, Weschke 41 W, Geigenberger P (2012) Subcellular analysis of starch metabolism in 42 developing barley seeds using a non-aqueous fractionation method. J Exp Bot 43 63: 2071-2087 44
www.plantphysiol.orgon April 17, 2020 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
31
Truenit E, Haseloff J (2008) A simple way to identify non-viable cells within living 1 plant tissue using confocal microscopy. Plant Methods 4: 15 2
Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical 3 adaptations by plants for securing a nonrenewable resource. New Phytol 157: 4 423-447 5
Varadarajan DK, Karthikeyan AS, Matilda PD, Raghothama KG (2002) Phosphite, 6 an analog of phosphate, suppresses the coordinated expression of genes under 7 phosphate starvation. Plant Physiol 129: 1232-1240 8
Versaw WK, Harrison MJ (2002) A chloroplast phosphate transporter, PHT2;1, 9 influences allocation of phosphate within the plant and phosphate-starvation 10 responses. Plant Cell 14: 1751-1766 11
Walker DA, Sivak MN (1986) Photosynthesis and phosphate: a cellular affair? Trends 12 Biochem Sci 11: 176-179 13
Yang H, Bogner M, Stierhof YD, Ludewig U (2010) H+-independent glutamine 14 transport in plant root tips. PLoS One 5: e8917 15
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell 16 system for transient gene expression analysis. Nat Protoc 2: 1565-1572 17
Zhang Z, Liao H, Lucas WJ (2014) Molecular mechanisms underlying phosphate 18 sensing, signaling, and adaptation in plants. J Integr Plant Biol 56: 192-220 19
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21
22
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Figure legends 1
2
Figure 1. Pi-dependent changes in FRET ratios of purified Pi sensors. A, Schematic of 3
Pi sensor indicating high FRET in absence of Pi and reduced FRET when bound to Pi. 4
Acceptor is eYFP for FLIPPi sensors and cpVenus for cpFLIPPi sensors. PiBP, Pi 5
binding protein. B, Values shown are means ± SE for three independent protein 6
preparations. Error bars smaller than the symbols are not shown. 7
8
Figure 2. Effect of pH on Pi-dependent FRET responses. Pi binding of cpFLIPPi-6.4m 9
was tested in pseudocytosol mdium adjusted to pH 7.0, 7.5 and 8.0, and Pi was adjusted 10
to the same pH. Binding affinity (Kd) values were estimated from nonlinear regression 11
analysis. Plotted values are means ± SE for three independent protein preparations. Error 12
bars smaller than the symbols are not shown. 13
14
Figure 3. Differential targeting of cpFLIPPi-6.4m. Protoplasts were isolated from leaves 15
of stably transformed Arabidopsis plants expressing cpFLIPPi-6.4m with or without an 16
N-terminal chloroplast transit peptide. Confocal microscoy was conducted using settings 17
for cpVenus excitation and emission (green) and chlorophyll autofluorescence 18
(magenta). Scale bar, 10 μm. 19
20
Figure 4. Live imaging of cytosolic Pi in root epidermal cells. A, Representative FRET 21
ratio images of cpFLIPPi-6.4m stably expressed in seedlings with sufficient Pi (control, 22
left), and after 24 hr Pi starvation (right). The same color scale was applied to ratio 23
values for both images. Size bar, 10 μm. B, Plotted FRET ratio values are means ± SE 24
for six independent seedlings. The difference between the two conditions is significant, 25
p<0.05, Student’s t-test. 26
27
Figure 5. Replenishment of cytosolic Pi after 24 hr starvation. Seedlings expressing 28
cpFLIPPi-6.4m were grown with sufficient Pi (0.5 mM, +Pi), starved for 24 hr (-Pi) then 29
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33
Pi was added to 0.5 mM and FRET ratios were monitored over time. The plotted values 1
are means ± SE for six independent seedlings. The first time point shown after 2
replenishment is 1 hr. * indicates significant difference (p<0.05, Student’s t-test) from 3
the –Pi treatment. 4
5
Figure 6. Effect of Pi supply on cytosolic contents of root epidermal cells. Seedlings 6
expressing cpFLIPPi-6.4m were grown with a sufficient supply of Pi (500 μM) then 7
transferred to medium containing the same or less Pi as indicated and grown for an 8
additional 24 hr before imaging. Plotted FRET ratio values are means ± SE for at least 9
six independent seedlings. * indicates significant difference (p<0.05, Student’s t-test) 10
from the 500 μM treatment. 11
12
Figure 7. Live imaging of Pi in plastids of root epidermal cells. A, Representative FRET 13
ratio images for cpFLIPPi-6.4m stably expressed in wild-type and pht4;2 seedlings. The 14
images were merged with the corresponding DIC images to distinguish cell boundaries. 15
The same color scale was applied to ratio values for both images. Size bar, 5 μm. B, 16
Plotted FRET ratio values are means ± SE for six independent seedlings. The difference 17
between the two genotypes is significant, p<0.05, Student’s t-test. 18
19
20
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34
Table I. Pi sensor affinity mutants 1 The cpFLIPPi-200μ sensor plasmid was subjected to a PCR-based mutagenesis. The sequences 2 shown correspond to the mutated region of the sensor. Pi binding constants were determined in vitro using 3 three independent protein preparations for each sensor. ∆Rmax indicates the maximum change in FRET 4 ratio between Pi-free and Pi-saturated sensor. 5 6
Sensor Kd (mM) ∆Rmax DNA sequence of PiBP cds Protein sequence cpFLIPPi-80μ 0.08 -1.06 49 TCCGtcGCtGGCtCGACCaccGCG 17 SVAGSTTA
cpFLIPPi-200μ 0.2 -1.08 49 TCCGGGGCGGGCGCGACCTTTGCG 17 SGAGATFA cpFLIPPi-1.6m 1.6 -0.91 49 TCCGccGatGGCtCGACCaTcGCG 17 SADGSTIA cpFLIPPi-3.5m 3.5 -0.43 49 TCCGGcGCtGGCtCGAaGTcTGCG 17 SGAGSKSA cpFLIPPi-4.8m 4.8 -0.76 49 TCCaccGatGGCGCGACCgccGCG 17 STDGATAA cpFLIPPi-5.3m 5.3 -0.71 49 TCCaGcGatGGCtCGtCCgtcGCG 17 SSDGSSVA cpFLIPPi-6.4m 6.4 -0.75 49 TCCGGGGCGGGCGCGgCCTTTGCG 17 SGAGAAFA cpFLIPPi-11m 11.0 -0.45 49 TCCatctCtGGCtCGtCCaTcGCG 17 SISGSSIA
7 8
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Figure 1. Pi-dependent changes in FRET ratios of purified Pi sensors. A, Schematic of Pi sensor indicating high FRET in absence of Pi and reduced FRET when bound to Pi. Acceptor is eYFP for FLIPPi sensors and cpVenus for cpFLIPPi sensors. PiBP, Pi binding protein. B, Values shown are means ± SE for three independent protein preparations. Error bars smaller than the symbols are not shown.
