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A mechanosensitive Ca2+ channel activity is dependent on the developmental regulator DEK1


Responses of cells to mechanical stress are thought to be critical in coordinating growth and development. Consistent with this idea, mechanically activated channels play important roles in animal development. For example, the PIEZO1 channel controls cell division and epithelial-layer integrity and is necessary for vascular development in mammals. In plants, the actual contribution of mechanoperception to development remains questionable because very few putative mechanosensors have been identified and the phenotypes of the corresponding mutants are rather mild. Here, we show that the Arabidopsis Defective Kernel 1 (DEK1) protein, which is essential for development beyond early embryogenesis, is associated with a mechanically activated Ca2+ current in planta, suggesting that perception of mechanical stress plays a critical role in plant development.


Multicellular development is dependent upon cell−cell communication. Recent research has highlighted the fact that, in addition to chemical signals, the perception of mechanical stress, at both the cell and tissue level, is a key factor underlying growth coordination and morphogenesis (e.g., ref. 1). However, while in animal systems mechanosensors with important roles in development have been identified2,3,4,5,6,7,8,9, the molecular basis for mechanoperception in plants remains enigmatic. As in animals, plants respond to mechanical stimuli by an elevation in cytoplasmic Ca2+ 10,11,12. Based on the knowledge of plasma-membrane mechanosensing in animal systems, the most probable trigger for Ca2+ release from internal cellular compartments (Ca2+-induced calcium release) is either the opening of plasma membrane-localized mechanosensitive Ca2+ permeable channels, or the opening of voltage-dependent Ca2+ channels in response to changes in membrane potential caused by mechanosensitive channels permeable to other ions12. Recent research in Arabidopsis has led to the identification of several plasma membrane-localized mechanosensitive ion channels. These include proteins similar to the bacterial mechanosensitive channel of small conductance (the MSL family13,14,15,16), the MCA1 protein that rescues the yeast Ca2+ channel mutant mid1, and its homolog MCA217,18,19,20. In addition, the membrane protein OSCA1 forms a hyperosmolarity-gated Ca2+ permeable channel required for osmosensing in Arabidopsis 21. However, although the MSL8 protein has recently been shown to be required for pollen grains to survive rapid rehydration during fertilization14, the very mild developmental phenotypes in single and multiple mutants of genes encoding the channels described above, suggests that they are unlikely to play a major role in mechanosensing during development. Furthermore, these proteins have not conclusively been shown to be responsible for any of the mechanosensitive Ca2+ currents which have been detected and extensively described by electrophysiologists over the past few decades in planta 20, 22,23,24,25,26. Since genes encoding voltage-sensitive Ca2+ channels similar to those identified in animal systems have not been found in plant genomes, it has been proposed that plants may have evolved novel systems for mediating mechanosensitive Ca2+ fluxes at the plasma membrane to control development (reviewed in ref. 12).

The Defective Kernel 1 (DEK1) protein is encoded by a highly conserved uni-gene found in all multicellular plant genomes sequenced so far, and it is absolutely required for both embryonic and post-embryonic development in angiosperms27,28,29,30,31. Null dek1 mutant embryos do not develop beyond the early globular stage. When plants with reduced DEK1 activity can be obtained, they show major developmental defects, notably in epidermal differentiation and adhesion30, 32, 33.

The DEK1 protein contains multiple predicted transmembrane (TM) spans interrupted by a loop, and an intracellular tail including a linker domain and a C-terminal domain showing similarity to animal calpains, a class of Ca2+-dependent cysteine proteases34. DEK1 localizes to the plasma membrane and to internal compartments32, 34. In their original model of DEK1, Lid et al.34 predicted the presence of 21 TM domains with an extracellular localization for the loop and cytoplasmic localization (subsequently confirmed in planta) for the C-terminus. However, in a more recent analysis, Kumar et al.35 proposed a consensus model with 23 TM domains and with a cytoplasmic localization for the loop. Structure-function studies have shown that the cytosolic CALPAIN domain of the protein can, alone, complement the embryo lethality of dek1 mutants suggesting that this domain, which is removed from the rest of the protein by an autolytic cleavage event, represents an active form of the DEK1 protein32. Consistent with this scenario, and with a role for DEK1 in maintaining epidermal integrity, overexpression of the CALPAIN domain of DEK1 leads to thickening of the outer epidermal cell wall in leaves, and increased deposition of pectins36, which are important for cell adhesion (reviewed in ref. 37). The epidermis is thought to be under tension during much of plant growth38. One plausible function of DEK1 could, therefore, be to perceive and respond to this tension to coordinate epidermal development and maintain epidermal integrity. As the epidermis plays a critical role in organ growth in plants (e.g., refs. 39,40,41,42), this, in turn, has major implications for plant development as a whole.

