PIEZO ion channel is required for root mechanotransduction in … · 2020. 8. 27. · 1 1 PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana 2 Seyed

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PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana 1

Seyed A. R. Mousavi1*, Adrienne E Dubin1, Wei-Zheng Zeng1, Adam M. Coombs1, Khai Do1, 2

Darian A. Ghadiri1, Chennan Ge2, Yunde Zhao2 and Ardem Patapoutian1* 3

4

1. Howard Hughes Medical Institute, Department of Neuroscience, Doris Neuroscience Center, 5

The Scripps Research Institute, La Jolla, California 92037, USA. 6

2. Section of Cell and Developmental Biology, University of California San Diego, La Jolla, 7

California 92037, USA. 8

*Correspondence to: Ardem Patapoutian ([email protected]); Seyed Ali Reza Mousavi 9

([email protected]) 10

11

Summary: 12

Plant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. 13

As in animal species, mechanosensitive ion channels in plants are proposed to transduce external 14

mechanical forces into biological signals. However, the identity of these plant root ion channels 15

remains unknown. Here, we show that Arabidopsis thaliana PIEZO (AtPIEZO) has preserved the 16

function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO 17

is highly expressed in the columella and lateral root cap cells of the root tip which experience 18

robust mechanical strain during root growth. Deleting PIEZO from the whole plant significantly 19

reduced the ability of its roots to penetrate denser barriers compared to wild type plants. piezo 20

mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, 21

supporting a role of AtPIEZO in root mechanotransduction. Finally, a chimeric PIEZO channel 22

that includes the C-terminal half of AtPIEZO containing the putative pore region was functional 23

and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest 24

that Arabidopsis PIEZO plays an important role in root mechanotransduction and establishes 25

PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant 26

kingdoms. 27

28

Main 29

Plants extend roots within the soil to access water and nutrients as well as provide stability for the 30

aerial parts of the plant. Underground barriers caused by drought and/or heterogeneous soil 31

components can exert mechanical resistance that alters root extension and penetration1-3. The root 32

cap at the very tip of the primary root is a dynamic organ that includes different classes of stem 33

cells which divide asymmetrically and is essential for growth through harder media and soils4. 34

Bending or poking root tips elicits a transient Ca2+ influx with short latency that is blocked by 35

lanthanides including Gd3+, a non-selective inhibitor of mechanically-activated (MA) cation 36

channels5-7. However, the molecular identity of putative ion channels underlying this response is 37

unknown. Only a few mechanosensitive ion channels have been described in plants8. MSL8, plays 38

a mechanosensory role in pollen9, MSL10 is involved in cell swelling8,10, and OSCA1 has mainly 39

been characterized for its role in osmosensation11. It has been proposed that MCA1, expressed in 40

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https://doi.org/10.1101/2020.08.27.270355

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the elongation zone but not the root cap, is a stretch-activated calcium permeable ion channel 41

involved in soil penetration; however, evidence for its being a bona-fide ion channel capable of 42

detecting mechanical force is lacking12-14. The genome of Arabidopsis thaliana encodes an 43

ortholog of the mammalian mechanosensitive ion channels PIEZO1 and PIEZO215. Given that 44

PIEZOs play prominent roles in multiple aspects of animal mechanosensation and physiology16-45 19, we investigated the role of AtPIEZO in plant mechanosensation. A recent study reported that 46

AtPIEZO regulated virus translocation within the plant, but its specific role in 47

mechanotransduction was not addressed20. Here we use genetic tools, electrophysiological 48

methods and calcium imaging to investigate the role of AtPIEZO in root mechanosensation. 49

To localize the expression of AtPIEZO in Arabidopsis, we used AtPIEZO promoters fused to the 50

reporter gene β-glucuronidase (GUS) and generated two AtPIEZOpro::GUSPlus constructs with 51

different promoter lengths, 823 bp and 2000 bp. Both constructs showed similar GUS expression, 52

with high levels observed in upper root, both primary and lateral root caps, and pollen grains 53

(Fig.1a, c, and Extended Data Fig. 1). We also detected GUS activity in the root vasculature and 54

in trichromes (plant hairs) (Fig.1b and Extended Data Fig. 1). Cross-sections of root tips revealed 55

expression in lateral root cap (LRC) cells and columella cells (Fig.1g,h), that are thought to be 56

important in detecting mechanical forces during root penetration into the soil4. When plants were 57

grown inside Murashige and Skoog medium (0.5X MS; 0.85% agar (8.5 g/l)) rather than on top of 58

it, higher GUS signal intensity was observed in the upper root and root cap of the seedlings 59

suggesting expression is enhanced when mechanical stress is applied to roots (Fig.1d,e). This 60

increase in GUS activity was confirmed by quantitative real time PCR; AtPIEZO expression was 61

3-fold higher in plants grown inside MS media (Fig.1f). 62

63

Next, to investigate the role of AtPIEZO in plant physiology and development, we generated two 64

piezo CRISPR/Cas9 knockout mutant lines: one in which the entire gene was deleted (referred to 65

as piezo-FL), the other in which the C-terminal half of the gene that encodes the putative channel 66

pore based on its homology to mouse PIEZO1 (mPIEZO1) was deleted (referred to as piezo-CT). 67

