1. Cell Biology
  2. Plant Biology
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FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis

  1. Rainer Waadt
  2. Kenichi Hitomi
  3. Noriyuki Nishimura
  4. Chiharu Hitomi
  5. Stephen R Adams
  6. Elizabeth D Getzoff
  7. Julian I Schroeder  Is a corresponding author
  1. University of California, San Diego, United States
  2. The Scripps Research Institute, United States
Research Article
Cite this article as: eLife 2014;3:e01739 doi: 10.7554/eLife.01739
9 figures, 1 video, 1 table, 4 data sets and 1 additional file

Figures

Figure 1 with 1 supplement
In vitro characterization of ABAleons.

(A) mTurquoise (cyan) is fused through a GP-linker to PYR1 (gold), which is separated by a flexible ASGGSGGTS(GGGGS)4 linker from ΔNABI1 (green) fused to cpVenus173 (yellow) through a PG linker. Structural features of the PYR1-ΔNABI1 complex including ABA (blue and red balls), ABI1 D413 (purple ball) and loops controlling access to the ABA binding site are highlighted. Dashed lines indicate linkers and unresolved structures. (B) Without ABA, ABAleon flexibility enables FRET from mTurquoise (mT) to cpVenus173 (cpV173). ABA triggered PYR1-ΔNABI1 binding increases the distance or orientation between the fluorescent probes, thereby reducing FRET efficiency. (C and D) Normalized (nu) emission spectra of (C) ABAleon1.1 and (D) ABAleon2.1 in absence (unbound) and in presence (bound) of ABA with indicated dynamic range (DR). (E) Emission ratios and (F) ΔR/ΔRmax plotted against increasing [ABA], with indicated ABA affinity K′d of each ABAleon calculated from the respective plot. (G) Time-dependent normalized emission ratios of ABAleon2.1 in response to 0 and 1 µM ABA. (H) Phosphatase activity assays of equimolar PYR1 and ΔNABI1 combinations and indicated ABAleons in presence of 0 and 5 µM ABA (mean ± SD, n = 4).

https://doi.org/10.7554/eLife.01739.003
Figure 1—figure supplement 1
ABA does not affect ABAleon absorbance and ABAleon emission is stable at physiological pH conditions.

(A) Normalized absorbance spectrum of ABAleon2.1 in absence (cyan) and in presence of 100 µM ABA (yellow). (B) pH titration of ABAleon1.1 emission ratios (left scale) in absence (cyan) and in presence of 10 µM ABA (yellow). Curves were fitted by a four parameter Hill equation and ratio change (red, right scale) was calculated by subtraction of the 10 µM ABA from the 0 µM ABA curve. Note the dramatic ABA-independent ratio change of ABAleon1.1 below pH 6.2. However in the physiological range (pH 7.2–7.8) ABAleon1.1 emission is stable.

https://doi.org/10.7554/eLife.01739.004
Figure 2 with 1 supplement
ABA-induced ABAleon2.1 responses in whole seedlings.

(A and B) Manually assembled ABAleon2.1 ratio images (A) before and (B) 2 h after application of 50 µM ABA. (C) Ratio image of untreated guard cells from lower epidermis of 33-day-old soil grown plants. Images were calibrated to the indicated calibration bar. Blue colors indicate low ABAleon2.1 emission ratios, corresponding to high ABA concentrations, and red colors indicate high ABAleon2.1 emission ratios corresponding to low ABA concentrations. Shown is a representative of four experiments.

https://doi.org/10.7554/eLife.01739.005
Figure 2—figure supplement 1
pRAB18-GFP expression in guard cells.

(AC) Confocal images of pRAB18-GFP expression in the intact lower epidermis of 27-day-old plants show highest expression (GFP emission) in guard cells. (A) Representative image of plants grown at 70 % relative humidity (RH) conditions, (B) 4 h after leaf floating in 50 µM ABA and (C) 2 days after plant transfer to 25 % RH conditions. Note that pRAB18-GFP expression appears in the epidermal cells only after ABA application (B). (D) Images were calibrated to background fluorescence (lowest value) and to the maximum value recorded in guard cells (highest value). (E) Quantified pRAB18-GFP emission in guard cells from the same analyses as in (AD) (means ± SEM, n = 3 with ≥ 34 guard cells/n).

https://doi.org/10.7554/eLife.01739.006
Figure 3 with 1 supplement
ABA-induced ABAleon2.1 responses in Arabidopsis tissues.