The activity of the CALPAIN domain has been shown to be Ca2+-dependent in vitro43. These findings are in line with results in animal systems where the activation of cytoplasmic calpains is associated with the Ca2+-dependent autolytic-removal of an N-terminal extension, which in some cases can inhibit calpain activity44,45,46. Because calpains have been shown to act downstream of mechanosensitive channels such as PIEZO in animals4, we tested the hypothesis, and demonstrated, that the TM domain of DEK1 is associated with Ca2+ dependent mechanoperception at the plasma membrane in plants.


A rapidly mechanically activated plasma membrane Ca2+ channel

Combining genetic and electrophysiological approaches, the Arabidopsis MSL9 and MSL10 proteins have been shown to mediate a mechanically activated channel activity in protoplasts derived from wild-type plant root cells13. Selectivity characterization indicated that anions permeate preferentially through the wild-type MSL channels13. Because in this work we used excised membrane patches from callus protoplasts, rather than whole root cell-derived protoplasts, we first investigated whether mechanically activated ion currents due to MSLs and other unidentified channels could also be detected in this material.

Reverse transcription quantitative-PCR (RT-qPCR) analysis confirmed the expression of MSL9 and MSL10 in wild-type (Columbia-0) callus tissue (Supplementary Fig. 1a). When Cl is provided at the cytosolic face of plasma membrane patches, a mechanically activated channel with a high conductance (46 ± 1.4 pS; ± indicates standard deviation, n = 6) is elicited upon application of positive pressure using a high-speed pressure clamp, as previously described in root protoplasts (Supplementary Fig. 1b)13. This activity, which is mainly due to MSL9 and MSL10, was completely abolished in protoplasts obtained from quintuple msl4 msl5 msl6 msl9 msl10 (mslΔ5) mutant callus (Supplementary Fig. 1c). In addition, to MSL activity, we observed a rapidly activated-inactivated current with a smaller conductance, elicited immediately after the pressure increase (Supplementary Fig. 1b). Unlike the situation for the MSL current, removing Cl from the cytosolic face of the membrane did not modify this rapid current (Fig. 1a, Supplementary Fig. 1b). The rapid current was still present in protoplasts derived from mslΔ5 mutant callus with or without Cl ions at the cytosolic face (Fig. 1a, Supplementary Fig. 1c). In addition this mechanically activated channel showed a similar conductance to that seen in Col-0 callus (Fig. 1b), with a reversal potential at positive voltage (E revWT = 47.5 ± 5.4 mV and E rev msl∆5 = 60.6 ± 11.5 mV; ± indicates standard deviation, n = 6) suggesting that this rapid current is most likely mediated by Ca2+ influx (dominant cation gradient, E Ca2+=128.7 mV). Consistent with this hypothesis, we found that it could be reversibly eliminated by replacing Ca2+ in the bath solution with the larger TEA+ cation (Fig. 1c, d). The pressure required to activate the current was higher than that required to activate MSL channels, suggesting distinct activation thresholds for these channels (Supplementary Fig. 1b, c). Thus, we can distinguish at least two independent mechanically activated channel activities in callus protoplasts: one dependent on MSLs activity and the second due to an unidentified Ca2+ channel which we called the rapid mechanically activated (RMA) channel.