AtPIEZO has 27% amino acid identity with mPIEZO1 with similar overall topology and 38 68

predicted transmembrane domains (Extended Data Fig.2). We confirmed the lack of AtPIEZO 69

transcripts in both mutants by PCR and RT-qPCR in samples harvested from the leaves as well as 70

the roots (Extended Data Fig. 3). We did not observe any significant growth difference between 71

WT and piezo mutants in roots or aerial parts when grown in MS media. 72

73

Based on the robust root expression of AtPIEZO, we sought to evaluate its role in root growth. We 74

grew A. thaliana seeds on the surface of the MS media and plates were positioned vertically (at a 75

90° angle). The length of the seedling roots of WT and the two mutants were not different when 76

grown on top of the MS media (Extended Data Fig. 4 a, b). However, when seedling roots grew 77

within the MS media, mutant roots were shorter compared to WT. To confirm the differences 78

observed for root penetration and growth inside the media, we challenged the roots at a 60° plate 79

angle to stimulate growth into the media. Again, we observed that the roots of both mutants were 80



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shorter than WT roots (Fig. 2a-b). To further investigate root penetration, plants were grown at 81

60° in MS media containing different agar concentrations (7, 8.5 (standard), and 10 g/l) mimicking 82

different levels of soil hardness. The root lengths of piezo mutants and WT plants were similar in 83

the lowest agar concentration (7 g/l; Extended Data Fig. 4c). However, at higher agar 84

concentrations (8.5 and 10 g/l), the average root lengths of both mutants were significantly shorter 85

by about 17% and 18%, respectively, than observed for WT. These data show that mutants had 86

shorter length than wild type in hard medium (Extended Data Fig. 4c). 87

88

To assess the response of roots to stiff materials that might be encountered during growth, we 89

challenged roots with barriers of varying stiffness consisting of 10, 12, 15, 18 and 21 g/l agar in 90

MS media (Fig. 2d). We plated A. thaliana seeds on the standard agar concentration in MS media 91

(8.5 g/l agar), 2 cm above the barrier. Within 4-5 days after germination, seedling roots of all 92

genotypes reached the barrier. At this point, three different scenarios were observed: 1) penetration 93

across the barrier, 2) root coiling and delayed penetration after growing at the interface surface, or 94

3) no penetration (Extended Data Fig. 5c-d). At a 10 g/l barrier, 80% of WT roots penetrated the 95

harder agar while only 74% and 73% of piezo-FL and piezo-CT, respectively, were able to 96

penetrate (n=9). As the agar concentration increased, the barrier penetration phenotype in the 97

mutants became more pronounced. For example, at 15 g/l, the penetration percentage for WT was 98

58% while only 29% of piezo-FL and 26% of piezo-CT roots penetrated (n=11) (Fig. 2d; 99

Supplementary Video 1). Furthermore, mutants showed a delayed penetration with excessive 100

coiling on the barrier surface (Extended Data Fig. 5d). 101

102

For the roots that penetrated the various barriers, root length inside the barriers was more variable 103

and shorter in the mutants compared to WT (Fig. 2e,f). For example, at a 12 g/l agar barrier 104

concentration, the root length of WT was 5.2±01.2 cm (n=28), while it was 3.4 ±1.3 cm and 105

3.1±1.2 cm for piezo-FL and piezo-CT, respectively (n=25-28) (Fig. 2f). The shorter root length in 106

mutants became more severe in 15 or 18 g/l agar. Although mutant root coiling at the barrier 107

interface delays root penetration and contributes to the decreased total root length, shorter roots 108

are observed for mutants seeded directly into media containing 8.5 and 10 g/l agar (Extended Data 109

Fig. 4c), indicating that the velocity of root growth is slowed in denser media. 110

111

These results implicate a role of AtPIEZO in mechanosensory processes in plants. To assess 112

whether AtPIEZO is a mechanosensitive ion channel like its animal homologs, we cloned the full 113

length coding sequence (7455bp) into a mammalian expression vector (see Methods). Transient 114

heterologous expression of PIEZO proteins from various animal species (including mammals and 115

flies) confer robust MA currents15-17. We heterologously expressed either native or codon-116

optimized AtPIEZO in HEK293T Piezo1 knockout (HEK P1KO) cells21. Neither native nor codon-117

optimized AtPIEZO revealed MA currents in two separate assays for mechanotransduction: poking 118

cells with a fire-polished glass pipette22 and stretching the membrane at the tip of the pipette in 119

cell-attached patch clamp recordings22. To determine whether the lack of response was due to 120



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improper trafficking to the plasma membrane, MYC-tags were inserted at five separate predicted 121

extracellular loops of the protein based on homology between transmembrane domains of 122

AtPIEZO and mPIEZO122-24 (Extended Data Fig 2). Immunostaining with an anti-MYC antibody 123

detected AtPIEZO expression in HEK P1KO cells, however, non-permeabilized staining revealed 124

that AtPIEZO did not traffic to the membrane (Extended Data Fig. 6). We next generated chimeras 125

between mPiezo1 and codon optimized AtPIEZO in an effort to traffic chimeras containing the 126

putative pore domain of AtPIEZO to the membrane. As the pore region of PIEZOs is located at 127

the C-terminus22-25, we generated 7 chimeras between mPiezo1 and AtPIEZO in which the C-128

terminus was derived from AtPIEZO and the N-terminus from mPiezo1 (Extended Data Fig. 2). 129

