M.9.1. Root imaging pH measurements
The method described in (Waadt et al. 2017) was used, with modifications. Arabidopsis thaliana Col-0 plants expressing the pHGFP fluorescent reporter were grown vertically in solid LAK 1 mM K pH 5.6 10 mM MES-Tris medium in a growth chamber (Conviron CMP 6010, Berlin, Germany). Three-day-old seedlings were transferred to microscope dishes (MatTek, Ashland, MA, USA) supplemented with LAK 1 mM K pH 5.6 10 mM MES-Tris and 0,7% low melting point agarose (Roth), and incubated vertically overnight in the growth chamber.
Before imaging, seedlings were placed horizontally and 90 μl of liquid LAK media 1 mM K pH 5.6 10 mM MES-Tris was added. Seedlings were gently pressed back into their agarose bed and incubated for 40–60 min for recovery. Imaging was performed with a Leica SP5II using a
×10 objective (HC PL Fluotar ×10/0.3 DRY). The wavelengths for reporter excitation were 405 and 488 nm, and emission was detected in a 500-550 range by HyD2 detector. Frames were acquired every 6 seconds during 40 minutes. After 4 minutes of imaging (stack 40) roots were treated with different treatments adding 10µl of the desire solution on the side of the root.
For image processing the following steps were conducted using Fiji (Schindelin et al. 2012):
background subtraction, gaussian blur, 32-bit conversion, threshold, ratio calculation and royal look up table. Image data were obtained from processed 32-bit images using Fiji. Entire images were quantified for presentations of global responses. Heat maps were generated by analyses of 64 adjacent regions of 16 × 178 pixels (24.2 × 268.2 μm). Normalized datasets (ΔR : R) were calculated as (R − R0)/R0, where R0 represent mean 4 min baseline values.
Normalized heat maps were obtained from registered movies and each region was normalized to its respective baseline. Graphs and heat maps were generated using OriginPro.
M.9.2. Seedlings pH measurements
Plants expressing the pHGFP fluorescent reporter were grown on LAK medium (pH 5.6 10 mM MES-KOH) as described in Section M.1.3.2. Three-day-old seedlings grown vertically in the growth chamber were transferred to new plate with the same media and supplemented with either KCl, NaCl or LiCl (in different concentrations) or no treatment (control plate), and grown for an additional 24 h. Microscopy analyses were performed using a Leica TCS LSI microscope equipped with a PLAN APO ×5.0 macro objective (Leica Microsystems, Wetzlar, Germany). pHGFP reporter was sequentially imaged with excitation wavelength of 405 nm
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and 488 nm, and emission wavelength of 500–550 nm. Seedlings were directly imaged from the plate without any manipulation. To image entire seedlings in x, y and z, z-stacks at multiple positions were acquired. Image processing and analysis was performed using Fiji (Schindelin et al., 2012). Fluorescence intensity values of z-stacks were summed up and individual tiles were stitched together using the Grid/collection stitching tool (Preibisch et al. 2009). After background subtractions, ratiometric image calculations proceeded as described by (Kardash et al. 2011).
M.9.3. BCECF to measure petals vacuolar pH
Vacuolar pH of Arabidopsis thaliana opened-flower petals was determined using the fluorescent cell-permeant dye BCECF AM (Molecular Probes). BCECF 2’,7’-Bis-(2-Carboxyethyl)-5-(and-6-)-Carboxyfluorescein Acetoxymethyl Ester is a pH sensitive dual-ratiometric dye that has been used to measure intracellular pH. The protocol used was based in Bassil et al. 2013 with modifications.
M.9.3.1. Loading of the dye.
Plants were grown as described in section M.3.2. At least 4 independent plants per genotype. When all plants presented completely opened flowers, petals were harvested and placed in a 96-well micro-plate: 8 petals per well, 6 wells per genotype. Loading of the dye was performed in liquid media containing 1/10 MS medium, 0.5% sucrose, and 10 mM Mes-KOH (pH 5.8) in the presence of 10 μM BCECF AM. First, petals were subjected to 15 minutes vacuum, and afterwards they were incubated 1 h at 22 °C in darkness. After that, the petals were washed two times for 10 min in the medium mentioned above. BCECF fluorescence was detected using a microplate reader.
M.9.3.2. In situ calibration
To obtain the calibration curve, dye loaded petals were incubated in each of the pH calibration buffers for no longer than 15-20 min. Then the solution was replaced by liquid medium containing 1/10 MS medium. The ratios for each pH incubation were plotted against pH to obtain the calibration curve. A sigmoidal regression (Boltzmann function) was fitted to
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describe the calibration curve and to calculate subsequent pH values from the equation describing the curve.
Calibration buffer
Ammonium acetate 50 mM
Mes-BTP (pH 5.2-6.4) or 50 mM-HEPES-BTP (pH 6.8-7.6) 50mM
M.9.3.3. pH measurement
Fluorescence intensity and absorbance values were acquired using (aquí falta lo que quieras que usaras para medir). Settings for calibration and measurement should be identical.
Samples were sequentially excited by two wavelengths: 440 nm and 495 nm, fluorescence emition was detected at 525 nm for each of the two excitation wavelengths. Three reads per well and weavelenght were done.
The data obtained was analysed in a Microsoft Excel sheet. First, after blank subtraction (petals without BCECF treatment), the Ratio 495/440 was calculated for the calibration wells dividing emission values excited with 495 nm by the emission values excited with 440 nm.
