FRET kinase sensor development reveals SnRK2/OST1 activation by ABA but not by MeJA and high CO2 during stomatal closure
- Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, United States
- Institute of Technology, University of Tartu, Estonia
- Horticulture Department, University of Wisconsin-Madison, United States
Figures
Figure 1 with 1 supplement
Structure and in vitro testing of SnRK2 protein kinase reporter, SNACS, and SnRK2 protein kinase activity.
(A) Domain structure of the SNACS protein: YPet and Turquoise GL are yellow and cyan fluorescent proteins. The full coding region of the phosphoserine/threonine binding 14-3-3 GF14phi protein …
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Figure 1—source data 1
Uncropped gel images for Figure 1C.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig1-data1-v2.docx
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Figure 1—source data 2
In vitro SNACS FRET ratio values in Figure 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
In vitro FRET assay of SNACS reporter.
The SNACS protein or the control SNACSS785A isoform was incubated in vitro with GST-OST1, GST-OST1D140A (inactive OST1), GST-CPK6 or GST-SnRK2.3 for 2 hr, respectively. The ratio was calculated from …
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Figure 1—figure supplement 1—source data 1
In vitro SNACS FRET ratio values in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Abscisic acid induces increases in the ratio of YPet to Turquoise GL fluorescence emission of the SNACS reporter in N. benthamiana.
(A) Time-resolved FRET ratio changes in response to ABA. SNACS was co-infiltrated with pUBQ10:OST1-HF in N. benthamiana. Leaf epidermises were perfused with assay buffer (5 mM KCl, 50 µM CaCl2, 10 …
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Figure 2—source data 1
SNACS FRET ratio values from each stomate in Figure 2.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Ratiometric image of 535/480 nm wavelength emissions show fluorescence in SNACS-expressing Arabidopsis epidermal cells (pseudo-colored fluorescence ratio scale is shown on the right, non-normalized).
Bar = 20 µm.
Figure 3 with 1 supplement
In vivo response of SNACS reporter of SnRK2 activity in response to ABA in Arabidopsis guard cells.
(A) Time-resolved FRET ratio changes in response to ABA (20 µM). FRET efficiency changes were recorded by measuring the ratio of fluorescence emissions at 535 nm/480 nm with an excitation wavelength …
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Figure 3—source data 1
SNACS FRET ratio values from each stomate in Figure 3.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
SNACS fluorescence appears to be observed mainly in the cytoplasm, with possible low expression in the nucleus or fluorescence may ‘bleed’ from the cytoplasm towards the nuclear region.
Stomata imaged at the SNACS YPet emission wavelength (535 nm; scale bar = 20 µm).
Figure 4 with 2 supplements
The amino acid AKS1 Ser-30 in the SNACS reporter is necessary for ABA-induced ratio increases in Arabidopsis guard cells.
Leaf epidermises from transgenic lines expressing SNACS were used for depicting the ratio of YPet to Turquoise GL emission produced by exciting Turquoise GL with 434 nm light. (A) ABA (20 µM) …
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Figure 4—source data 1
SNACS FRET ratio values from each stomate in Figure 4.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
EtOH controls for SNACSS785A in Col-0 and in pUBQ10:OST1-HF expressed in the ost1-3 genetic background.
The response of the SNACSS785A to EtOH (0.02%) treatment in Col-0 (A) and in pUBQ10:OST1-HF expressed in the ost1-3 genetic background (B). The ratio of YPet to Turquoise GL emission was normalized …
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Figure 4—figure supplement 1—source data 1
SNACS FRET ratio values from each stomate in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Combined and averages of single stomate imaging data from experimental sets including experiments in Figure 4.
Red lines show the average ratio changes of all stomata traces. Grey lines denote each single stomate ratio change. For visualization of individual stomate time courses see colored traces in Figure 4…
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Figure 4—figure supplement 2—source data 1
SNACS FRET ratio values from each stomate in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig4-figsupp2-data1-v2.xlsx
Figure 5 with 1 supplement
SnRK2 activity is needed in guard cells for ABA-induced increases in the FRET ratio of SNACS.