Fig. 1
figure 1

A mechanically activated current permeable to Ca2+ is present at the plasma membrane. a Representative membrane patches from Col-0 (left) and an msl quintuple mutant msl4;msl5;msl6;msl9;msl10 (mslΔ5, right), exposed to increased positive pressure steps in an outside-out patch configuration show a rapidly activated, rapidly inactivated current in ionic conditions favorable for Ca2+ current recording. We have named the channel responsible for this activity the RMA channel (see text). Time constants are means ± SE (n = 6); b Under the same conditions, single channel I/V curves show similar RMA channel conductance in Col-0 (solid square) and in the mslΔ5 mutant (open square). Values are means ± SE (n = 6); c Representative single channel recordings showing that the RMA current is reversibly eliminated by exchanging Ca2+ ions with non-permeant TEA+ ions. d Open probability (for n channels, (nP(o)) is severely decreased in TEA+ bath solution. A paired t-test was used to compare means (**P < 0.01). Values are means ± SE. For all experiments, the membrane potential was held at −196 mV. Ionic conditions are described in the Methods section

To understand the nature of the RMA-mediated current we tested a range of pipette and bath solutions. The RMA channel conductance is unaffected when Ca2+ ions are replaced with Ba2+ ions in the bath solution (Fig. 2a, b) suggesting that the RMA channel is similarly permeant to divalent Ca2+ and Ba2+ ions, as is classically found for Ca2+ channels. To test whether the RMA channel shows characteristics previously described for mechanically activated channels in plants, we investigated the effect of Gd3+ ions, which are known to affect mechanically activated channel activity in both animal and plant systems23, 47,48,49,50,51,52. RMA activity was almost totally blocked by the presence of Gd3+ ions, and activity could subsequently be restored by washing out Gd3+ (Fig. 2c). The presence of La3+ ions, a known blocker of Ca2+ channels53,54,55 strongly reduced the mean open time of the RMA channel (Fig. 2d, e) but did not modify the channel conductance. We conclude that the RMA channel activity fulfils the key criteria previously attributed to mechanically sensitive Ca2+ channel activities recorded at the plant plasma membrane.

Fig. 2
figure 2

The mechanically activated RMA current is preferentially permeable to Ca2+ and affected by both the mechanosensitive channel inhibitor Gd3+ and the calcium channel blocker La3+. a Representative single channel recordings in response to positive pressure, in an outside-out patch configuration with Ca2+ (top) or Ba2+ (bottom) as the permeant bivalent cation in the bath. The dotted lines indicate the open (O) and closed (C) channel state; b Single channel I/V curves show similar conductance with permeant cations Ca2+ (solid square) and Ba2+ (open square). Values are means ± SE (n = 6); c The mechanically activated RMA current is inhibited by Gd3+ and restored after wash-out (left, n = 6) and the corresponding open probability (nP(o), right). Values are means ± SE; d Representative recording showing the effect of La3+ (left, n = 6) on the RMA current; e La3+ ions affects the open state of the RMA channel and reducing the mean open time. Bars represent the two time constant τ1 and τ2 of the open state with (white bars) or without (black bars) La3+. A paired t-test was used to compare means (P < 0.01). Erev(Ca2+) = 47.5 ± 5.4 mV; Erev(Ba2+) = 36.5 ± 6.1 mV. Values are means ± SE (open events, n ≥ 100). For all experiments, the membrane potential was held at −196 mV. Ionic conditions are described in the Methods section

