Supplementary Materials and Methods
Plant growth and treatment
Arabidopsis thaliana, ecotype Col-0, seeds were sterilized distributed over Petri dishes
containing sterile MS medium pH 6. 2. The plates were put at 4°C for two days for seeds
stratification. Afterwards, they were placed into a growth room and positioned vertically
to allow the roots to grow outside of the medium. 6-days old seedlings were pre-treated
for 1 or 24 hours with one of the following solutions diluted in Basic measuring solution
(BSM): Adenosine 3’5’- cyclic monophosphate sodium salt monohydrate (cAMP; 10µM),
guanosine 3’5’- cyclic monophosphate sodium salt (cGMP; 10µM), or the cell permeant
analogues 8-Br-cAMP (10µM) and 8-Br-cGMP (10µM). The BSM contained 0.1 mM
KCl and 0.2 mM CaCl2 and the pH was adjusted to 6 with TRIS base and 2-(Nmorpholino) ethanesulfonic acid (MES). Seedlings were transferred into 55 mm x 15mm
Petri dishes with 4 mL of BSM containing the second messengers or only BSM for the
controls. The roots are completely immersed into solution and aerial parts of the
seedlings were supported with a plastic mesh.
Measurement of ion fluxes with the Non-invasive microelectrode ion flux estimation
technique (MIFE)
Root ion fluxes of protons H+, Potassium K+ and Calcium Ca2+ were measured using
MIFE system (University of Tasmania) a technique that is further detailed elsewhere [1,
2].
Electrode fabrication
Non-filamentous borosilicate glass capillaries (1.5 mm O.D. x 0.86 mm) were introduced
into a vertical pipette puller and pulled out with a diameter of ca. 2 µm. The pulled
electrodes were stored in an aluminium covered rack in a vertical position before
silanising. For silanising, the electrode blanks were placed uncovered in a rack with tips
upright and base down and keep in the oven set at 250 ºC overnight. Then a steel cover
was placed over the electrode blanks and 65 µL of tributylchlorosilane added on the rack
under the cover using a micropipette. Ten minutes later, the lid was removed and the
electrodes baked for further 30 minutes. The cooled silanised blank electrodes where
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taken into a electrode filling station and back-fill the electrodes with appropriate backfilling solution using the syringe and nylon needle. Then, the electrode’s tips were frontfilled with different Liquid ion exchangers (LIX) (Fluka of Sigma-Aldrich) by being put
briefly into contact with the LIX-containing tube to achieve the column length of ~ 100
µm. The electrodes were labeled and placed into BSM. The reference electrode was made
by galvanizing a silver wire in a 0.25 N HCl solution for 15 minutes. Then a glass
capillary was filled with 1M KCl in 2% Agar, the silver wire was placed into the
capillary and sealed with Parafilm.
Calibration of the electrodes
Three electrodes were mounted in the MIFE holder and connected to the reference
electrode. The electrodes were immersed in BSM. The MIFE CHART software was used
to run the calibration. Three calibrations standards for the same ion were used: pH (5, 6,
7), KCl (0.05 mM, 0.1 mM, 0.2 mM) and CaCl2 (0.1 mM, 0.2 mM, 0.4 mM). After
running the average routine, the equation that relates electric potential (mV) and ion
concentration (mM) was calculated and the values stored in an AVC file.
Sample preparation
To immobilize the sample custom-made 4 mL rectangular chamber and specimen holder
were used. The seedling roots were placed on the middle of the holder and wrapped with
Parafilm. The immobilized seedling placed into the measuring chamber that was
subsequently filled with BSM solution containing the desired concentration of cyclic
nucleotides. The chamber stayed under dim green light for one hour.
Determination of changes in ion fluxes with MIFE
The measuring chamber was mounted in the MIFE multi holder. The three
microelectrodes were positioned in the same plane using a microscope and 3D
micromanipulators. Then, the tips were taken 40 µm above the root surface. The MIFE
software was used to record ion fluxes. A stepper motor was used to change electrode’s
position each 5 seconds. The ion fluxes were measured for 5 to 10 minutes until they
reach steady-state. Stress solutions were added to the chamber, and mixed gently with a 5
mL pipette. The response was recorded for 30 minutes.
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Stress treatments
The response of pre-treated and control plants in response to stress solutions was tested.
The following solutions were prepared in BSM: Salt stress was imposed with NaCl at 200
mM, oxidative stress with Hydrogen peroxide (H2O2) at 5mM and a mixture of Sodium
Ascorbate 1mM and Copper chloride (CuCl2) 0.3 mM.
Data analysis
The MIFEFLUX program to calculate the ion fluxed based on the calibration and flux
recordings files. The FLX files were opened and analyzed with Excel. The net ion fluxes
before and after stress and peak values of 6-8 independent samples were averaged and
compared by T-Student test.