Time-resolved ABAleon2.1 responses to 10 µM ABA in (AC) guard cells of 45-day-old plants and (DF) the hypocotyl, (GI) the root differentiation-, (JL) maturation- and (MO) elongation-zone of 5-day-old seedlings. (A, D, G, J, M) Time course of mTurquoise (mT, solid lines) and cpVenus173 emission (cpV, dashed lines) and (B, E, H, K, N) the corresponding normalized emission ratios colored according to the analyzed regions boxed in the in initial t = 0 min images (C, F, I, L, O). Each analysis is a representative of 3–4 experiments. Note, that there is a slight sample drift, which causes cpVenus173 emission increases in (A).

https://doi.org/10.7554/eLife.01739.007
Figure 3—figure supplement 1
ABAleon2.1 but not the empty FRET cassette responds specifically to ABA.

Time-resolved responses of ABAleon2.1 in (AC) the hypocotyl and (DF) the root maturation zone, and of (GI) the empty FRET cassette in the hypocotyl. (A, D, G) Time course of mTurquoise (solid lines) and cpVenus173 emission (dashed lines) and (B, E, H) the respective normalized emission ratios colored according to the analyzed regions (blue, yellow and red boxes) given in the t = 0 min ratio images (C, F, I). (C, F, I) Emission ratio images from indicated time points after solvent control (0.05 % EtOH), buffer or ABA application calibrated to the scale given in the final ratio images. Shown are representative analyses of one to three experiments.

https://doi.org/10.7554/eLife.01739.008
Accelerated ABAleon2.1 responses in roots of the pyl4ple mutant.

Normalized 10 µM ABA-induced ABAleon2.1 emission ratio changes in the root maturation zone of Col-0 (A, C cyan line) and pyr1-1/pyl1-1/pyl2-1/pyl4-1 (pyl4ple) (B, C yellow line). (A and B) Data points from single measurements fitted by the respective four parameter logistic curve. (C) Combined data from four experiments in (A and B) fitted by the respective four parameter logistic curve. (D) t1/2 values (means ± SEM, n = 4) calculated from the fitted curves in (A and B).

https://doi.org/10.7554/eLife.01739.010
ABAleon2.1-expressing plants show an ABA hyposensitivity.

From left to right, Col-0 wild type, ABAleon2.1 (line 3), ABAleon2.1 (line 10), YFP-PYR1 and abi1-3/YFP-ABI1. (A) Analyses of cpVenus173/YFP fluorescence emission in the leaf epidermis. Numerical fluorescence intensity values in the images represent means ± SEM of n = 4 images. (B and C) 7-day-old seedlings germinated and grown on 0.5 MS media supplemented with (B) 0 and (C) 0.8 µM ABA. (D and E) 9-day-old seedlings 5 days after transfer to 0.5 MS media supplemented with (D) 0 and (E) 10 µM ABA. (FH) 7 day time course of (F and G) seed germination and (H) cotyledon expansion in presence of (F) 0 and (G and H) 0.8 µM ABA normalized to the seed count of each replicate (means ± SEM, n = 4 technical replicates with 49 seeds/n). (I) Fresh weight of seedlings from (D and E) normalized to the 0 µM ABA control conditions (means ± SEM, n = 5 technical replicates with seven seedlings/n). (J) Stomatal aperture of 20-23-day old seedlings exposed to 10 µM ABA normalized to the 0 µM ABA control conditions (means ± SEM, n = 3 with ≥ 24 stomata/n).

https://doi.org/10.7554/eLife.01739.011
Figure 6 with 1 supplement
Visualization of long-distance ABA transport.

(A) ABAleon2.1 seedlings were transferred to microscope dishes, which were divided into two isolated experimental chambers by a horizontal block of modeling clay. (B, E, F) Shoot-to-root, (C, G, H) hypocotyl-to-root and (D, I, J) root-to-hypocotyl ABA transport after application of 50 µM ABA. (BD) Time-dependent normalized emission ratios (means ± SEM, n = 3) in the hypocotyl (cyan) and root (yellow) were quantified in three regions indicated by boxes in the initial images (EJ). The calibration bar in the final t = 180 min image indicates the scale of the emission ratios. Decreasing ratios indicate ABA accumulation. Shown are representative analyses of 3–4 experiments.

https://doi.org/10.7554/eLife.01739.012
Figure 6—figure supplement 1
Solvent control for long-distance ABA transport.

(A) Time-dependent normalized ABAleon2.1 emission ratios (mean ± SEM, n = 3) in the hypocotyl (cyan) and root (yellow) in response to 0.05 % EtOH as solvent control for ABA were quantified in three regions indicated by boxes in the initial images of (B and C). The calibration bar in the t = 180 min image indicates the scale of the emission ratios. Shown are representative analyses of four experiments.

https://doi.org/10.7554/eLife.01739.013
Figure 7 with 2 supplements
In vitro analyses of ABAleon2.1 mutants.

