Plant material, chemicals, and experiments. V. faba ssp. minor cv. Bobas (Danko, Sobiejuchy 2
88-400, Żnin Sobiejuchy 2, Poland; www.danko.pl).
88-400 Żnin seeds (20) were germinated for 3 days in Petri dishes (15 cm in diameter and 3 cm high) on two blotting papers with distilled water (the seeds were half submerged) in a dark breeding room. For analyses 6 of the 3-d-old seedlings with nearly equal root length (2.0 ± 0.3 cm) were transferred into a glass container (8 cm in diameter and 4 cm high) with two blotting papers moistened with 10 cm3 of water (Ctrl) or adequate solutions of chemicals and cultivated at 23 ± 1 °C and at 92% ± 2% of relative humidity exactly for 72 h and then used for analyses. The types and sources of factors originally used in the studies are presented in Table 1.
First, the impact of CK perception and metabolism regulators as well as Ca2+channel inactivators at two selected concentrations (Table 1) without Kin on CD and cytosolic Ca2+ amounts in faba bean seedling root cortex was tested. Then the concentration of the factor which did not induce, or slightly induced cell death compared to the other one was used to analyse its respective influence on vitality and amount of cytosolic Ca2+ in cortex cells during Kin-induced CD.
Quantification of cell viability. Analyses of viability of cells were made in cortex of untreated plants (Ctrl) and those treated with PI-55 (5 μM and 10 μM, series PI-55-5 and PI-55-10), Ad (50 μM and 100 μM, series Ad-50 and Ad-100), Ado (10 μM and 50 μM, series Ado-10 and Ado-50), CPPU (5 μM and 10 μM, series CPPU-5 and CPPU-10), EGTA (10 μM and 50 μM, series EG-10 and EG-50), CsA and La2+ (5 μM and 25 μM, series CsA-5 and CsA-25 and 25 La-5 and La-25), Kin (46 μM 0.1 M HCl solution) with 5 μM PI-55 (series Kin/Pi-55-5), Kin with 100 μM Ad (series Kin/Ad-100), Kin with 50 μM Ado (series Kin/Ado-50) as well as Kin with 5 μM CPPU (series Kin/CPPU-5), Kin with 10 μM EGTA (series Kin/EG-10), Kin with 25 μM CsA (series Kin/CsA-25) and Kin with 5 μM La2+ (series Kin/La-5).
Viability (Figure 1S). of cells was also analysed after Sorafenib treatment, a Raf-like kinase inhibitor, at 1.0 µM concentration with treated and untreated by Kin V. faba ssp. minor seedlings (Figure 2S).
Additionally, the effects of solvents, used for PI-55 and CsA, Kin as well as for CPPU preparation, i.e., the 0.05% DMSO (Avantor), 0.01 N HCl (Avantor) and 0.1% methanol and mixture of HCl and DMSO as well as HCl with methanol (Avantor) in distilled water, respectively, on viability of cortex cells were tested and the average values were used to complete the figures. Moreover, effects of the mentioned solvents on fluorescence intensity of CTC/cytosolic Ca2+ complexes used to determine the relative amounts of Ca2+ were assessed and the results of studies were referred to the respective Ctrl values.
To detect and measure the intensity of CD (percentage number of dying cells) in root cortex, apical fragments of roots were cut off from the seedlings, washed twice with 0.1 M PHB (Na phosphate buffer pH 7.4; Avantor), stained with the mixture of 100 μg cm−3 acridine orange (AO; Sigma-Aldrich) and 100 μg cm−3 ethidium bromide (EB; Sigma-Aldrich) in PHB, washed in PHB two times and fixed with 2% solution of glutaraldehyde in PHB. Then, the handmade thin sections of long axes of 2-cm apical part of roots were analysed and photographed under the blue light of fluorescence filter (B2A) of Optiphot-2 (Nikon, Japan), epi-fluorescence microscope equipped with DXM1200 digital camera and Act-1 (Precoptic, Poland) software.
