Mechanism of kinetin-induced death of Vicia faba ssp. minor cortex cells

Cell death (CD) takes part in the control of all the steps of plant development. It may be induced by endogenous or exogenous factors. This paper presents the results related to mechanism of CD regulation induced by kinetin (Kin) in root cortex of Vicia faba ssp. minor. To explain the process 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55), adenine (Ad), 5'-amine-5'-deoxyadenosine (Ado) and N-(2-chloro-4-piridylo)-N’-phenylurea (CPPU) were applied to (i) block cytokinins (CKs) receptors and toinhibit the activity of (ii) kinases, (iii) oxidases and (iv) phosphoribosyltransferase, respectively. ethylene signaling MAP kinases, CKs receptor and enzymes of their metabolism in induction of Kin-ECD is proposed.


Introduction
Cell death (CD) is a developmentally and environmentally (biotically or abiotically) induced process in all organisms 1 . During plant tissue and organs differentiation, i.e., developmental CD (dPCD), according to Galluzzi et al. (2018) 2 such type of CD is mentioned as programmed CD (PCD). It occurs via modi cation of cell functions or cell elimination, control of formation of vegetative (e.g., xylem and phloem tissues) and generative (e.g., embryos) 1 organs. One of the factors, which applied exogenously induced cell death, is Kin 3,4,5 . Remaining consistent with suggestion of Galluzzi et al. (2018) 2 that CD induced by exogenous factors is a Regulated CD (RCD), authors of the paper will use the term "kinetin-exogenously induced-CD" (Kin-ECD).
to 3rd h, from 6th to 18th and from 24th to 96th h, respectively 5,6 . In the speci cation (signaling) phase of Kin-ECD, the total and cytosolic levels of calcium ions (Ca 2+ ) in the cortex of apical parts of faba bean seedling roots and in cortex cells respectively, as well as the amount of ACC in these fragments, were the greatest 3 . Whereas in the executive phase, the reactive oxygen species (ROS) amounts were the greatest and in the degradation phase of the process the activity of the ROS metabolism enzymes and the amount of free sugars were the greatest 3,9 . The studies which results are presented in the paper were undertaken to prove the hypothesis suggested in Kunikowska et al. (2013) 7 . Authors of that paper suggested that Kin induced CD after its conversion with phosphoribosyl transferase to corresponding monophosphates which are mentioned as the purine speci c ligands 15,16 for one or two histidine kinases (HKs) CK receptors. This hypothesis is based on the data described in several papers showing that exogenously applied free CK bases into organisms are rapidly converted into their nucleosides and nucleotides 16,17,18 . Then they triggered apoptosis in human leukemia cell lines (HL-60 cell line) 16,19 . Moreover, treatment of the cell cultures of Arabidopsis thaliana with benzyl adenine (BA) induced CD only in the presence of HK4 receptors 20 although BA had low a nity to them 21 . In A. thaliana HK3 had about 10-fold lower a nity to isopentenyladenine and its riboside, but higher a nity than HK4 to dihydrozeatin and zeatin and isopentenyl adenine/cytokinin ribosides and cis-zeatin 26,27 . This fact con rmed that CK ribosides and their monophosphates are purine ligands allegedly inducing CD via HK4 20 . This receptor can cooperate with HK3 one 14,20,22,23,24 , which together with HK2 are plasmaand endoplasmic reticulum (ER)-localized transmembrane proteins with extracellular and intracellular domains. These facts were evidenced in A. thaliana 23 , Zea mays 14 and in other plants 24 .
The main steps of cytokinin transport and signaling cascade involve purine permeases (PUP; a transporter of free cytokinins) equilibrating nucleoside transporters (ENT), subfamilies of HK-cytokinin receptors 14 HPTs, small (app. 16 kDa) monomeric proteins, together with RR, initiate cytokinin signaling. The A-type RRs are stabilized by their phosphorylation and they are negative regulators of cytokinin response, but Btyp of RRs, which can bind to DNA and start expression of cytokinin-sensitive genes after their phosphorylation 14,22−29 . We proposed that monophosphates interacting with HK4 or, eventually with HK3, whose presence in faba bean we suggested, evoking e ux of Ca 2+ , activated ETH-dependent cell death process regulators. This hypothesis was based on the data showing that (i) ETH and CKs interacted at the level of signal transduction 29,30 as well as Kin (ii) elevated ACC amount 3 , and (iii) evoked e ux of Ca 2+ to cytosol 3 , nally forming aerenchyma 3,6 .