(A) Structural model of the PYR1(gold)-ABA(purple)-ABI1(green) complex, indicating mutations in ABAleon2.1 that were analyzed in (BG): H60P monomer-inducing, V83H in Pro-Cap and H115A in Leu-Lock of PYR1 (grey balls) and D413L phosphatase-inactivating in ABI1 (purple ball). Emission spectra of (B) ABAleon2.11, (D) ABAleon2.13 and (F) ABAleon2.15 in the absence (unbound) and presence of (+)-ABA (bound) with indicated dynamic range (DR). (B) ABAleon2.11 exhibited no clear response to ABA. (C) (+)-ABA titrations of ABAleon2.11 compared to ABAleon2.1 suggest saturation of ABAleon2.11 in the absence of ABA. (E and G) ΔR/ΔRmax plots of ABAleon titrations with (E) naturally occurring (+)-ABA and (G) its enantiomer (−)-ABA, which binds more weakly. The respective apparent ABA affinities (K′d) are indicated.

https://doi.org/10.7554/eLife.01739.014
Figure 7—figure supplement 1
(+)- and (−)-ABA titrations of selected ABAleons.

(A) Structural model of the PYR1(gold)-ABA(purple)-ABI1(green) complex with indicated mutations in PYR1 (grey balls) and ABI1 (purple balls) of ABAleon2.1 (ABI1D413L), ABAleon2.11 (PYR1H60P), ABAleon2.12 (PYR1F61L), ABAleon2.13 (PYR1V83H), ABAleon2.14 (PYR1L87F), ABAleon2.15 (PYR1H115A), ABAleon2.16 (PYR1E141Q) and ABAleon2.17 (ABI1E142Q). (B and E) Emission ratios, (C and F) ratio changes and (D and G) ΔR/ΔRmax of ABAleon1.1, ABAleon2.1, ABAleon2.13 and ABAleon2.15 plotted against (BD) increasing (+)-ABA or (EG) (−)-ABA concentrations. The color code and the respective mutations in the analyzed ABAleons are given above the graphs. Values in the graphs give (B and E) the dynamic range and (C and F) the apparent affinities (K′d) calculated form a four parameter logistic fit or (D and G) a three parameter sigmoidal Hill fit.

https://doi.org/10.7554/eLife.01739.015
Figure 7—figure supplement 2
Purification of ABAleons after expression in E. coli.

(AD) Purification of recombinant ABAleon2.1. (A) anti-GFP immuno-detection, (B) PageBlue stain of SDS-gel after blotting, (C) Instant Blue stain of indicated fractions after gel filtration (GF) run and (D) normalized absorbance at 280 nm (protein; cyan) and 516 nm (cpVenus173; yellow) measured during GF-run. Ex, extract; FT, flow through; W, wash; E, elution; W2, Amicon filter wash; E2, Amicon filter elution; GF, gel filtration with numbered fractions. (E) anti-GFP immuno-detection and (F) PageBlue stain of 1 µg empty FRET cassette (F3) and ABAleon proteins after purification.

https://doi.org/10.7554/eLife.01739.016
Figure 8 with 1 supplement
ABA-induced ABAleon2.1 (line 10), ABAleon2.13, ABAleon2.14 and ABAleon2.15 responses in the root maturation zone.

10 µM ABA-induced normalized emission ratio changes in the root maturation zone of (A, E dark blue line) ABAleon2.1 (line 10), (B, E cyan line) ABAleon2.13, (C, E yellow line) ABAleon2.14, and (D, E orange line) ABAleon2.15. (AD) Data points from single measurements fitted by the respective four parameter logistic curve. (E) Combined data from three to four experiments in (AD) fitted by the respective four parameter logistic curve. (F) t1/2 values (means ± SEM, n = 3–4) calculated from the fitted curves in (AD).

https://doi.org/10.7554/eLife.01739.018
Figure 8—figure supplement 1
ABA-induced ABAleon2.1 (line 10), ABAleon2.13, ABAleon2.14 and ABAleon2.15 responses in the root maturation zone (examples).

Responses to 10 µM ABA in the root maturation zone of (AC) ABAleon2.1 (line 10), (DF) ABAleon2.13, (GI) ABAleon2.14 and (JL) ABAleon2.15. (A, D, G, J) Time course of mTurquoise (solid lines) and cpVenus173 emission (dashed lines) and (B, E, H, K) the corresponding normalized emission ratios colored according to the analyzed regions boxed in the in initial t = 0 images (C, F, I, L). Each analysis is a representative of 3–4 experiments.

https://doi.org/10.7554/eLife.01739.019
ABAleon2.1 reports ABA concentration changes in response to low humidity, salt and osmotic stress.