Estimation of the numbers of living, dying and dead cortex cells was carried out according to the method described in Doniak et al. (2016)3, Byczkowska et al. (2013)6 and Kunikowska et al. (2013)8 using the specially prepared calibration curve which presents relations between fluorescence intensity (FI) of dyes and amount of nuclear chromatin. Measurements of FI were carried out using the Scion Image (Scion Corporation) software. During measurements, each stained nucleus was separately outlined using the threshold function, then the values of fluorescence intensity in arbitrary units were read and compared to the calibration curve Byczkowska et al. (2013)6.
This method uses the properties of EB-migration through damaged plasma and nuclear membrane, which amount in nuclei increases proportionally with the CD-induced permeabilisation. OA-migration through all types of membrane do not depend on their conditions. Thus, the changing colour of nuclear chromatin ranging from green to orange-red is related to increasing fluorescence intensity (FI; Figure 3S; A)5.
Bright-orange (Figure 3S; A,A1) and orange-red (Figure 2S; A,A2) colours indicate dead cells (FI values > 46 a.u.), yellow (Figure 3S; A,A3) and yellow-orange (Figure 3S; A,A4) indicate dying ones (FI values 34–55 a.u.), while green-yellow (Figure 2S; A,A2) green and indicates living cells (RFI values 10–35 a.u.) whereas (Figure 3S; A,A1),
FI values of viability were reported as indices. Data represent the mean ± SE of two replicates of three independent experiments (n = 3) from each of about 450–550 cells.
Estimation of cytosolic calcium ions content. Analyses of cytosolic Ca2+ amounts were made in cortex of untreated plants (Ctrl) and those treated with PI-55 (5 μM and 10 μM, series PI-55-5 and PI-55-10), Ad (50 μM and 100 μM, series Ad-50 and Ad-100), Ado (10 μM and 50 μM, series Ado-10 and Ado-50), CPPU (5 μM and 10 μM, series CPPU-5 and CPPU-10), EGTA (10 μM and 50 μM, series EG-10 and EG-50), CsA and La2+ (5 μM and 25 μM, series CsA-5 and CsA-25 and 25 La-5 and La-25), Kin (46 μM 0.1 M HCl solution) with 5 μM PI-55 (series Kin/Pi-55-5), Kin with 100 μM Ad (series Kin/Ad-100), Kin with 50 μM Ado (series Kin/Ado-50) as well as Kin with 5 μM CPPU (series Kin/CPPU-5), Kin with 10 μM EGTA (series Kin/EG-10), Kin with 25 μM CsA (series Kin/CsA-25) and Kin with 5 μM La2+ (series Kin/La-5).
To measure cytosolic Ca2+ amount in the cortex cells, 2-cm long apical parts of faba bean roots (between the 4th and the 20th mm from apex) were fixed with 2 % solution of glutaraldehyde (POCH) in PHB for 1 h and stained with 100 μM chlortetracycline (CTC; Merck-Sigma) and longitudinal handmade (about 300-400 μm thick) sections were prepared. Then analyses were carried out under B2A filter of an epi-fluorescence microscope (Figure 3S; B,B1–B6), photos were taken and total green fluorescence intensity (TFI) of Ca2+-CTC complexes was cytophotometrically measured using the Scn Image software3.
During measurements, each stained cell was separately outlined using the threshold option, then the values of fluorescence intensity in a.u. were read and used for calculation of the Ca2+ amounts. The decreasing amount of Ca2+ was related to the values of green TFI of Ca2+-CTC complexes (Figure 3S; B,B1–B6). Data represent the mean ± SE of two replicates of three independent experiments (n = 3) from about 500-600 cells.
Estimation of ETH amount, Raf-like kinase and MEK2 activities and protein amount. ETH measurements were carried in Erlenmeyer flasks before sample preparations. Erlenmeyer flasks were sealed with aluminum foil (to keep seedlings in the dark) and tightly closed with cap with the clogged pipette tips. After 30 min of incubation, handheld ETH analyzer (SCS56, Storage Control System, United Kingdom), equipped with a pump. was connected to pipette tips via flexible tube directly before measurement (Figure 2S). Then the pump was turn on and measurements were conducted for 30 sec. Results between 20th to 30th sec, when the values reached the plateau, the five readings from monitor were written in a spreadsheet of MS.Excel and taken to calculate the ETH amount in ppm per six seedlings.