DEK1 trans-membrane domains are required for RMA activation

Loss of DEK1 activity in Arabidopsis leads to early embryo lethality (Supplementary Fig. 2a)29, 30. By expressing the cytoplasmically localized CALPAIN domain under a constitutive promoter (the RPS5A promoter) in dek1 mutant alleles32, we could restore post-embryonic growth and thus investigate the contribution of the DEK1 TM domain to RMA activation. This was done in both the dek1-2 mutant background (which has a T-DNA insertion toward the beginning of the TM encoding regions (Supplementary Fig. 2b, c)32) and in the dek1-3 mutant background (which contains a T-DNA downstream of the TM encoding regions of the DEK1 gene (Supplementary Fig. 2b, c)32). Calli were generated from both dek1-2 CALPAIN-OE and dek1-3 CALPAIN-OE lines and genotyped (Supplementary Fig. 3a−c). In callus from CALPAIN complemented dek1-3 mutants (dek1-3 CALPAIN-OE), we detected wild-type levels of the transcript corresponding to the endogenous TM-span encoding region of DEK1 (Supplementary Figs. 2c, 4a, b). This allele therefore has the potential to encode a truncated version of the DEK1 protein, with an intact set of TM spans. However, in callus from complemented dek1-2 mutants (dek1-2 CALPAIN-OE), the TM domain-encoding region of the DEK1 gene is physically disrupted (Supplementary Figs. 2c, 4a, c) and an aberrant transcript is produced downstream of the T-DNA insertion (Supplementary Fig. 4b). This transcript is unlikely to be translated to produce a functional truncated protein as the dek1-2 allele shows a null phenotype. Overexpression of the CALPAIN transcript was detected in both backgrounds (Supplementary Fig. 4b). We also generated an antibody against the CALPAIN domain and verified overexpression at the protein level (Supplementary Figs. 5a, b, 6).

Because the dek1-2 CALPAIN-OE background cannot produce the intact N-terminal region of DEK1, we next tested the potential effect of DEK1 TM domain disruption on the RMA channel activity. In the majority of responsive patches obtained from dek1-2 CALPAIN-OE calli we observed a complete absence of the RMA current upon membrane stretching (Fig. 3a, Supplementary Fig. 7). Only a residual current was detectable in most patches of this background. In patches from dek1-3 CALPAIN-OE calli the RMA channel activity was still detectable, but showed 6–8 times more rapid inactivation kinetics (Fig. 3a, b, Supplementary Fig. 7) compared to those observed in callus from Col-0 plants.

Fig. 3
figure 3

RMA activation depends on the trans-membrane region of the essential DEK1 protein. a Mechanosensitive current kinetics in response to pulse pressure (as illustrated) in dek1-2 CALPAIN-OE, dek1-3 CALPAIN-OE, Col-0 CALPAIN-OE, dek1-2 DEK-GFP and dek1-3 DEK-GFP. Time constants are means ± SE (n = 6); b Inactivation times are fitted with an exponential function, and time constants (τinact) are represented in a box plot distribution for different lines. Number in brackets represent the number of time constants. *significantly different from Col-0 line, Anova on ranks (P < 0.05). For all experiments the membrane potential was held at −196 mV. Ionic conditions are described in the Methods section

The production of viable dek1 mutant plants, as mentioned before, requires the expression of at least the CALPAIN domain of DEK1. In our lines, the CALPAIN domain is overexpressed in the callus system at the RNA level (Supplementary Fig. 4). To confirm that the loss of the RMA channel activity in the dek1-2 CALPAIN-OE background is not a consequence of CALPAIN overexpression, we generated calli from Col-0 plants over-expressing the CALPAIN domain (Supplementary Figs. 4d and 5)18. This material was submitted to electrophysiological analysis. The RMA current was still present in patches from this material, and behaved similarly to the current in Col-0 patches. (Fig. 3a, b, Supplementary Fig. 7). Therefore, we conclude that the CALPAIN domain overexpression per se is not responsible for the loss of RMA channel activity in the dek1-2 CALPAIN-OE background.

Loss of the DEK1 TM domains does not affect MSL activity

Next, we investigated whether the removal of the DEK1 TM domains could cause a perturbation of other mechanoresponsive channel activities at the plasma membrane by testing whether MSL channels can be activated in the dek1-2 CALPAIN-OE line. We found that the MSL activity in dek1-2 CALPAIN-OE protoplasts was still detectable upon membrane stretching, and its characteristics were comparable to that of Col-0 (Supplementary Fig. 8). Thus, loss of the DEK1 TM domains leads to a specific perturbation of the mechanosensitive RMA channel activity in our system.