Using an extracellular Myc tag on the N-terminal mouse-derived sequence, we observed that one 130

of the chimeras mPiezo1/AtPIEZO (CH) with 49% mouse and 51% AtPIEZO trafficked to the 131

membrane of HEK P1KO cells (Fig. 3a and Extended Data Fig. 6). The structural elements 132

required for MA current including the pore, anchor and beam is derived from AtPIEZO. Stretch-133

activated currents (SAC) were observed in 40% of the cell-attached patches recorded from HEK 134

P1KO cells expressing the CH; 76% of patches from mPiezo1-expressing cells revealed SAC. The 135

maximum current elicited (Imax; Fig. 3c), negative pressure thresholds (Fig. 3f) and P50 values (Fig. 136

3g) were similar for mPiezo1 and CH (Extended Data Fig. 7). Interestingly, inactivation of SAC 137

from CH-expressing cells was abrogated and currents were maintained throughout the entire 138

250ms stretch stimulus (Fig.3e vs d; Extended Data Fig. 7). The lower proportion of SAC-139

expressing patches in CH is consistent with that observed for mPiezo222,25). The reversal potential 140

of SACs mediated by mPiezo1 and CH were similar (Fig. 3h, i; stretch-induced current is shown 141

in brown), consistent with CH being a non-selective cation channel. Thus, the chimera containing 142

the pore-containing C-terminus of AtPIEZO is activated by a mechanical stimulus, suggesting that 143

the native AtPIEZO is indeed a non-selective ion channel in plant cells. 144

145

We next investigated whether the observed root growth phenotype could be attributed to 146

compromised PIEZO channel activity in root tips challenged with mechanical forces. To 147

accomplish this, we monitored calcium influx in response to mechanical stimulation in vivo using 148

a GFP based Ca2+ indicator (GCaMP3) expressing transgenic line26. First, we applied a localized 149

stimulation by a blunt glass pipette to the root cap of WT plant in increments of 20 µm (Fig. 4a, 150

b). The transient and localized Ca2+ signals appeared in the columella cells and LRC cells starting 151

at an indentation of ~60 µm, while peak Ca2+ signals were observed at 80 µm of indentation (n=12) 152

(Fig. 4c, d; Supplementary Video 2). At 100 µm indentation and beyond, Ca2+ signals propagated 153

between neighboring cells bidirectionally in a manner similar to a wound-mediated response and 154

were not studied here26,27 (Supplementary Video 3). We next generated a model in which PIEZO 155

was knocked down specifically in columella cells by using the artificial PIEZO-targeting 156

microRNA driven by the PIN3 promoter28 (Fig. 4a). This approach provides an internal control 157

within each root tip: mechanical stimulation-dependent Ca2+ responses in columella cells (where 158

AtPIEZO is knocked down) can be compared with neighboring WT LRC cells. Using this strategy, 159

we observed normal responses in LRC cells but significantly reduced responses in the columella 160



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cells at 80 µm of indentation (n=9, Fig. 4e-h; Supplementary Video 4). indeed, the area under the 161

curve and the peak of GCaMP3 signals were significantly decreased in the columella but not LRC 162

cells of piezo knock-down plants compared to WT plants (4i,j).These data indicate that mechanical 163

stimuli can induce calcium transients in columella cells through AtPIEZO. 164

Plant roots sense physical properties of the soil to either avoid it or penetrate it3. Here, we report 165

that Arabidopsis PIEZO activity is required for proper root penetration in compacted environments 166

imposing mechanical stresses. PIEZO proteins from numerous animal species are established 167

physiologically relevant MA cation channels15-17. We present evidence to suggest that AtPIEZO 168

is functionally conserved as a mechanosensitive ion channel in plant roots. Using calcium imaging 169

we identify at least one cell type in the root cap (columella cells) that requires AtPIEZO to respond 170

to a mechanical stimulus with increased calcium transients. Mutants in other components of 171

calcium signaling pathways such as clm24 (tch2) show similar growth defects to those reported 172

here for piezo29. The receptor-like kinase FERONIA maintains cell wall integrity through a direct 173

interaction between its extracellular domain and components of the cell wall; it has been proposed 174

to activate a calcium permeable channel whose identity is unknown30. AtPIEZO protein might 175

directly alleviate mechanical pressure in columella cells by protecting cell wall integrity and/or 176

by transducing Ca2+ signals to other parts of the root such as the elongation zone. Our findings will 177

enable future research to understand the molecular and cellular pathways involved in 178

mechanotransduction within roots. Our results also suggest that other MA ion channels contribute 179

to barrier penetration since the root growth deficits observed in piezo mutants are incomplete. In 180

summary, we provide evidence that AtPIEZO acts as a mechanosensitive ion channel in root tips: 181

it has appropriate expression in the root, is required for responses to acute mechanical stimulations 182

and for proper root growth, and it forms an ion channel. These results demonstrate that AtPIEZO 183

mediates a mechanosensory function in Arabidopsis, highlighting a conserved function of PIEZOs 184

from plants to mammals. 185

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Method: 190

Plant material and growth conditions 191

Arabidopsis thaliana (Col-0) were soil-grown on a 14h light/10h dark cycle (100 μE s−1 m−2), 192

70% humidity at 22 °C. For seedling propagation, seeds were surface-sterilized with 70% ethanol 193

for 15 min and washed 5 times with sterile distilled water. Seeds were grown on plates 194

containing half-strength Murashige and Skoog solid medium (0.5X MS) (Sigma-Aldrich, 195