Authors of the paper would like to nd the answer to question related to assumption whether MAP kinases being the crucial elements of ethylene-dependent signaling pathways are engaged in Kin-ECD.
Thus, the effect of plant Raf-like kinase (CTR1) inhibitor (Sorafenib) 3738 on vitality of cortex cells as well as MEK2 and Raf-like kinase activities were measured using its substrates, i.e., Syntide 38 andMek2 39 .
To nd the source of cytosolic Ca 2+ taking part in Kin-ECD, the stream of its destinations were studied by the effects of the of all (ALM-Ca 2+ ) 40 , ER-41 and mitochondria-42 membrane channel inhibitors i.e., ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA), lanthanum chloride (LaCl 3 ) and cyclosporine (CsA) respectively on vitality of cortex cells and cytosolic amount Ca 2+ .

Results
Analysis of the double stained nuclei showed that after 72 h in Ctrl about 4% of cells were dying or dead, while in the same time, after Kin treatment number of these cells signi cantly increased to about 48% ( Fig. 1A,C -5A,C). The cytophotometric analysis showed that the amounts of cytosolic Ca 2+ differed between series, and were about 10 000 a.u. and 14 000 a.u. in Ctrl and in the Kin-treated cells, respectively Effect of PI-55, inhibitor of cytokinin perception, and of Ad, Ado and CPPU, cytokinin metabolism inhibitors, on Kin-ECD and the amount of cytosolic Ca 2+ . It was showed that PI-55-5 did not induce cell death compared to Ctrl, while PI-55-10 did. The number of dying cells in the latter case was about 20%. In Kin/PI-55-5 cell death was almost completely inhibited in comparison with Kin ( Fig. 1A). At the same time, the amounts of Ca 2+ in PI-55-5 and PI-55-10 were signi cantly greater, about 2.5-and 2.0-fold, than in Ctrl and about 2.0-and 1.5-fold than in Kin, respectively. In Kin/PI-55-5, the amounts of Ca 2+ were signi cantly greater, about 3.0-and 2.5-fold, than in Ctrl and Kin, respectively (Fig. 1B).
Both Ad-50 and Ad-100 did not increase the number of dying cells compared to Ctrl. However, to study its effect on Kin-ECD, the Ad-100 was used. In Kin/Ad-100 the number of Kin-ECD-dying cells was like in Ctrl ( Fig. 2A). While the amounts of Ca 2+ in Ad-50 and Ad-100 were signi cantly greater, about 2.0-fold, in comparison with Ctrl and about 1.3-fold in comparison with Kin. In Kin/Ad-100 variant the amount of Ca 2+ was like in Kin (Fig. 2B).
Ado-50 did not increase the number of dying cells compared to Ctrl, but Ado-10 increased their number to 12%, thus Ado-50 was used to study its impact on Kin-ECD. It was showed that in Kin/Ado-50 variant the number of dying cells was like in Ctrl (Fig. 2C). While in Ado-10 and Ado-50 variants the Ca 2+ amounts were signi cantly greater, by about 2.0-and 3.0-fold, in comparison with Ctrl, respectively, and by about 1.5-and 2.5-fold, in comparison with Kin variant respectively, while in Kin/Ado-50 series, the amount of Ca 2+ was like in Kin (Fig. 2D).
CPPU-5 did not increase the number of dying cells compared to Ctrl but in CPPU-10 the number of dying cells was like in Kin. Thus, to study its effect on Kin-ECD, CPPU-5 was used. In Kin/CPPU-5 the number of Kin-ECD-dying cells was signi cantly lower, about 30%, in comparison with Kin (Fig. 3A). Analyses of the levels of Ca 2+ in CPPU-5 and CPPU-10 showed that its amount were signi cantly greater, about 2.0-and 1.5-fold, in comparison to Ctrl, and about 1.5-and 1.3-fold greater, in comparison to Kin, respectively, while in Kin/CPPU-5 the amount of Ca 2+ was signi cantly greater, about 1.6-fold, in comparison with Kin and like in CPPU-5 (Fig. 3B).