Arabidopsis cell suspension culture
Cells derived from roots of Arabidopsis thaliana (ecotype Columbia 0) were prepared as
described elsewhere [3]. Briefly, cells were grown in 100 mL of Gamborg’s B5 [4] basal
salt mixture (Sigma-Aldrich, St Louis, MO) with 2,4-dichlorophenoxyacetic acid (2,4-D;
1 mg mL-1) and kinetin (0.05 μg mL-1) in 250 mL sterile flask. Cells were grown in a
growth chamber (Innova® 43, New Brunswick Scientific Co., NJ) with shaking at 120
rpm, and subcultured every 10 days. Photosynthetic light of the growth chamber was set
for 12 h light/12 h dark cycles at 23°C. Cells were then treated with 10 μM of 8-bromocyclic guanosine monophosphate (8-Br-cGMP) and four biological replicates were
collected at 0 (untreated), 60 min (untreated) and 60 min post-treatment. Treated and
untreated cells were harvested by draining off the media using Stericup® filter unit
(Millipore, Billerica, MA), immediately flash frozen in liquid nitrogen and stored at 140°C until further use.
Microsomal protein extraction
Approximately 1 g of cells was ground to a fine powder in liquid nitrogen and subjected
to microsomal extraction following protocol described elsewhere [5]. The powder was
incubated in a sucrose buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 2 mM DTT, 0.25 M
sucrose and 1 × protease inhibitor cocktail tablet) and centrifuged at 8’000 x g for 15 min.
The supernatant was then subjected to ultracentrifugation using the Beckman Coulter
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Optima L-100K ultracentrifuge (CA, USA) at 100’000 x g for 1 h. The supernatant
(cytosolic fraction) was pipetted out leaving the pellet (microsomal fraction) untouched
in the centrifuge tube. The pellet was washed once in sucrose buffer and centrifuge
further at 100’000 x g for 1 h. The final pellet was suspended in sucrose buffer and the
microsomal fraction corresponding to the pellet was aliquoted into separate 1.5 mL
microcentrifuge tubes and either used immediately or stored at -80°C.
In parallel, approximately 50 mg of cells were weighed into a 2.0 mL microcentrifuge
tube and subjected to hard tissue membrane protein extraction using the Mem-PER
eukaryotic membrane protein extraction reagent kit (Pierce, Thermo Scientific, IL, USA).
Protein extraction was performed according to the manufacturer’s recommendations
except that buffer volumes were adjusted according to the quantity of starting material.
One-dimensional polyacrylamide gel electrophoresis (1-DE) and in-gel trypsin digestion
Approximately 15 μg of the microsomal protein extract was reduced with 4 × SDS buffer
containing 0.002% (w/v) bromophenol blue and electrophoresed in 12% SDSpolyacrylamide gel at 100 V for 30 min. The gel was stained with Coomassie brilliant
blue and destained until a protein versus background ratio appropriate for visualization
was obtained. Gel bands were assessed for equal loading and each gel lane was entirely
cut and divided into four fractions. Protein band sections were then subjected to in gel
trypsin digestion prior to LC-MS/MS analyses.
Protein identification by LTQ-Orbitrap
Digested peptides were resuspended in 5% (v/v) ACN and 0.1% (v/v) formic acid (FA),
and analyzed on an LTQ Orbitrap mass spectrometer (Thermo-Scientific, Bremen,
Germany), operated as described elsewhere [6].
All spectra were submitted to a local MASCOT (Matrix Science, London, UK) server
and searched against Arabidopsis thaliana in the TAIR database (release 10), with a
precursor mass tolerance of 10 ppm, a fragment ion mass tolerance of ± 0.5 Da, and strict
trypsin specificity allowing up to one missed cleavage, carbamidomethyl modification on
cysteine residues as a fixed modification. Proteins were considered positive if Mascot
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score was ≤ 95% confidence limit corresponding to a score ≥ 26. Data was further
analyzed with Scaffold (Oregon, USA) version 4.0.4 allowing for a 0.1% false
discovery rate (FDR) confidence threshold. Further, Scaffold was used for quantitative
analysis of the identified proteins from the treated samples against the control. Fold
changes were calculated based on normalized values of the total unique spectral counts.
Proteins with a >1.5-fold change (p  0.05) were considered as significantly changing.
The significant differences of proteins between the samples were manually assessed by
checking the matched peptide(s) and replication level across samples.
All proteomics methods are extensively detailed elsewhere [6-8].
Functional enrichment analysis
All identified differentially regulated proteins were considered for gene ontology (GO)
enrichment analysis using Babelomics 4.2 (Fatigo+, http://babelomics4.bioinfo.cipf.es/,
September 2013). More detailed protocols for the bioinformatics analyses are to be found
elsewhere [9-11].
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