M5524), 0.5 g/L MES hydrate (Sigma- M2933), pH adjusted to 5.7, and then supplemented with 196

8.5 g/l agar (Sigma-Aldrich, A1296). Plates were placed in a growth chamber vertically or as 197

indicated. 198

Hard agar media and barrier plates 199

First, we poured 160 ml of higher agar concentration in 0.5X MS media into 200

120mmx120mmx15mm plates. After the plates had cured, we cut the solidified media with a 201

sharp sterile blade into four parts. The first and third segment are removed and replaced with 202

standard MS media containing agar at 8.5 g/L concentration with 5 mm thickness. 203

Generation of AtPIEZOPro:GUSPlus plants 204

In order to determine tissue specific AtPIEZO expression, we used GUSPlus protein under the 205

AtPIEZO promoter. Since, there is an annotated gene close to AtPIEZO in the opposite 206

orientation, two different GUSPlus lines containing promoter sizes of 823 bp and 2000 bp were 207

generated. In order to faster screen the transgenic seeds, we first inserted the FAST 208

(fluorescence-accumulating seed technology) cassette31 into pCAMBIA-0380 plasmid 209

(CAMBIA), referred to as pCAMBIA-FAST. The FAST cassette was amplified from 210

VSP2Pro:GUSPlus plasmid32 using forward 211

atgttgggcccggcgcgccgagatctTCTAGTAACATAGATGACACC and reverse 212

tggctgcaggtcgacgTCTAGAGGTACCCGGGATCCAGTGTATGTAGGTATAGTAACATG 213

primers. pCAMBIA-380 was cut by EcoRI and BamHI restriction enzymes (ThermoFisher). 214

Both insert and plasmid were ligated using the Gibson assembly kit (New England Biolabs). The 215

AtPIEZO promoters were amplified using forward, 216

ttgttcatgttactatacctacatacactgtggaaagaaagtaaaggattag and reverse, 217

agtagccatgTGGAAACTTTTGTCTTAACG primers and GUSPLUS amplified from 218

VSP2Pro:GUSPlus plamid using forward, aaagtttccaCATGGCTACTACTAAGCATTTG and 219

reverse gtcagatctaccatggtggactcctcttaaCAATTCACACGTGATGGTG primers. pCAMBIA-220

FAST was cut using BamHI and HindIII. Both inserts and plasmid were ligated by the Gibson 221

assembly kit. Agrobacterium tumefaciens (GV3101) competent cells were transformed with this 222

plasmid. Seeds that expressed red fluorescence protein (RFP) were selected by fluorescence 223

microscopy. The T3 generation was used for GUS staining experiments. 224

225

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GUS staining and cross sectioning 227

Transgenic Arabidopsis plants expressing β-glucuronidase (GUS) were stained following the 228

protocol described by Jefferson33. 5-10 day old seedlings or flowers were collected. Samples 229

were stained with 1 mM X-Gluc (Thermo Fisher Scientific, R0851) in a pH 7.0 phosphate buffer 230

containing 10 mM EDTA, 0.1 mM potassium ferricyanide, 10% (v/v) Triton X-100 at 37 °C 231

overnight. The tissue was destained with serial dilution of 25%, 50%, 75% and 95% (v/v) 232

ethanol. For root cross sectioning, the GUS stained roots were submerged in a box containing 233

Tissue-Tek O.C.T compound (Sakura FineTek USA) and then frozen in liquid nitrogen. 234

Different layers of the root were sectioned at 15 µm thickness and placed on coverslips. 235

Generating piezo mutants and genotyping 236

Since homozygote seeds from Salk T-DNA lines were unavailable, we used CRISPR/Cas9 gene 237

editing technology to generate piezo-FL (full length deletion) and piezo-CT (C-terminal deletion) 238

knockout mutants34-36. Both piezo mutants were generated in the pNano65 Ca2+ indicator 239

background37 and these lines and pNano65 WT were used for all experiments except Ca2+ 240

imaging experiments. Guide RNA 4 and 6 were used for generating a whole deletion in the 241

PIEZO gene, and guide RNA 4 and 5 were used for generating a C-terminal deletion from the 242

LGYL motif to the end of the PIEZO gene (1253aa to 2485aa). The target sequences for the 243

whole deletion in PEIZO were target 4 CCCTCTGCTCTAGCCGCGTACA and target 6 244

AGGTGCAATATAGTGAACGAGG (PAM sites were underlined). Targets for the partial 245

mutants were target 4 and target 5 CCCAGGTGTAGAATGTCATACT. Primers for 246

genotyping: Piezo-GT1 TCTGCCACATTCCCACTCAG, Piezo-GT2 247

GGTTTAGCCATTTCTCGGCG, Piezo-GT3 CGGGAGTGTTGGCTTGGTAT, Piezo-GT4 248

CCGCCACGTAAGTTAGCTCT. The piezo-FL mutants were genotyped with the primer pair 249