ABAleon2.1 emission ratios in response to (A and B) low humidity and (CH) 4–6 h treatments with 0.01 % EtOH (control), 100 mM NaCl, 300 mM sorbitol and 10 µM ABA in (C and D) guard cells, (E and F) the root maturation- and (G and H) elongation zone. (A, C, E, G) Representative emission ratio images with indicated calibration bars. (B and D) Normalized emission ratios in guard cells (means ± SEM, n = 3 with ≥ 24 guard cell pairs/n). (F and H) Normalized emission ratios analyzed from two boxed regions (cyan and yellow) color-coded in the left images of (E and G) (means ± SEM, n = 8–10 seedlings).

https://doi.org/10.7554/eLife.01739.020

Videos

Video 1
10 µM ABA-induced ABAleon2.1 responses in Arabidopsis.

Video of 10 µM ABA-induced ABAleon2.1 responses in (A) guard cells, (B) the hypocotyl, (C) the root maturation- and (D) elongation-zone. ABA was applied at timepoint 00:00:00 of the indicated timescale. Single videos represent data of analyses in Figure 3. Emission ratio changes to blue color indicate ABA concentration increase. (B) Emission ratio changes in the hypocotyl propagate gradually from the hypocotyl base towards the shoot.

https://doi.org/10.7554/eLife.01739.009

Tables

Table 1

Biochemical properties of ABAleons

https://doi.org/10.7554/eLife.01739.017
ABAleonMutations/DeletionsRminRmaxDR [%]K’d (3 parameter Hill) [nM]K’d (4 parameter logistic) [nM]
empty FRETΔ(PYR1-ΔNABI1)2.502.51−0.69
ABAleon1.10.870.98−14.83266 ± 55332 ± 45
ABAleon2.1ΔNABI1D413L0.910.97−8.9879 ± 29114 ± 32
ABAleon2.2Δ[(GGGGS)3] linker0.981.04−8.14121 ± 38156 ± 47
ABAleon2.3Δ(GGSGGTS) linker0.940.99−7.5372 ± 1884 ± 22
ABAleon2.11PYR1H60P, ΔNABI1D413L0.910.91−2.39
ABAleon2.12PYR1F61L, ΔNABI1D413L1.051.12−8.2987 ± 20107 ± 22
ABAleon2.13PYR1V83H, ΔNABI1D413L0.910.97−7.098600 ± 71002900 ± 1500
ABAleon2.14PYR1L87F, ΔNABI1D413L0.890.91−2.801200 ± 1200
ABAleon2.15PYR1H115A, ΔNABI1D413L0.921.01−10.09488 ± 45510 ± 41
ABAleon2.16PYR1E141Q, ΔNABI1D413L0.971.02−7.30194 ± 46229 ± 48
ABAleon2.17ΔNABI1D413L,E142Q0.780.82−4.9035 ± 1048 ± 8
  1. Biochemical properties of the empty FRET cassette and ABAleons with indicated mutations or deletions compared to the wild type ABAleon1.1. Shown are minimum (Rmin) and maximum emission ratios (Rmax), the dynamic range (DR) calculated as RminRmaxRmin100 and the apparent ABA affinity (K′d) calculated from a three parameter Hill fit or a four parameter logistic fit.

Data availability

The following previously published data sets were used
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  2. 2
  3. 3
  4. 4

Additional files

Supplementary file 1

(A) Oligo-nucleotides used in this work. List of oligo-nucleotides with indicated Arabidopsis GeneBank ID (AGI) number of the gene or construct, the oligo-nucleotide name and 5′-3′-sequence, the restriction sites included in the oligo-nucleotide and the description for what it was used for. In the oligo-nucleotide sequences the restriction sites are indicated by italic letters and mutations or non gene-coding nucleotides are indicated by lower case letters. (B) Plasmids and constructs used and generated in this work. List of plasmids and constructs with indicated Arabidopsis GeneBank ID (AGI) number of the inserted gene or construct, the promoter included in the plasmid, the clone name, the restriction sites, which were used for cloning, the vector backbone of the clone with incorporated selection markers for bacteria and plants and the clone information. The clone information includes additional information about restriction sites, promoters, inserts and point mutations. Amp, ampicillin; Bar, BASTA; Hyg, hygromycin; Kan, kanamycin; XFP, fluorescent protein. (C) Transgenic Arabidopsis lines used and generated in this work. List of transgenic Arabidopsis thaliana plants with indicated ecotype, Arabidopsis GeneBank ID (AGI) of the mutated genes, gene names, mutant names and IDs, the name of the transgenic lines, the construct used for transformation, plant selection marker and description. Bar, BASTA; Hyg, hygromycin; Kan, kanamycin.

https://doi.org/10.7554/eLife.01739.021

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