Means and standard deviations of technical replicates were calculated.
The calibration curve was created plotting pH values of the buffers used in the calibration against the respective values of Ratio 495/440 calculated. Using OriginPro, a sigmoidal regression fit (Boltzamn fit) was performed with the calibration data. The obtained equation was used to calculate the pH of the problem samples. To that end, the Ratio 495/440 values of the problem samples were calculated after blank subtraction as before mentioned obtained s directl, and by interpolating these values in the equation, the pH was obtained.
M.9.4. BCECF to measure yeasts vacuolar pH
M.9.3.1. Loading of the dye.
To measure vacuolar pH in yeast, the protocol described by Ali et al., 2004 was used, with the proper modifications to make the pH measurements in a microplate reader (Brett et al.
2005b). Yeasts were grown overnight in AP pH 6.0 medium without histidine and uracil.
Culture in exponential phase of growth (OD600 0.5-0.6) were harvested, washed two times and finally resuspended in AP medium without aminoacids, to a final OD600 of 0.2-0.3. Three independent colonies per mutant allele and control were used. Each smaple was incubated
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with 50 µm de 2’,7’-bis-(2-carboxietil)-5-(y-6)-carboxifuoresceein acetoximetil ester (BCECF-AM; Molecular Probes, Eugene, OR) at 28ºC, with gentle shaking.
After 20 minutes, the culture was centrifugated 10’ at 5,000 rpm and washed three times with AP medium without aminoacids and without BCECF (incubating during 10 min with the new medium after each centrifugation). Finally, yeast cells were resuspended in 100 μl of AP medium without aminoacids and without BCECF end immediately the fluorescence was measured.
Fluorescence measurements were performed using a Varioskan LUX Multimode Microplate Reader (ThermoFisher Scientific) at 20 ºC, that were shaked before each measurement.
M.9.3.2. pH measurement
Fluorescent intensity and absorbance values were recorded using a Varioskan LUX Multimode Microplate Reader (ThermoFisher Scientific). Settings for calibration and measurement were identical. Samples were sequentially excited by two wavelengths: 450 nm and 490 nm; emition fluorescence was detected at 535 nm for each of the two excitation wavelengths. Absorbance was also measured with 600 nm weavlength. Three reads per well and weavelenght were done. Measurements were repeated three times for each culture, washing the cells in between with AP medium without aminoacids and without BCECF.
M.9.3.3. In situ calibration
At the end of each experiment, a calibration curve of fluorescence intensity versus pH was obtained. Each of the 200 μl culture used were treated with the following experimental medium:
MES 50 mM
HEPES 50 mM
KCl 50 mM
NaCl 50 mM
Ammonium acetate 200 mM
NaN3 10 mM
2-deoxyglucose 10 mM
Cyanide m.chlorophenylhydrazone 50 μM
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Buffers were titrated to eight different pH values (5.2, 5.6, 6.0, 6.4, 6.8, 7.2, 7.6, 8.0) using 1 M NaOH.
To estimate accurately acid pH values below 5.0, the James-Kracke (1992) approximation was performed. According to this, in a pH close to neutrality, BCECF fluorescence measured as H+ activity, or hwat is the same, H+ concentrations using the formula:
𝐻+ = 𝐾𝑎𝑥(𝑅𝑚𝑎𝑥−𝑅)
(𝑅−𝑅𝑚𝑖𝑛) 𝑥 𝐹𝑏𝑎𝑠𝑒 450
𝐹𝑎𝑐𝑖𝑑 450 (1)
Being: Ka the acid dissociation constant, R the ratio of the emited fluorescence by BCECF excited at 490 and 440 (Ratio 490/450), Rmax is the maximun Ratio 490/450 value, obtained in alkaline conditions, Rmin is the minimum Ratio 490/440, in acid conditions. Fbase450/Facid450 is the ratio of the fluoreacence at 450nm in acid and basic conditions (James-Kracke 1992)
The logarithmic transformation of the ecuation is:
𝑝𝐻 = 𝑝𝐾𝑎− 𝑙𝑜𝑔 (𝐹𝑏𝑎𝑠𝑒 440
𝐹𝑎𝑐𝑖𝑑 440) − log ((𝑅𝑚𝑎𝑥−𝑅)
(𝑅−𝑅𝑚𝑖𝑛)) (2) In the isosbestic point Fbase450/Facid450 is 1, reason why:
𝑝𝐻 = 𝑝𝐾𝑎− log ((𝑅𝑚𝑎𝑥−𝑅)
(𝑅−𝑅𝑚𝑖𝑛)) (3)
Where Rmax and Rmin are obtained from the most alkaline and most acid pH buffers measured.
Th values obtained for each culture treated with different buffers were background subtracted and normalized to cell density. The pH calibration curve was obtained by plotting log ((𝑅𝑚𝑎𝑥−𝑅)
(𝑅−𝑅𝑚𝑖𝑛)) against pH. The resulting equation was used to obtain the pKa.
M.9.3.4. Data analysis.
The data obtained was analysed in a Microsoft Excel sheet. After blank subtraction (yeast without BCECF tratment) and cell density normalization. To obtain pH values, the ratio 490/440 was calculated in each case, and the values obtained were extrapolated from the calibration curve.
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