DEK1 function influences root growth inhibition by Gd3+ ions

Based on the mild developmental defects observed in the dek1 CALPAIN-OE lines32, 56, the CALPAIN domain of DEK1 is likely the “active” domain of the protein. The DEK1 CALPAIN domain is autolytically cleaved from the full length DEK1 protein32, and its activity has been shown to be Ca2+ dependent in vitro 43. Because the release of the CALPAIN domain upon DEK1 cleavage could be triggered by Ca2+ influx via the RMA channel, blocking the RMA channel might affect development in wild-type plants, but would have a reduced impact in the dek1-2 CALPAIN-OE line, where the expression of a “pre-cleaved” version of the DEK1 CALPAIN would partially bypass the need for the RMA channel activity. Considering the sensitivity of the RMA current to Gd3+, and the absence of this current in CALPAIN complemented dek1-2 mutants we tested whether CALPAIN complemented dek1-2 mutants might show such reduced sensitivity to Gd3+. Gd3+ dramatically inhibits Arabidopsis root growth57. We generated a dose sensitivity curve of wild-type Col-0 seedlings to concentrations of Gd3+ between 0 and 200 μM (Supplementary Fig. 9). We found strong growth inhibition (40–50%) in wild-type seedlings even at the lowest concentrations tested (30 and 60 μM) (Supplementary Fig. 9). This inhibition was alleviated in CALPAIN complemented dek1-2 plants (Supplementary Fig. 10), formally relating DEK1 function to Gd3+ sensitivity in planta.

Reintroducing full-length DEK1 restores RMA current

We hypothesize that the loss of RMA current observed in dek1-2 CALPAIN-OE callus is caused by the absence of an intact DEK1 transmembrane region. We propose that in the dek1-3 CALPAIN-OE plants a truncated DEK1 protein, which contains the DEK1 transmembrane spans but which cannot confer normal inactivation kinetics on the RMA current is produced (Supplementary Fig. 2c). To test both these hypotheses we investigated the RMA channel activity in both the previously published and characterized dek1-3 line complemented with the full length GFP-tagged DEK1 protein expressed under the RPS5A promoter (dek1-3 DEK1)30, and in a dek1-2 mutant background into which the same, complementing full length DEK1-encoding construct had been introduced by crossing (dek1-2 DEK1). In the majority of patches obtained from these lines, we recorded an RMA channel activity with inactivation kinetics and current amplitude resembling those of wild-type plants (Fig. 3a, b; Supplementary Fig. 7). The properties of this restored current were tested in the complemented dek1-2 DEK1 mutant callus and found to be strikingly similar to those of the RMA current (Supplementary Fig. 11). Our results show that reintroducing the full length DEK1 into dek1 mutant backgrounds leads to the restoration of an RMA current similar to that observed in wild-type callus.


In summary, we show that the mechanosensitive activity of a Ca2+-permeable channel present in the plasma membrane of Arabidopsis callus-derived protoplast requires the TM domains of the DEK1 protein. It has previously been demonstrated that the CALPAIN domain of DEK1 is released from the plasma membrane by an autolytic cleavage event32, and that the CALPAIN activity is enhanced by Ca2+ ions43. We therefore propose a model in which DEK1 activity leads to transient elevation of cytoplasmic Ca2+ concentration during mechanical stimulation, which is locally transduced by autolytic cleavage (and thus activation) of the CALPAIN domain32. Based on the phenotype of lines with reduced DEK1 activity, this mechanotransduction pathway is likely required for the maintenance of cell−cell contacts and epidermis integrity33, consistent with embryo lethality in loss of function alleles. In turn, DEK1-dependent epidermis integrity is required for the propagation of mechanical signals between neighboring cells, in a feedback loop. Given the absolute requirement for DEK1 in both coherent embryogenesis and post-embryonic growth29,30,31, 33 (summarized in Supplementary Fig. 12), our work suggests a key contribution of mechanoperception to plant development. The absence of cell migration in plant tissues may explain why the perception of tension between adjacent cells plays such an essential role in development in this kingdom.