Leaf epidermises from transgenic lines expressing SNACS were used for analyzing the ratio of YPet to Turquoise GL emission in guard cells produced by exciting Turquoise GL with 434 nm light. The …
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Figure 5—source data 1
SNACS FRET ratio values from each stomate in Figure 5.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Combined and averages of single stomate imaging data from experimental sets shown in Figure 5.
Red lines show the average ratio changes of all stomata traces. Grey lines denote each single stomate ratio change. For visualization of individual stomate time courses see colored traces in Figure 5…
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Figure 5—figure supplement 1—source data 1
SNACS FRET ratio values from each stomate in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 2 supplements
Effects of kinase inhibitors on SNACS fluorescence emission ratios in guard cells.
SNACS responses in guard cells were analyzed in the pUBQ10:OST1-HF-expressed in the ost1-3 genetic background. The ratio of YPet to Turquoise GL emission was normalized to the average value over the …
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Figure 6—source data 1
SNACS FRET ratio values from each stomate in Figure 6.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
W-7 does not have a clear effect on ABA-induced OST1/SnRK2.6 activation in plant cells.
Mesophyll cell protoplasts prepared from stable transgenic UBQ10:OST1-HF/ost1-3 Arabidopsis plants were incubated with 10 µM K252a, 50 µM W-7, or DMSO (control) for 20 min. Then, protoplasts were …
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Figure 6—figure supplement 1—source data 1
Uncropped gel images for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig6-figsupp1-data1-v2.docx
Figure 6—figure supplement 2
Combined and averages of single stomate imaging data from experimental sets including experiments in Figure 6.
Red lines show the average ratio changes of all stomata traces. Grey lines denote each single stomate ratio change. For visualization of individual stomate time courses see colored traces in Figure 6…
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Figure 6—figure supplement 2—source data 1
SNACS FRET ratio values from each stomate in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig6-figsupp2-data1-v2.xlsx
Figure 7 with 1 supplement
MeJA did not induce consistent FRET ratio changes of the SNACS SnRK2 activity reporter in guard cells under the imposed conditions.
The SNACS reporter was analyzed in guard cells in leaf epidermises of plants expressing pUBQ10:OST1-HF in the ost1-3 genetic background. The ratio of YPet to Turquoise GL emission was normalized to …
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Figure 7—source data 1
SNACS FRET ratio values from each stomate in Figure 7.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Combined and averages of single stomate imaging data from experimental sets including experiments in Figure 7.
Red lines show the average ratio changes of all stomata traces. Grey lines denote each single stomate ratio change. For visualization of individual stomate time courses see colored traces in Figure 7…
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Figure 7—figure supplement 1—source data 1
SNACS FRET ratio values from each stomate in Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig7-figsupp1-data1-v2.xlsx
Figure 8 with 1 supplement
CO2 elevation neither induces SNACS FRET ratio increases nor affects ABA concentration in guard cells.
Fluorescence ratios of guard cells in intact leaf epidermises were analyzed in the pUBQ10:OST1-HF expressed in the ost1-3 genetic background (A and B). The ratio of YPet to Turquoise GL emission was …
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Figure 8—source data 1
SNACS FRET ratio values from each stomate in Figure 8.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Combined and averages of single stomate imaging data from experimental sets including experiments in Figure 8.
Red lines show the average ratio changes of all stomata traces. Grey lines denote each single stomate ratio change. For visualization of individual stomate time courses see colored traces in Figure 8…
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Figure 8—figure supplement 1—source data 1
SNACS FRET ratio values from each stomate shown in Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig8-figsupp1-data1-v2.xlsx
Figure 9 with 3 supplements
CO2 elevation causes robust stomatal closing responses in two ABA receptor quintuple mutant and in ABA receptor sextuple mutant plants.