Piezo-GT1 and Piezo-GT2, which generates a PCR product of 1300 bp in mutants. This primer 250

pair could not amplify WT genomic DNA due to the large size of the fragment. To determine 251

zygosity of piezo-FL mutants, we used the Piezo-GT1+ Piezo-GT4, which amplifies a 949bp 252

product from WT DNA, but could not amplify a band in the homozygous mutant. For the C-253

terminal deletion mutant piezo-CT, we used Piezo-GT1 and Piezo-GT3, which would generate a 254

fragment of about 1000bp in mutants. PCR of Piezo-GT1+ Piezo-GT4 was performed for 255

determine the zygosity of piezo-CT as well. 256

Generating piezo knockdown mutant 257

We used atMIR390 microRNA38 to knockdown PIEZO in columella cells. We synthesized 258

atMIR390 microRNA that included the PIEZO targeting sequence of 259

TGCAGTTGCTCGGTCTTCCGA and amplified using the forward 260

TTTTTGTCCCTTCAAGTATAGGGGGGAAAAAAAGGTAG and reverse 261

tcttaaagcttggctgcaggGAGACTAAAGATGAGATCTAATC primers. PIN3 promoter was 262

amplified using the forward 263

cggcgcgccgaattcccgggAATTTTATTGCATATAGTGTGTTTATTAAATG and reverse 264

TTTTTTCCCCCCTATACTTGAAGGGACAAAAATGGAAAAC primers. pCAMBIA-1380 265



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plasmid was cut by BamHI and SalI restriction enzymes, and the cut plasmids and both 266

fragments were ligated using the Gibson assembly kit. Agrobacterium tumefaciens, GV3101 267

competent cells were then transformed by this plasmid. GCaMP3 plants were transformed using 268

Agrobacterium tumefaciens bacteria. MS media containing 10mg/l Hygromycin B was used for 269

screening the transgenic seeds. Two different independent T3 transgenic lines used for Ca2+ 270

imaging. 271

Quantitative RT-PCR 272

The upper roots of the Arabidopsis plant grown either inside or on top of the surface of the MS 273

media were harvested and total RNA was extracted using the RNeasy plant mini extraction kit 274

(Qiagen). 1 µg of the total RNA was copied into cDNA with The SuperScript III First-Strand 275

Synthesis according to the manufacturer’s instructions (ThermoFisher). Quantitative RT-PCR 276

was performed on 50 ng of cDNA in final volume of 10µl according to the SsoAdvanced 277

Universal SYBR® Green supermix (BioRad). Ubiquitin-conjugating enzyme (UBC21) 278

At5g25760 was used as a reference gene. Four biological replicates, which were mixture of 3-5 279

plants, were used for each experiment. Primers used were: UBC21 (At5g25760) forward 280

CAGTCTGTGTGTAGAGCTATCATAGCAT, reverse AGAAGATTCCCTGAGTCGCAGTT, 281

AtPIEZO (AT2G48060) forward ACGCTCTGATATCCAAATGGT, and reverse 282

ACTTCATCCGTCTGATCCTC. 283

Cloning AtPIEZO 284

The full length of 7455 bp of AtPIEZO were amplified in two parts from Arabidopsis total RNA 285

extracted from roots and leaves. The AtPIEZO gene was cloned into pCDNA3-1 IRES2-eGFP 286

Zeo+ (AtPIEZOGFP), a mammalian expression vector. We also generated a codon-optimized 287

AtPIEZO for expression in mammalian cell culture (HEK P1KO cells). 288

Generating Myc tagged plasmids 289

The site of Myc tag insertion were chosen based on homology alignments between mPIEZO1 290

and AtPIEZO and the tag shown to work for these experiments in the extracellular loop of 291

mPIEZO1. The position of the Myc tag is highlighted in Extended Data Fig. 2. The sequence of 292

Myc tag (GAACAAAAACTTATTTCTGAAGAAGATCTG) were insterted in the primers. The 293

Myc tag insertion was cloned into pCDNA3-1 IRES2-eGFP Zeo+ (AtPIEZO GFP). Myc tag 1-3 294

were inserted in codon optimized AtPIEZO and Myc 4 and 5 inserted in the native codon of 295

AtPIEZO. All plasmids were generated by the Gibson assembly kit (NEB) according to the 296

manufacturer’s instructions. The sequence of the primers in the following table: 297

primer name direction sequence

Myc tag1 forward gaacaaaaacttatttctgaagaagatctgctgctgaccagcctggtcgctc

Myc tag1 reverse cagatcttcttcagaaataagtttttgttcctgcagagccagaaagtacatg

Myc tag2 forward gaacaaaaacttatttctgaagaagatctgctgtacttctatctgggatataac

Myc tag2 reverse cagatcttcttcagaaataagtttttgttcgtcgataaatccgctcagcc



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298

Generating Chimeras 299

The chimeras between mPiezo1 and AtPIEZO were generated by swapping specific regions 300

highlighted in Extended Data Fig. 2. The N-terminus of all chimeras was derived from mPiezo1 301

and C-terminus was derived from a mammalian codon optimized AtPIEZO. The junction 302

between chimeras was chosen based on conserved amino acid regions between mPiezo1 and 303

AtPIEZO. We amplified the fragments using Q5 polymerase (NEB). pCDNA3-1 IRES2-eGFP 304

Zeo+ plasmid was cut by BamHI and EcoRI, followed by ligation of plasmid and fragments 305

using the Gibson assembly kit (NEB). The ligated plasmids were transformed into XL-gold 306

competent cells and colonies screened. All positive chimera clones were verified by full-length 307