This study, together with previously published results from both our work and the work of others32, 56 supports the idea that the CALPAIN domain of DEK1 is the effector component of the protein in terms of mechanotransduction. Whether the transmembrane portion of DEK1 forms a mechanically activated Ca2+ channel per se, or it is the mechano-sensor associated with an independent Ca2+ channel remains to be determined. However, the published Ca2+ dependence of CALPAIN catalytic activity, combined with our electrophysiological results and the fact that CALPAIN complemented dek1 null mutants develop relatively normally, suggests that once activated by cleavage, the CALPAIN domain of DEK1 can respond to changes in cytoplasmic Ca2+ levels mediated by other Ca2+ channels. These could include proteins such as MCA1 and MCA2, or potentially the plant PIEZO protein, which has yet to be functionally characterized. The presence of such channels is consistent with our finding that CALPAIN complemented dek1-2 plants retain some Gd3+ sensitivity in planta.

Interestingly, intracellularly localized calpains have been proposed to act downstream of mechanosensitive ion channels in animals, to regulate a variety of cellular processes, including cell-to-cell adhesion58,59,60,61. In this context, the association of a calpain protease with a domain influencing a mechanosensitive Ca2+ channel activity in the membranes of multicellular plants may reflect evolutive convergence between these two kingdoms.

In animals, the characterization of mechanosensitive channels Piezo1 and Piezo2 has revealed novel roles of mechanoperception in development and physiology. For instance, respiration in lungs relies on Piezo2-expressing sensory neurons, which use mechanical signals to sense airway stretching62. Axon growth and trajectory was also shown to depend on Piezo1-dependent perception of the mechanical environment of neurons63. The association of DEK1, a key developmental regulator, with a mechanosensitive channel activity paves the way to the identification of new roles of mechanical signals in plant development.


Plant material

Arabidopsis (Arabidopsis thaliana) Columbia-0 (Col-0) wild-type seeds were obtained from Nottingham Arabidopsis Stock Centre (NASC, School of Biosciences, University of Nottingham, United Kingdom). Plants expressing the pRPS5A:CALPAIN-HIS in a wild-type background (CALPAIN-OE), pRPS5A:CALPAIN-GFP, or pRPS5A:DEK1 in the dek1-3 mutant background and pRPS5A:CALPAIN-GFP in the dek1-2 background have been previously described18, 30. The pRPS5A:DEK1 construct was transferred from the dek1-3 to the dek1-2 background by crossing, and plants were genotyped as described in Supplementary Fig. 3.

Plant and callus growth conditions

For in vitro cultures, seeds were surface-sterilized with chlorine gas, sown on square plates, and stratified for 2 or 3 days in the dark at 4 °C. After stratification, seeds were germinated in a growth chamber under a 16-h light/8-h dark cycle at 21 °C.

Callus generation

Surface-sterilized seeds were sown on “initiation medium” containing 4.3 g/L Murashige and Skoog salts (MS, Sigma-Aldrich), 2% sucrose, 10 mg/L myo-inositol, 100 µg/L nicotinic acid, 1 mg/L thiamine-HCl, 100 µg/L pyridoxine-HCl, 400 µg/L glycine, 0.23 µM kinetin, 4.5 µM 2,4-D, 1% Phytagel, (pH 5.7). For callus generation, seeds were cultured in a growth chamber for 15 days. Calli were then transferred onto “maintenance medium” containing 4.3 g/L MS salts (Sigma-Aldrich), 2% sucrose, 10 mg/L myo-inositol, 100 µg/L nicotinic acid, 1 mg/L thiamine-HCl, 100 µg/L pyridoxine-HCl, 400 µg/L glycine, 0.46 µM kinetin, 2.25 µM 2,4-D, 1% phytagel, (pH 5.7), and sub-cultured every 15 days onto fresh “maintenance medium”.

Protoplasting protocol

Calli were digested for 15 min at 22 °C under hyperosmotic conditions (2 mM CaCl2, 2 mM MgCl2, 1 mM KCl, 10 mM MESs (pH 5.5), 0.2% cellulysin (Calbochem), 0.2% cellulase RS (Onozuka RS, Yakult Honsha Co.), 0.004% pectolyase Y23 (Kikkoman Corporation), 0.35% bovine serum albumine and mannitol to 600 mOsmol. For enzyme removal, the preparation was washed twice with 2 mM CaCl2, 2 mM MgCl2, 10 mM MES (pH 5.5), and mannitol to 600 mOsmol. For protoplast release, the preparation was incubated with 2 mM CaCl2, 2 mM MgCl2, 10 mM MES (pH 5.5), and mannitol to 280 mOsmol. The suspension was filtered through a 50 µm nylon mesh.


