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 thepH was adjusted to 6 with TRIS base and 2-(N- morpholino) 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
taken into a electrode filling station and back-fill the electrodes with appropriate back- filling solution using the syringe and nylon needle. Then, the electrode’s tips were front- filled 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.
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-bromo- cyclic 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
Coulter 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% SDS-
polyacrylamide 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
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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2. Ordonez NM, Shabala L, Gehring C & Shabala S (2013) Noninvasive microelectrode ion flux estimation technique (MIFE) for the study of the
regulation of root membrane transport by cyclic nucleotides. Meth Mol Biol 1016, 95-106.
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