DNA sequencing. 308

Electrophysiological characterization of Arabidopsis PIEZO (AtPIEZO), mPiezo1 and the 309

chimera. 310

HEK P1KO cells were transiently transfected with AtPIEZO, mPiezo1, mPiezo1/AtPIEZO 311

chimera (CH), or vector using Lipofectamine 2000 per manufacturer’s instructions and allowed 312

to settle on 12 mm diameter PDL-coated coverslips as previously described21. 313

Electrophysiological recordings were made 2-3 days after transfection using cell-attached patch 314

clamp methods to determine their responsiveness to membrane stretch using a High Speed 315

Pressure Clamp HSPC-1 (ALA Instruments)22,39. Pipettes had resistances of 1.4 – 2.6 MΩ when 316

filled with 130 mM NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 TEA-Cl, and 10 HEPES (pH 7.3 with 317

NaOH). Cells were exposed to in a high KCl bath solution during recordings to depolarize the 318

membrane potential (140 KCl, 1 MgCl2, 10 glucose, 10 HEPES (pH 7.3 with KOH)). Negative 319

pressure steps (250 msec in duration) were applied after 6 sec at +5 mmHg followed by 20ms at 320

0 mmHg; steps of increasing negative pressure in -5 mmHg increments were applied every 15 321

sec. For SAC recordings, the Multiclamp700A feedback resistor used was either 5 or 50 GΩ 322

depending on the size of the currents. To determine the apparent reversal potential (Vrev), a 323

stretch stimulus eliciting a submaximal response was applied during a voltage ramp protocol in 324

order to record SAC currents between ± 60mV. The apparent Vrev determined from this voltage 325

ramp protocol was validated in the same patch with a typical voltage step protocol. Whole cell 326

recordings were made as described16 for AtPIEZO-transfected cells 2-3 days after transfection. 327

In vivo Ca2+ imaging 328

A. thaliana plants expressing genetically-encoded Ca2+ (GCaMP3) that obtained from Dr. 329

Edward E. Farmer (university of Lausanne) were used for in vivo imaging. Images were 330

Myc tag3 forward cagatcttcttcagaaataagtttttgttccctttcatacttgacggtctc

Myc tag3 reverse gaacaaaaacttatttctgaagaagatctgagcgtcgaccccctggatctg

Myc tag4 forward taccgaacaaaaacttatttctgaagaagatctgcagcaacacgctgctggcttgg

Myc tag4 reverse ctgcagatcttcttcagaaataagtttttgttcggtaggatatatgctccaagc

Myc tag5 forward aatgaaagaacaaaaacttatttctgaagaagatctgcaaggcgaggcaacgagtaactc

Myc tag5 reverse gccttgcagatcttcttcagaaataagtttttgttctttcattgatatagaagcatgatc



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collected with a Nikon Instruments A1R+ confocal mounted onto an inverted Ti-E microscope. 331

An S Plan Fluor ELWD 20x objective NA 0.45 was used to acquire images at 1 frame/sec 1024 332

x 512 scan area (frame rate 0.946 msec/frame), 0.62 microns/pixel, pinhole 1.4AU, laser power 333

0.2 microwatts out of the objective. A Coherent 488nm solid-state laser was used for excitation, 334

with a Chroma 525/50 emission filter. Nikon Elements software was used for timelapse intensity 335

measurements. GCaMP3 imaging was recorded 30-35 s before applying mechanical stimulation. 336

A day before Ca2+ imaging, 5-7 day old seedling was transferred onto a 60mmx24mm coverglass 337

covered by 1-2mm of MS media in 0.6% low melting agarose (IBI scientific). The GCaMP3 338

plants were excited using a mercury lamp, 488nm laser and emission filter of 525/50nm with 339

andore 897EMCCD camera. The GFP signals of several regions of interest (ROI) such as 340

columella cells and lateral root cap cells analyzed using the NIS-Elements imaging software. 341

Representative images are shown in Figure 4, after adjusting brightness and contrast for clarity in 342

publication. We used (ΔF/F), the equation ΔF/F = (F − F0)/F0 for analyses the fluorescence 343

changes. F0 is baseline fluorescence that calculated from 10s before stimulation and F is 344

fluorescence of the recording. We estimated the area under curve using the equation 345

(y1+y2)/(2*(t2-t1)), where y is the value of (ΔF/F) and t is the each time point of GCaMP3 346

recording. 347

348

Mechanical stimulation to root 349

For plant in vivo Ca2+ measurements, mechanical stimulation was achieved using a fire-polished 350

glass pipette (tip diameter 10-12 μm) positioned at an angle of 80° to the recorded cells. 351

Downward movement of the probe was driven by a Clampex controlled piezo-electric crystal 352

microstage (E625 LVPZT Controller/Amplifier; PhysikInstrumente). The probe had a velocity of 353

1 μm.ms−1 during the ramp segment of the command for forward motion and the stimulus was 354

held for 150 ms before releasing the stimulus. To assess the mechanical responses of a cell, the 355

probe was first placed as close to the cell as possible (this distance could vary from plants to 356

plants). We optimized the mechanical stimulation and found that 4 series of mechanical steps in 357

20 μm increments in every 15s lead to a transient and local Ca2+ responses. Longer or more steps 358

led to Ca2+ fluxes traveled bidirectionally into both sides of the region being poked resemble of 359

damage/wounding Ca2+ signaling. 360

361

Statistics 362

All results in main figures and extended data with error bars are represented as mean ± s.d. 363

according to standard methods using Microsoft Excel or GraphPad Prism. The P values were 364

generated with Student’s one-tail unpaired t-tests. For qRT–PCR experiment, four technical 365

replicates were used (three technical replicates). The biological replicates were indicated as ‘n’ in 366

the figure legends. 367

368

369



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Acknowledgements 466

We would like to thank Dr. Simon Gilroy, and Dr. Edward E. Farmer and Dr. Elliott Meyerowitz 467

for seeds. We also thank Dr. Kathryn R. Spencer for helping in vivo Ca2+ imaging. Dr. Elizabeth 468