Effect of the EGTA, La 2+ and CsA inhibitors of ALM-Ca 2+ -, ER-Ca 2+ -, MIT-Ca 2+ -dependent channels, respectively, on Kin-ECD and the amount of cytosolic Ca 2+ . EGTA-10 and EGTA-50 did not increase the number of dying cells compared to Ctrl. To study its effect on Kin-ECD, EGTA-10 was used. It was showed that in comparison with Kin in Kin/EGTA-10 the number of dying cells was signi cantly lower, about 38% ( Fig. 4A). The amounts of Ca 2+ in EGTA-10 and EGTA-50 were signi cantly greater, about 1.5-and 2-fold, in comparison with Ctrl, respectively, and about 1.3-and 1.5-fold greater, in comparison with Kin, respectively, while in Kin/EGTA-50 the amount of Ca 2+ was like Ctrl (Fig. 4B).
La-5 and La-25 did not increase CD index compared to Ctrl. Therefore, La-5 was used to study its effect on Kin-ECD. It was observed that in comparison with Kin in Kin/La-5 the number of Kin-ECD-dying cells was signi cantly lower; there were only 10% of them (Fig. 4C). The amounts of Ca 2+ in La-5 and La-25 were signi cantly greater, about 2-and 1.5-fold, in comparison with Ctrl and Kin, respectively. In Kin/La-5 the amount of Ca 2+ was signi cantly greater, about 1.5-fold, in comparison with Kin (Fig. 4D).
CsA-5 signi cantly increased the number of Kin-ECD-dying cells in comparison with Ctrl while CsA-25 did not. Therefore, CsA-25 was used to study its effect on Kin-ECD. It was observed that in comparison with In Ctrl series amount of ETH was about 5 ppm while after treatment with Kin for 72 h its amount was statistically signi cantly greater by about 30 %. Treatment with Sorafenib of Ctrl seedlings (Sorafenib series) showed that ETH amount per six seedlings was about 20 % lower compared to Ctrl. In combination of 1 mM of Sorafenib with Kin (Sorafenib-Kin series), the amount of ETH was about 30 % and 40 % greater compared to Ctrl and Sorafenib series respectively and was like in Kin (Fig. 6B).
Analyses of kinase activities showed that activities of Raf-like kinase, with Syntide substrate, and MEK2, with Mek2 substrate, in Ctrl series was about 2.5 mg and 1 mg of ATP per mg of protein, respectively. After treatment with Kin for 72 h their activities were about 20 % greater and 20 % lower respectively compared to Ctrl series (Fig. 6C).
These facts allowed us to suggest that CD depends on an organism and the concentration of CKs which was decided on su cient amount of free bases of CKs important for synthesis of their ribosides and their phosphorylated derivatives both in animal (HL-60) 16,19 as well as plant cells, e.g., in Arabidopsis sp.
suspension cell culture 20 and in Gerbera sp. callus where the free zeatin became conjugated with ribose or ribose phosphate to produce the physiologically-active compounds, i.e., zeatin-9-riboside and zeatin-9ribotide 52, respectively.
The studies of Kin-ECD showed that Kin in faba bean roots induced two responses 3 . The rst one is related to development of the protective mechanisms, acting against death. It is known that Kin is an antioxidative factor which strongly inhibits oxidative and glycoxidative protein-damage 11 . Such antioxidative effect is observed in apical parts of faba bean seedlings during Kin-ECD as the increase and then decrease in ROS amount as the results of increase in activities of the enzymes of their metabolism 9 . CK in Kin form is the factor which can delay the senescence of detached Raphanus sativus L leaf discs 52 . It inhibits the leaf senescence via activation of cytokinin receptor (AHK3), the type-B response regulator (ARR2) and the cytokinin response factor (CRF6) 53 .
The second responses 3 of Kin in faba bean roots is related to induction of ECD 5,8,9 in which Kin may be degraded to adenine and ribosylated to its ribosides by adenosine phosphorylase. This enzyme was identi ed in wheat (Triticum aestivum) germs 49 . To obtain phosphorylated forms of CKs, the adenosine kinase catalysed formation of the adenosine monophosphate from adenosine. Adenosine kinase was found both in human 16 and in tobacco BY-2 cells 17 as well as in Arabidopsis 36 . The next step of adenine metabolism is related to conversion of 5'-monophosphate of zeatin to its i.e., trans or cis forms. The fact that Ad and Ado, adenosine phosphorylase 31,32−34 and adenosine kinase 16,35,36 inhibitors, respectively, completely suppressed Kin-ECD in root cortex con rmed occurrence of such enzyme in faba bean roots and worked according to their functions.