To estimate Raf-like and MEK2 kinase activities, the one third of the length of apical parts of roots were homogenised and reextracted in 0.04 M Tris-HCl pH 7.5 buffer (Sigma-Aldrich) containing 20 mM MgCl2, 10 µg ml-1 BSA (bovine serum albumin; Sigma-Aldrich) and 1 mM PMSF (phenylmethylsulphonyl fluoride; Sigma-Aldrich) in 1.5 ml Eppendorf-like tubes with the plastic mortar and centrifuged at 5 000 g for 10 min8.
The reaction mixture for kinase activity analyses was prepared by sequentially adding to 2-ml tubes: the extract (20 ml), extraction buffer (1035 ml), of ATP (5 ml), of Syntide or Mek2 (25 µl; 1 µg protein per 1 ml of buffer) as substrates and of Kinase-Glo Reagent (50 ml; Promega), containing Ultra-Glo™ Luciferase and luciferin. After mixing, the luminescent signals were measured in semi-micro fluorometer cell with Teflon Stopper by Fluorescence/Luminescence Spectrophotometer F – 2500 (Hitachi) at 458 nm, every for 2.5 min with 30 s intervals. The kinase activities were calculated as the difference between the luminescence of the samples without substrates and the luminescence of the amount of ATP not utilized for kinase substrates phosphorylation. The kinase activity was expressed in relative lights unites (RLU), indicating RLU amount of ATP utilized by kinases in 1 mg of protein,
To estimate the protein amounts, the apical parts of roots were homogenised with 100 mM Tris-HCl (pH 7.4) buffer in Eppendorf-like tubes using a plastic pestle (4–8 °C), then centrifuged at 5000 × g for 10 min (4 °C) and residues were re-extracted. Combined supernatants were used to measure protein amounts in the reaction mixture containing, in 2-ml Eppendorf-like tubes, extract (10–100 µl), extraction buffer (90–0 µl) and Coomassie Brilliant Blue G-250 reagent5 (1.4 ml). Absorbance was measured at 595 nm after 10 min of incubation (Amersham Biosciences Ultrospec 1100 Pro UV-VIS spectrophotometer with semi-micro cell).
To calculate protein amount, standard, i.e., BSA dissolved in PHB, was prepared in a range of 5 to 100 micrograms of protein in 100 µl volume and measurements were carried out according to above description. The prepared calibration curve was used to read the amount of protein in the sample and final calculations.
Reagent to protein determination was prepared by oneself with Coomassie Brilliant Blue G-250 (100 mg) diluted in 95 % ethanol (50 ml) and 85% H3PO4 (100 ml) and 950 ml of distilled water and saved in dark bottle.
Statistics and software. Three biological replicates, at least in tri-, duplicate and more random samples were analysed. The samples were prepared at least from six plants. The results of measurements were statistically verified by Mann–Whitney U test and/or Student’s t-test using the MS.Excel of the Microsoft 365® Software (licensed) by independent step by step analyses of each column of results. It allowed to show the significant differences between results at P ≤ 0.05. Calculations, all charts and table have been prepared using MS.Excel of the Microsoft 365® Software.
To estimate the vitality of cells, by counting the number of alive, dying, and dead cells, the Optiphot-2 epi-fluorescence microscope (Nikon) with a blue filter (B2A) equipped with a digital camera (DXM1200) and objectives (10x, 20x, 40x) Act-1 software (Precoptic, Poland; http://www.precoptic.pl) and Scion Image (Scion Corporation) software (open source; http://www.scioncorp.com) were used.
The CorelDraw Graphics Site X7 EduLic (https://www.coreldraw.com/pl/product/coreldraw)
or Inskape (open source; https://inkscape.org/release/inkscape-1.0.1) were used to prepare figures and images planes in tiff extensions.
The BioRender (https://biorender.com) software was used to prepare the Figure 7.