Abscisic acid dynamics in roots detected with genetically encoded FRET sensors
- Carnegie Institution for Science, United States
- Universität Heidelberg, Germany
Figures
Figure 1 with 1 supplement
ABA responses of potential FRET sensors expressed in yeast and tested in yeast cell lysates or as purified proteins.
(A) Diagram of cloning strategy based on pDR FLIP Destination vectors encoding FRET fluorescent protein pairs and PAS Entry clones encoding sPAS or dPAS ABA sensory domains. Also shown is an example …
Figure 1—figure supplement 1
Fluorescence emission curves and ABA response of potential FRET sensors expressed in yeast and tested in yeast cell lysates or as purified proteins (excitation wavelength = 428 nm).
(A) Sensor designs with single Potential ABA Sensory domains (sPAS) and double Potential ABA Sensory domains (dPAS) combined with one fluorophore pair (from pDR FLIP39). (B) dPAS sensor designs …
Figure 2
Fluorescence emission spectra of two sensors and individual fluorescent proteins expressed in yeast strain 23344C and protease deficient yeast strain BJ5465 (excitation wavelength = 428 nm).
Proteins were analyzed in washed cells and in the supernatant or pellet of cell lysates.
Figure 3
ABACUS1 design and fluorescence response to ABA and related compounds.
(A) Hypothetical model of ABACUS1 bound to ABA. The structure shown for dPAS110 is derived from a crystal structure of ABA bound to PYL1 and ABI1 (PDB: 3JRQ) (Miyazono et al., 2009), and the …
Figure 4
ABA response titration curves for wild-type ABACUS1 (ABACUS1-80µ) and four different point mutants.
DxAm/DxDm values for purified sensor samples plus ABA were normalized to corresponding mock treated samples. Kd values were determined using Prism software (GraphPad) and are calculated based on the …
Figure 5
ABA import by AIT1/NRT1.2 in protease deficient yeast expressing ABACUS1 variants.
ABA was added to yeast cells suspended in 20 mM MES buffer (pH4.7) and ABACUS1 fluorescence was measured after 5 min. DxAm/DxDm values for yeast samples plus ABA were normalized to corresponding …
Figure 6 with 1 supplement
Expression pattern and ABA responses of ABACUS1 in Arabidopsis roots.
(A) Expression pattern and cytosolic localization of ABACUS1-2µ fluorescence (AxAm) in root cells in the root tip (left), in the elongation zone (center), and in the differentiation zone (right). (B)…
Figure 6—figure supplement 1
ABA titrations of ABACUS1 roots from Figure 6B.
Traces show FRET (DxAm), CFP (DxDm), and YFP (AxAm) fluorescence intensities of ABACUS1-2µ, ABACUS1-80µ and ABACUS1-nr roots in response to six 15-min pulses with increasing concentration of …
Figure 7 with 1 supplement
ABA responses and plant growth of ABACUS1 plant lines (homozygous, T3 generation).
(A) Fluorescence response of ABACUS1 in roots to 5 µM (±)-ABA. All lines were imaged simultaneously while growing in the same RootChip16. Because ABACUS1 is stereospecific for (+)-ABA, the effective …
Figure 7—figure supplement 1
Expression and Plant growth of ABACUS1 plant lines (homozygous T3 generation).
(A) Top: 10 day old seedlings grown vertically on ½ × MS medium agar plates. Bottom: Fluorescence image of the same seedlings. (B) 3 week old ABACUS1 plant lines (homozygous T3) grown on soil in a …
Figure 8
Architecture of the RootChip16.
(A) Photograph of the RootChip16 with control layer microchannels and micromechanical valves stained in blue, flow layer with observation chambers in green, and 5-days old seedlings growing on …
Figure 9
Dynamics of ABA uptake and elimination in roots as measured by ABACUS1-2µ.
(A) Spatial differences in the dynamics of ABA response. Traces showing ratio (DxAm/DxDm) changes in different root zones of an ABACUS1-2µ expressing root in response to three 15-min pulses of 5 µM …
Figure 10
Pattern of ribosome-associated transcripts for AIT1 and ABCG40 transporter genes.
Data are derived from microarray studies of RNA bound to polysomes (http://efp.ucr.edu/) (Mustroph et al., 2009). Cell-type-specific expression is based on coexpression with any of the six genes …
Figure 11
ABACUS1 response to salt and ABA treatment of detached leaves.
DxAm/DxDm ratios for Arabidopsis leaves after 24 hr of suspension of the petiole in ¼ × MS medium or the same medium supplemented with 150 mM NaCl or 100 µM (±)-ABA. Average ratio are shown for (A) …
Figure 12
ABA response of nuclear localized ABACUS.
(A–F) Confocal scanning microscopy images of the roots (A and D), hypocotyl (B and E) and region around the shoot apical meristem (C and F) of seedlings expressing either the cytosolic (A–C) or the …
Videos
Video 1
The video shows an ABACUS1-2µ Arabidopsis root, growing in a RootChip16, exposed to pulses of increasing concentrations of ABA.
The RATIO and YFP videos show the root throughout the experiment whereas the central charts show the average ratio (in magenta) and intensity (in yellow) of three regions (ROIs shown as overlaid on …
Tables
Table 1
Guide to sensor component variants used for screening (for details see Supplementary file 1).
| Destination vector N-term | Gateway site | dPAS Entry clone | Gateway site | Destination vector C-term | |||
|---|---|---|---|---|---|---|---|
| pDR FLIP | FRET acceptor | N-term Linker | dPAS N-term | dPAS Linker | dPAS C-term | C-term Linker | FRET donor |
| 30 | Aphrodite.t9 | attB1 invariant | HAB1 · ABI1aid · ABI1cd · PYR1 · PYL1 · PYL4 · PYL5 · PYL6 · PYL7 · PYL8 · PYL9 · PYL10 | L12 Flexible · L52 Spring · L65 Flexible · L71 α-helix · L118 Spring & α-helix | HAB1 · ABI1aid · ABI1cd · PYR1 · PYL1 · PYL4 · PYL5 · PYL6 · PYL7 · PYL8 · PYL9 · PYL10 | attB2 invariant | mCerulean |
| 32 | Aphrodite.t9 | t7.eCFP.t9 | |||||
| 34 | Aphrodite.t9 | t7.TFP.t9 | |||||
| 35 | Aphrodite.t9 | mTFP.t9 | |||||
| 36 | Aphrodite.t9 | Cerulean | |||||
| 37 | Citrine | Cerulean | |||||
| 38 | edCitrine | edCerulean | |||||
| 39 | edAphrodite.t9 | t7.ed.eCFP.t9 | |||||
| 42 | Citrine | mCerulean | |||||
| 43 | edAphrodite.t9 | edCerulean | |||||
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Abbreviations: t7, t9: 7 or 9 amino acid terminal truncations; aid: ABA interaction domain; cd: catalytic domain; ed, m: enhanced dimerization or monomeric variant
Additional files
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Supplementary file 1
Details for pFLIP Destination vectors, PAS Entry clones, L linkers, primers, and ABACUS plant lines.
- https://doi.org/10.7554/eLife.01741.020