(A, B) Time-resolved stomatal conductances of PYR/RCAR receptor quintuple (pyl-11458 and pyl-12458) and sextuple (pyl-112458) mutants. Air CO2 concentration was increased from 400 ppm to 800 ppm at …
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Figure 9—source data 1
Stomatal conductance values of individual plants and half response times.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig9-data1-v2.xlsx
Figure 9—figure supplement 1
PYR/RCAR ABA receptor pyr1 pyl2/4/5/8 quintuple mutant (pyl-12458) and pyr1 pyl1/2/4/5/8 (pyl-112458) sextuple mutant show larger absolute stomatal closing responses during the first 20 min under elevated CO2 compared to wild-type (Col-0) (light blue bars).
Changes in stomatal conductance (gs) after the first 20 min CO2 elevation (gs20) in absolute units (left Y axis scale mmol m−2 s−1) and relative units (right Y axis scale in %: (gs0-gs20)/gs0). gs0 =…
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Figure 9—figure supplement 1—source data 1
Absolute and relative changes in stomatal conductance values used in Figure 9—figure supplement 1.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig9-figsupp1-data1-v2.xlsx
Figure 9—figure supplement 2
Stomata of ABA receptor quintuple mutant (pyr1/pyl1/pyl4/pyl5/pyl8) and guard cell-targeted (B) PYL1 and (C) PYL4 ABA receptor complemented plants show stomatal CO2 responses similar to wild-type (Col-0) controls.
Time-resolved stomatal conductance responses to changes in light and CO2 concentration in Col-0 (WT; A–D). (A) pyr1/pyl1/pyl4/pyl5/pyl8 quintuple mutant (pyl-11458). (B) pyr1/pyl1/pyl4/pyl5/pyl8/pGC1…
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Figure 9—figure supplement 2—source data 1
Stomatal conductance values of individual plants used in Figure 9—figure supplement 2.
- https://cdn.elifesciences.org/articles/56351/elife-56351-fig9-figsupp2-data1-v2.xlsx
Figure 9—figure supplement 3
Genotyping of ABA receptor multiple mutants and guard cell-targeted ABA receptor complemented plants.
(A) Col-0 (WT) and ABA receptor higher-order mutants were confirmed by primers targeting WT or mutant alleles. Primer pairs: PYR1 (PYR1-F + PYR1-R), PYL1 (PYL1-F + PYL1-R), PYL1 mutant (PYL1-F + …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis) | AKS1 | Tair (https://www.arabidopsis.org/) | Tair ID: At1g51140 | |
| Gene (Arabidopsis) | 14-3-3, GF14phi | Tair (https://www.arabidopsis.org/) | Tair ID: At1g35160 | |
| Strain, strain background (Escherichia coli) | BL21-CodonPlus (DE3) | Agilent Technologies | Model: 230245 | Electro-competent cells |
| Strain, strain background (Agrobacterium tumefaciens) | GV3101 | Other | Widely distributed | |
| Antibody | Anti-FLAG M2 Mouse monoclonal | Sigma-Aldrich | RRID:AB_262044 | x5,000 |
| Chemical compound, drug | K-252a | Sigma-Aldrich | Cas No. 99533-80-9 | |
| Chemical compound, drug | W-7 | Sigma-Aldrich | Cas No. 61714-27-0 | |
| Chemical compound, drug | MeJA | Bedoukian Research, Inc | Ct. 06810–4192 | |
| Software, algorithm | MetaFluor software | MetaFluor(https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy) | RRID:SCR_014294 | version 7.0r3 |
| Software, algorithm | Fiji software | Fiji (https://imagej.net/Fiji) | RRID:SCR_002285 | |
| Software, algorithm | GraphPad Prism software | GraphPad Prism (https://graphpad.com) | RRID:SCR_015807 | version 7.0 |
Additional files
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Supplementary file 1
Transgenic lines used in this study.
Detailed information on the transgenic lines is provided including the plasmid, promoter, and genetic background.
- https://cdn.elifesciences.org/articles/56351/elife-56351-supp1-v2.docx
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Supplementary file 2
Primer sequences for genotyping.
Primers used to genotype higher order ABA receptor mutants (Figure 9—figure supplement 3).
- https://cdn.elifesciences.org/articles/56351/elife-56351-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56351/elife-56351-transrepform-v2.pdf