Ad inhibited formation of AMP in human 27 although in A. thaliana not 35 . Its application to faba bean seedlings inhibited the CD induced by Kin. This fact suggested that the Ad inhibiting activity of APRTs broke the CKs synthesis pathway therefore lower amount of CK monophosphates, which are suggested as ligand for CKs receptors, were released in the CK-dependent pathway.
Inhibition of adenosine phosphorylase and cytokinin kinases activities might enhance the inhibitory effect on Kin-ECD because the results showed that an inhibitor of cytokinin oxidase, i.e., CPPU, suppressed Kin-ECD. Since adenine, which is a direct product of CKs degradation by cytokinin oxidase and inhibitor of adenine phosphoribosyl transferase activities 27,31 acted against Kin-ECD 5 . It indicated that the level of Kin or other CK riboside monophosphates, as the results of the activity of ATPR, may decreased. Thus, its CD-inducing effects can be decreased.
It is suggested that riboside monophosphates of CKs could activate faba bean HK4 receptors in ERmembrane 20,45,46 . The monophosphorylated forms of cytokinin ribosides have greater a nity for HK4 16,20,21 than HK3 and free cytokinin bases however both receptors can interact with each other 21 . This might result from the fact that PI-55 completely inhibited the death of cortex cells. After dimerisation and autophosphorylation of HKs, the phosphate group is translocated from histidine to aspartic acid of a regulatory domain of the receptor. Then histidine of faba bean homologues of A. thaliana HPT carriers and type B of RR 15,20 can activate the cytokinin-dependent response (Fig. 7). The earlier results showed that during Kin-ECD the level of ATP decreased 4 . It suggests that decreased level of ATP amount after Kin treatment was related to the fact that ATP was used as the phosphate group for synthesis CKs riboside monophosphates and/or with the mitochondria destruction, observed in apical parts of faba bean roots as the reduction of their number and structure malformation caused by ROS 9 , but also on lower activities of cellular dehydrogenases 15 and histone kinase activities 4 . It may be assumed that, during Kin-ECD the cytotoxic N 6 -furfuryladenosine (Kin-riboside) may be synthetized and depleted ATP amounts, like in human cancer cell lines 48 .
The extensive studies delivered important results suggesting that ETH may be the effector of Kin-induced death signal transduction pathway 3 . The loss of HK4 function in ahk4 mutant decreased ETH response to CKs, indicating that the A. thaliana HK4 receptor is probably a primary contributor to ETH biosynthesis responding to CKs 29,30 . Therefore, A. thaliana HPT can transduce signals from CKs ( Fig. 7) 15,23 to ETH receptors (ethylene triple response, ETR1 and 2; 1; ethylene insensitive 4, EIN4; ethylene response sensor 1 and 2 ERS1 and 2) in ER membranes , 28,29,38,40,56 . Moreover, CKs can post-transcriptionally increase the activity of ACS4,5 (ACC synthases 4,5) gene products 29,56 , leading to increase in activities of these enzymes in ethylene synthesis, thus it can synergistically increase ETH amount (Fig. 6,7). This suggestion was con rmed by the fact that in the Kin-treated seedlings in apical parts of faba bean roots, the ACC 12 and ETH amounts increased. It can activate EIN2, ER-and nuclear-ETH-dependent membrane receptors and induce ETH response elements (ERE) 28,29 . The fact that after Kin-3,7 and ACC-induced 12 death of cortex cells of faba bean roots aerenchyma was formed 3,7,12 also con rmed the hypothesis, because ETH seemed to be direct hormonal factor involved in the process 3 .
It cannot be excluded that another ETH-CKs pathway in Kin-ECD exists. The MAP kinases are considered regulators of ETH signal transduction pathway. One of them in plants is CTR1, a structural equivalent of RAF kinase. It is assumed that CTR1 acts in the ETH signaling that is MAP-kinase-dependent cascade.