S. Haswell, Haley DeGuzman, Tess Whitwam, Shang Ma, Ivan Radin, Kara Marshall, Yunxiao 469

Zhang, Swetha Murthy and Viktor Lukacs for discussion and critical reading of the manuscript. 470

Funding acknowledgments 471

Early and advanced mobility fellowship from Swiss National Science Foundation (S.A.R.M.). 472

YZ was supported by NIH GM114660, and CG was supported by a scholarship from the China 473

Scholarship Council. 474

Author contributions: 475

S.A.R.M., A.E.D. and A.P. conceived and designed experiments and wrote the manuscript. A. E. 476

D. and W.Z.Z. recorded and analyzed electrophysiology data. C. G. and Y. Z. generated the 477

CRISPR-Cas9 piezo mutants. A. C. performed live labeling. K. D. and D. G. performed cloning 478

and generated transgenic plants. 479

Corresponding authors 480

To whom correspondence may be addressed. Email: [email protected] and 481

[email protected] 482

Competing interests 483

The authors declare no competing financial interests. 484

485





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Figure legends 486

487

Figure1. Expression pattern of AtPIEZO::GUSPlus reporter line in Arabidopsis root. a, 488

Expression pattern of the GUS reporter protein under 2000 bp of the AtPIEZO promoter in a 7 489

days old seedling. b- c, Expression in the upper root and root tip when the plant is grown on the 490

surface of standard MS media. Red arrowhead indicates that AtPIEZO is no longer expressed in 491

the oldest root cap cells that are most distal and are known to be sloughed off. d-e, Expression in 492

the upper root and root tip grown inside the MS media. Black arrows indicate the cross section of 493

root displayed in panels g and h. f, qRT-PCR for AtPIEZO in upper root of plants grown on the 494

surface (“surface”) or within the MS media (“inside”). **P < 0.01, N=4 (mean±s.d.). g-h, Cross 495

sections of the root cap in GUS reporter lines which indicate expression in columella and LRC 496

cells. 497



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498

Figure 2. Penetration of roots into hard media is compromised in piezo mutants. a, Root 499

length of Arabidopsis roots grown 9 days vertically at 60° to stimulate growth into the MS 500

media. Black arrowheads indicate roots within the MS media, white arrowheads indicate root 501

growth on the surface of MS media. b, Root length of WT and piezo mutants 9 days after 502

germination (N=30-36; mean±s.d). c. Representative plate of 18 day old Arabidopsis seedlings 503

challenged by root barriers. The more visible roots have not penetrated the barrier and are on the 504

surface of the MS media. WT and mutants were grown vertically at 90° on normal MS media 505

(0.5X MS + 8.5 g/l agar) before reaching the barrier (0.5X MS + 12 g/l agar). d, The percentage 506

of roots that penetrate barriers of different concentrations of agar as indicated (n=8-11 plates) 507

and each plate consists of 7-12 seedlings. e. Representative plate of 2 week-old Arabidopsis 508

seedlings challenged by root barriers indicating the difference root growth length in the denser 509

media (imaged brightness was adjusted for clarity). White arrowheads indicate roots within the 510

MS media, black arrowheads indicate root growth on the surface of MS media or roots that did 511

not penetrate the barrier. f, Root lengths of 2 weeks old seedlings that have penetrated the 512

barriers (indicated) (N=25-28, mean±s.d.). **P < 0.01, ***P < 0.001. 513



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514

515

516

Figure 3. A chimeric channel that includes putative AtPIEZO pore sequences is activated 517

by negative pressure applied to cell-attached patches. a, The region of AtPIEZO in the 518

chimera (CH) is highlighted in the mPIEZO1 structure, grey is mPiezo1 sequence (577-1185aa) 519

and red is AtPIEZO sequence (1228-2485aa). The first 577 amino acid of mPIEZO1 are not 520

resolved in the structure. b, Stimulus-response curves are shown for all mPiezo1 (black) and 521

mPiezo1/AtPIEZO chimera (CH, red) stretch-activated currents (SAC, Vpipette = +80mV in cell-522

attached configuration). Small high threshold responses are occasionally observed in HEK P1KO 523

cells transiently transfected with the empty IRES-GFP vector control (blue). The maximal 524

current observed from vector-transfected cells was -5.4 pA (dotted line) and this value is used as 525

a cutoff for identifying mPiezo1- and CH-mediated SAC. c, Imax is shown for mPiezo1 (black), 526

CH (red), and control cells (blue). d and e, SACs recorded in a patch from a cells expressing 527

either mPiezo1 (d) or CH (e); current amplitudes increase with increasing negative pressure 528

(shown below each family of currents). f, The negative pressure (mmHg) at which the first 529

response to stretch is observed (threshold) when patches are challenged with -5mmHg 530

increments (threshold) is plotted. g, The pressure producing half-maximal currents (P50, 531

determined using GraphPad Prism) is shown. h and i, A stretch stimulus eliciting a submaximal 532

response is applied during a voltage ramp protocol in order to record SAC currents between ± 533