FRET
acceptor eCFP
PiBP
Pi acceptor eCFP
PiBP A
0.5
1
1.5
2
2.5
0 4 8 12 16 20
FLIPPi-200µ
cpFLIPPi-200µ
FRET
ratio
(cpV
enus
/eC
FP)
Pi (mM)
B
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Figure 2. Effect of pH on Pi-dependent FRET responses. Pi binding of cpFLIPPi-6.4m was tested in pseudocytosol medium adjusted to pH 7.0, 7.5 and 8.0, and Pi was adjusted to the same pH. Binding affinity (Kd) values were estimated from nonlinear regression analysis. Plotted values are means ± SE for three independent protein preparations. Error bars smaller than the symbols are not shown.
1.2
1.4
1.6
1.8
2.0
0 5 10 15 20
FRET
ratio
(cpV
enus
/eC
FP)
Pi (mM)
Kd (mM)
pH 7.0 4.9 ± 0.3
pH 7.5 6.4 ± 0.4
pH 8.0 9.6 ± 0.5
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Figure 3. Differential targeting of cpFLIPPi-6.4m. Protoplasts were isolated from leaves of stably transformed Arabidopsis plants expressing cpFLIPPi-6.4m with or without an N-terminal chloroplast transit peptide. Confocal microscopy was conducted using settings for cpVenus excitation and emission (green) and chlorophyll autofluorescence (magenta). Scale bar, 10 µm.
Plas
tid-lo
caliz
ed
cpFL
IPPi
-6.4
m
cpVenus Chlorophyll Merged
Cyt
osol
-loca
lized
cp
FLIP
Pi-6
.4m
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Pi starved 24 hr Control 1.0 1.2 1.4 1.6 1.8 2.0 2.2
FRET
ratio
(cpV
enus
/eC
FP)
Figure 4. Live imaging of cytosolic Pi in root epidermal cells. A, Representative FRET ratio images of cpFLIPPi-6.4m stably expressed in seedlings with sufficient Pi (control, left), and after 24 hr Pi starvation (right). The same color scale was applied to ratio values for both images. Size bar, 10 µm. B, Plotted FRET ratio values are means ± SE for six independent seedlings. The difference between the two conditions is significant, p<0.05, Student’s t-test.
B
Pi starved 24 hr Control A
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Figure 5. Replenishment of cytosolic Pi after 24 hr starvation. Seedlings expressing cpFLIPPi-6.4m were grown with sufficient Pi (0.5 mM, +Pi), starved for Pi for 24 hr (-Pi) then Pi was added to 0.5 mM and FRET ratios were monitored over time. The plotted values are means ± SE for six independent seedlings. The first time point shown after replenishment is 1 hr. * indicates significant difference (p<0.05, Student’s t-test) from the -Pi treatment.
1
1.2
1.4
1.6
1.8
2.0
2.2
+Pi -Pi 4 8 12 16 20 24
FRET
ratio
(cpV
enus
/eC
FP)
Hours after Pi replenishment
* *
* *
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Figure 6. Effect of Pi supply on cytosolic contents of root epidermal cells. Seedlings expressing cpFLIPPi-6.4m were grown with a sufficient supply of Pi (500 µM) then transferred to medium containing the same or less Pi as indicated and grown for an additional 24 hr before imaging. Plotted FRET ratio values are means ± SE for six independent seedlings. * indicates significant difference (p<0.05, Student’s t-test) from the 500 µM treatment.
1.0
1.2
1.4
1.6
1.8
2.0
2.2
0 10 25 50 100 500
FRET
ratio
(cpV
enus
/eC
FP)
Pi in growth medium (µM)
* *
*
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WT pht4;2 A
0.0
0.5
1.0
1.5
2.0
2.5
WT pht4;2
FRET
ratio
(cpV
enus
/eC
FP)
B
Figure 7. Live imaging of Pi in plastids of root epidermal cells. A, Representative FRET ratio images for cpFLIPPi-6.4m stably expressed in wild-type and pht4;2 seedlings. The images were merged with the corresponding DIC images to distinguish cell boundaries. The same color scale was applied to ratio values for both images. Size bar, 5 µm. B, Plotted FRET ratio values are means ± SE for six independent seedlings. The difference between the two genotypes is significant, p<0.05, Student’s t-test.
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