Results showed that Sorafenib suppressed the Kin-ECD because the number of living cells in Kin series were at the same level as in the Ctrl. However, the Raf-like kinase activity in root of Kin series was greater than in Ctrl series while the MEK2 activity was lower compared to Ctrl series. Sorafenib, an inhibitor of ATP-competitive kinase can affect activity of other kinases, e.g., ERK (extracellular signal-regulated kinase) 50 one. These facts clearly indicated that in faba bean roots both kinases exist.
The results showed that decrease in the number of dying cells is dependent on the effect of Raf-like kinase activity increase and of MEK2 activity inhibition. Moreover, the results con rmed that Kin-ECD is the process controlled by cooperation of ETH-dependent MAP kinases singling pathway. The crosstalk between Kin and ETH might take place at the level of ETH and CK receptors because ETH receptors are members of HK family proteins 57 . It was proved by the appearance of the triple ethylene response (cell length and width changes as well as root apical hook formation) 3,7,59 in the faba bean seedlings after Kin treatment which features were inhibited by 2,5-norbornadiene (NBD), the ETR3 and ETR4 receptors inhibitor 3 .
The results presented by Scharein and Groth (2011) 59 con rmed the existence of ETH-CKs crosstalk. The phosphorylation between ETR1 and AHP1 complexes play a crucial role in this process. When both are either in phosphorylated or non-phosphorylated states, the a nity between them decreased. On the other hand, the a nity between the two partners is greater when one of them is phosphorylated and the other one is not. The additional reason that this process might exists in faba bean is the fact that both ETH and CK signaling pathways work using the transduction factors being the two-component systems 15,57 .
The present results showed that the amounts of cytosolic Ca 2+ in the root cortex cells of faba bean seedlings in PI-55-5 and CPPU-5 were similar to Kin/PI-55-5 and Kin/CPPU-5 but in Ad-100 and Ado-50 they were greater, in comparison with Kin/Ad-100 and Kin/Ado-50; the levels of cytosolic Ca 2+ in Kin/PI-55-5 and Kin/CPPU-5 were greater than in Kin while in Kin/Ad-100 and Kin/Ado-50 were similar to Kin series.
The fact that Ca 2+ migrated from ER, which is important for Kin-ECD induction, was suggested previously 3 . The present research showed that application of La 2+ , inhibitors of ER-membrane Ca 2+ channels 40,60 , in a nitrate 3 or chloride form, inhibited the process. The results of the present paper showed that migration of Ca 2+ via plasma membrane-(from outside the cell) or mitochondria-channels was also (2013) 7 , but it seems that their amounts are important for activation Kin-ECD enhancing ACC and ETH synthesis via calcium-dependent ACC synthase (ACS) enzymes.
The fact that in Kin/EGTA-5 the amount of Ca 2+ was like in Ctrl con rmed that induction of Kin-ECD depended on direct migration and on the type of Ca 2+ channels. Thus, when in mitochondria 61 and in ER membranes CNGCs (cyclic nucleotide gated channels) 40,62 were blocked by CsA or La 2+ , Ca 2+ were transported to cytoplasm via plasma membrane channels leading to elevation of cytosolic Ca 2+ (Fig. 7). It is con rmed by the results showing that EGTA (an inhibitor of all Ca 2+ channels) 39 applied to the culture solution blocking plasma membrane channels lowered the in ux of Ca 2+ into cytosol.
Thus, it is possible that ETH initiated expression of protein kinases and/or their activities, especially of H1 and core histones 4 and induced ETH-positive feedback via Ca 2+ -dependent ACC synthase (Fig. 7) important for the regulation of ETH synthesis. Moreover, phosphatidyl inositol 4,5 bisphosphate and inositol 1,4,5 trisphosphate may increase ETH amount 11,58 .
The results indicting that ethylene is involved in Kin-ECD control were also con rmed by the studies revealing that inhibitors of ACC synthesis and its conversion to ETH suppressed Kin-ECD 3 . Moreover, Kin-ECD triggered aerenchyma formation accompanied ETH-dependent triple-response 3,7,58 .
The results of studies related to male sex determination of Anemia phyllitidis gametophytes, where ETH is the gibberellin secondary signal, additionally con rm that reorganization of metabolism and of cell wall structure in plant depends on ETH 59 .