Patch-clamp experiments were performed at room temperature with a patch-clamp amplifier (model 200 A, Axon Instruments, Foster City, CA) and a Digidata 1322 A interface (Axon Instruments). Currents were filtered at 5 kHz, digitized at 20 kHz, and analyzed with pCLAMP8.1 and Clampfit 10 software. During patch-clamp recordings, cells were held at a holding potential (corrected from liquid junction potential) of −16 or −6 mV depending on the composition of the pipette solution and pressure was applied with a high speed pressure-clamp system (ALA Scientific Instrument, NY), allowing the application of precise and controlled pressure pulses in the pipette15, 64. For Ca2+ and Ba2+ current recordings, bath solutions contained: 50 mM CaCl2 or 50 mM BaCl2, respectively, and 5 mM MgCl2, 10 MES-Tris (pH 5.6); while pipettes were filled with: 150 mM CsMES, 2 mM MgCl2, 5 mM EGTA, 4.2 mM CaCl2, and 10 mM Tris-HEPES (pH 7.2), supplemented with 5 mM MgATP. To remove Ca2+, a solution containing: 100 mM TeaCl, 5 mM MgCl2 and 10 mM MES-Tris (pH 5.6) was used.

For Cl current recordings, bath solution contained (mM): 50 CaCl2, 5 MgCl2, 10 MES-Tris (pH 5.6) and pipettes were filled with (mM): 150 CsCl, 2 MgCl2, 5 EGTA, 4.2 CaCl2, and 10 Tris-HEPES (pH 7.2), supplemented with 5 MgATP. For inhibitor treatments, 0.25 mM LaCl3 or 0.25 mM GdCl3 were added to the bath solution, osmolarity was adjusted with mannitol to 450 mOsmol for the bath solution and to 460 mOsmol for the pipette solution using an osmometer (Type 15, Löser Meβtechnik). Gigaohm resistance seals between pipettes (pipette resistance, 0.8–1.5 MΩ) (coated with Sylgard (General Electric) pulled from capillaries (Kimax-51, Kimble Glass)) and protoplast membranes were obtained with gentle suction leading to the whole-cell configuration, then excised to an outside-out configuration. The current inactivation kinetics were fitted with a mono-exponential function: F(t) = A × e-t/τ + C, where A is the coefficient, τ is the time constant, and C represents the maximum current intensity.

Genomic DNA extraction and genotyping

Plant callus DNA was extracted using a rapid CTAB isolation technique65. dek1-2 and dek1-3 mutants were genotyped using the primers shown in Supplementary Table 1 and Supplementary Fig. 3. The following thermal profile was used: 95 °C for 5 min, 35 cycles of 95 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min, and a final extension step of 5 min at 72 °C.

RNA extraction and quantitative gene expression analysis

Callus material was collected and snap-frozen in liquid nitrogen for gene expression analysis. For each experiment, at least two independent biological replicates were used. Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich). Total RNAs were digested with Turbo DNA-free DNAse I (Ambion) according to the manufacturer’s protocol. RNA concentrations were measured with a NanoDrop ND-1000 UV−Vis spectrophotometer (NanoDrop Technologies). One microgram of total RNA was reverse transcribed (RT) using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s instructions. PCR reactions were performed in optical 96-well plates in the StepOne Plus Real Time PCR System (Applied Biosystems). Five microliters of a 1:10 dilution of cDNA was amplified in 20 µL of reaction mix. The following thermal profile was used: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 30 s. Amplicon melting curves, were recorded after cycle 40 by heating from 60 to 95 °C with a ramp speed of 1 °C min−1. Expression levels of each gene, relative to EIF4A, were determined using a modification of the Pfaffl method33. Technical triplicates were performed for each biological sample. Primers used for RT-quantitative (q)PCR analysis are shown in Supplementary Table 1.

Antibody production

Anti-IIaIIb rabbit polyclonal antibodies were generated against a peptide from the predicted catalytic domain (RGDKQFTDQEFPPNC) of the Arabidopsis DEK1 protein using the PolyExpress Custom Polyclonal Antibody Production Package Service (GenScript).

Western blot analysis

Callus material for each genotype was snap-frozen in liquid nitrogen, thoroughly ground in pre-cooled porcelain mortars, and proteins were extracted using an extraction buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1% deoxycholate, 1 mM EDTA and 1× protease inhibitor cocktail P9599 (Sigma-Aldrich). Samples were incubated on ice for 1 h, centrifuged for 15 min at 14,000×g at 4 °C to remove cell debris, and protein concentrations in supernatants were determined using a Bio-Rad protein assay (Bio-Rad). Equal amounts of proteins were loaded and resolved on 7.5% polyacrylamide/0.1% SDS gels. Proteins were transferred onto nylon membranes using an iBlot II dry blotting system (Invitrogen). Membranes were blocked over night at 4 °C using 1× PBS/0.2% Tween-20 (Sigma-Aldrich) with 5% non-fat milk (Regilait), incubated for 2 h at room temperature with rabbit polyclonal anti-IIaIIb (Genescript) or mouse monoclonal anti-alpha tubulin clone B-5-1-2 (Sigma-Aldrich, catalogue number T5168) primary antibodies at dilutions of 1:1000 and 1:2000, respectively. Secondary horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Promega, catalogue numbers S3731 and S3721, respectively) were used at a dilution of 1:45,000. Blots were incubated with Super Signal West Femto reagents (Thermo Scientific) according to the manufacturer’s instructions, and then exposed to Super RX film (Fujifilm). Membranes were first incubated with anti-IIaIIb antibodies, and then re-probed with anti-alpha tubulin antibodies.

Gadolinium sensitivity experiments

Seeds were surface-sterilized with chlorine gas, sown on square plates (in a single row in the upper part of the plates), and stratified for 2 days in the dark at 4 °C. After stratification, seeds were germinated in a growth chamber under a 16-h light/8-h dark cycle at 21 °C, with plates kept in a vertical position. Plates containing 1/5 strength (0.86 gr/L) MS medium (Duchefa) pH = 5.7, 1% sucrose and 0.8% phytoagar, were supplemented with gadolinium (III) chloride hexahydrate (Sigma-Aldrich). Primary root length was scored after 5 or 6 days of growth with ImageJ software. Different genotypes were sown side by side on the same plate and in multiple combinations to buffer possible positional effects.

Data availability

The authors declare that all data supporting the findings of this study are available within the manuscript and its Supplementary Files or are available from the corresponding authors upon request.


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This work was funded by the European Research Council consolidator grant #615739 “MechanoDevo” to O.H. and the ANR Chaire d’excellence #ANR-10-CHEX-0011 “Mechanograine” to G.C.I. as well as a team grant from the Fonds de Recherche du Quebec-Nature et Technologies to R.S.-N. and A.G. E.D.N. was supported by a PhD studentship from the BBSRC. R.G. was also supported by the European Research Council consolidator grants #307387 “PhyMorph”. We would like to thank Professor Mike Blatt (University of Glasgow, UK), Dr. Anne-Aliénor Very (BPMP Montpellier, France), Prof. Arezki Boudaoud (ENS de Lyon, France) and Dr Kim Johnson (University of Melbourne, Australia) for their help and for insightful discussions. We would like to thank Claire Lionnet of the Platim microscopy facility and the RDP growth facility team (coordinated by Alexis Lacroix), logistics team, and secretarial team for their invaluable technical support.

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D.T., R.G., and A.D. performed the experiments and prepared the figures. E.N. carried out important groundwork. G.C.I., O.H., R.S.-N., A.G., and J.-M.F. supervised the work. The paper was written by G.C.I. with active input from R.G., O.H., J.-M.F., and D.T.

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Correspondence to Olivier Hamant or Gwyneth C. Ingram.

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Tran, D., Galletti, R., Neumann, E.D. et al. A mechanosensitive Ca2+ channel activity is dependent on the developmental regulator DEK1. Nat Commun 8, 1009 (2017).

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