Conclusions
The fact that some of the results of the paper are partially based on usage of factors such as Sorafenib, Syntide and Mek2, the inhibitor of RAF kinase 37,50 as well as Raf-like and MEK2 substrate related to the animals 37 , plants 35 and bacteria 31 it was the crucial for explanation Kin-ECD progress.
First and foremost is the fact that Kin is the factor playing important role in the controlling of differentiation both in plants and animals 6 . Secondly, CTR1 kinase (in plants called Raf-like 38 ) is a homologue of RAF kinase, existing in animals 37 . Additionally, in plants, animals and bacteria the similar histidine kinases forming the transmembrane receptors for ETH and CKs werefound 28,56 . Therefore, taking all results into account, we can conclude that the Kin-dependent signal by the phosphorylated forms of Kin or other CKs ribosides may activate CD-dependent HK4 (strongly suggested as the cell death receptor activated by CKs) 20,21 receptors and generate pathways via HPT 20,46 enhancing expression of ACS4,5 genes 15,55,56 and activities of Ca 2+ -dependent ACSs 58 , increasing ACC and then ETH amounts (Fig. 7).
Then the signal is transferred onto speci c ETH-dependent genes and it induces: (i) the expression of elements initiating the executive phase of Kin-ECD among them serine and caspase-like proteolytic 8 and nucleolytic 4,5 machinery as well as (ii) cell wall compound metabolism 3 which nally lead to complete degradation of some cells in the degradation phase of Kin-ECD 5,8 resulting in aerenchyma formation 3,7 . Kin generates two signals, the death one results in cells selection, via ETH, and the pro-life one against death in other cells, via activation of ROS enzyme metabolisms and via maintaining the appropriate concentration of cytosolic Ca 2+ and/or its destination 40,60 .
During the morphologic and metabolic studies, we demonstrated that perception and metabolism of CK inhibitors completely suppressed Kin-ECD, but they did not reduce the Kin-elevated amount of cytosolic Ca 2+ in the cortex of apical roots of faba bean seedlings. Moreover, ER-and mitochondria-membrane Ca 2+ channel inhibitors reduced Kin-elevated amount of cytosolic Ca 2+ in the cortex cells of seedling roots while the total-membrane ones did not.
The fact that CKs and ETH can crosstalk is crucial for the initiation of transducing CD execution signals. ACC-synthases and -oxidases elevate ETH which causes cortex cell degradation and aerenchyma formation in Ca 2+ -dependent pathway.
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 cm 3 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 Ca 2+ channel inactivators at two selected concentrations (Table 1) without Kin on CD and cytosolic Ca 2+ 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 in uence on vitality and amount of cytosolic Ca 2+ in cortex cells during Kin-induced CD. 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 uorescence intensity (FI; Figure 3S; Bright-orange ( Figure 3S; A,A1) and orange-red ( Figure 2S To measure cytosolic Ca 2+ amount in the cortex cells, 2-cm long apical parts of faba bean roots (between the 4 th and the 20 th mm from apex) were xed 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 lter of an epiuorescence microscope ( Figure 3S; B,B1-B6), photos were taken and total green uorescence intensity (TFI) of Ca 2+ -CTC complexes was cytophotometrically measured using the Scn Image software 3 .
During measurements, each stained cell was separately outlined using the threshold option, then the values of uorescence intensity in a.u. were read and used for calculation of the Ca 2+ amounts. The decreasing amount of Ca 2+ was related to the values of green TFI of Ca 2+ -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 asks before sample preparations. Erlenmeyer asks 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 exible tube directly before measurement ( Figure 2S). Then the pump was turn on and measurements were conducted for 30 sec. Results between 20 th to 30 th sec, when the values reached the plateau, the ve 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 MgCl 2 , 10 µg ml -1 BSA (bovine serum albumin; Sigma-Aldrich) and 1 mM PMSF (phenylmethylsulphonyl uoride; Sigma-Aldrich) in 1.5 ml Eppendorf-like tubes with the plastic mortar and centrifuged at 5 000 g for 10 min 8 .
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 or Inskape (open source; https://inkscape.org/release/inkscape-1.0.1) were used to prepare gures and images planes in tiff extensions.
The BioRender (https://biorender.com) software was used to prepare the