60mV and determine the apparent reversal potential (Vrev). 534



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Figure 4. AtPIEZO and calcium influx 535

in response to mechanical indentation 536

to the Arabidopsis root tip. a, AtPIEZO 537

expression was knocked down in the root 538

cap using an artificial microRNA under 539

the PIN3 promoter (PIN3::amiRNA-540

PIEZO) such that AtPIEZO is specifically 541

knocked down in columella cells (CC) 542

(blue) but AtPIEZO is still expressed in 543

lateral root cap (LRC) cells (red). Black 544

dashed circle indicates the area of 545

stimulation by the blunt-tip pipette and the 546

region of interest (ROI) for Ca2+ signal 547

intensity measurement. b, Displacements 548

of the root cap by the stimulating pipette. 549

Mechanical stimulation were initiated 30-550

35 s after GCaMP3 signal recording; A 551

ramp (1µm/ms) and hold (150 ms) 552

stimulus was applied in 20 µm increments 553

every 15 sec. c and d, Representative 554

image of the GCaMP3 Ca2+ response in a 555

WT root tip before (c) and after (d) a 80 556

µm mechanical stimulation (images are 557

from the Supplementary Video 2). e and 558

f, Representative image of the Ca2+ 559

responses in AtPIEZO knockdown 560

PIN3::amiRNA-PIEZO before and after a 561

80 µm mechanical stimulation (images are 562

from the Supplementary Video 4). g, Ca2+ 563

responses in the LRC cells and columella 564

cells in response to mechanical 565

stimulation in 7 day old seedlings; N=12 566

(mean±s.d.). Arrowheads indicate the 567

mechanical stimulation (µm). h, Ca2+ 568

responses after mechanical stimulation in 569

AtPIEZO knockdown PIN3::amiRNA-PIEZO root cap in 7 old days seedlings. Ca2+ transients 570

are significantly reduced in columella cells compared to LRC cells; N=9 (mean±s.d.). i, Area 571

under curve from starting the 80µm mechanical stimulation until the GCaMP3 signals returned 572

to base line (total of 15s). j, Maximum peak of fluorescence in WT and PIN3::amiRNA-PIEZO 573

(piezo knockdown) (N=9-12, mean ±s.d.). **P < 0.01, ***P < 0.001. 574



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575

576

Extended Data Figure 1. Representative images of the expression pattern of AtPIEZO 577

promoter activities in AtPIEZO::GUSPlus reporter line. a, The expression pattern of 578

AtPIEZO promoter activity in the line with the 823 bp AtPIEZO promoter (AtPIEZO 579

(short)::GUSPlus). b-e, The expression pattern of AtPIEZO promoter activity in the line with 2000 580

bp AtPIEZO promoter (AtPIEZO (long)::GUSPlus) in leaf that indicates AtPIEZO expression in 581

trichome, laterial root cap, flower and pollen. 582

583



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Chimera4 TM1 Chimera5 584 AtPIEZO -----MASFLVGFLLPSL||LLAAALINWSVISFLDLIAFL||LVHYIAPEIGYRFQRRHWLLW 55 585 mPiezo1 MEPHVLGAGLYWLLLPCT||LLAASLLRFNALSLVYLLFLL||LLPWLPGP------------- 47 586 :.: * :***. ****:*:.:..:*:: *: :**: :: 587 TM2 Myc1 588 AtPIEZO PIFIFSFAVFLAQVVYLVIWAALGQDWDTPDTGWMRVIGFMILKSWRNPTVMYFLALQLL 115 589 mPiezo1 ------------------------S----------------------------------- 48 590 . 591 TM3 592 AtPIEZO TSLVALADIYSSRFGFARWRDTWWSHFSGIFEHLGSHLRVASCLLL--PAVQLAVGICNP 173 593 mPiezo1 --------------------------RHSIPGHTGRLLRALLCLSLLFLVAHLAFQICLH 82 594 .* * * **. ** * ..:**. ** 595 596 AtPIEZO SWVSLPFFIGSCAGLVDWSLTSNVSGLFRWWRVLYIYAGFNIVLLYLYQLPINFSDMIRW 233 597 mPiezo1 TVPHLDQFLGQNGSL--WVKVSQHIGVTRL----DLKDIFN---------------TTRL 121 598 : * *:*. ..* * .*: *: * : ** * 599 TM4 600 AtPIEZO IASFIGLFRISLETEGPDICSGLFLVLFYIMLSYV-RSDLEDMDF-------IMSTSENN 285 601 mPiezo1 VAPDLGVLL------ASSLCLGLCGRLTRKARQSRRTQELDDDDDDDDDDEDIDAAPAVG 175 602 :* :*:: . .:* ** * . .:*:* * * :: . 603 TM5 604 AtPIEZO L-AERLLPPKYSFFIRESRAGVRHTNVLLRGAVFKTFSI--------NFFTYGF-PVSLF 335 605 mPiezo1 LKGAPALATKRRLWLASRFRVTAHWLLMTSGRTLVIVLLALAGIAHPSAFSSVYLVVFLA 235 606 * . * * ::: . . * :: * .: . : . *: : * * 607 TM6 TM7 608 AtPIEZO ALSFWSFHFA-------SLCAFGLLAYVGYI--IYAFPSLFQLHRLNGLLLVFILLWAVS 386 609 mPiezo1 ICTWWSCHFPLSPLGFNTLCVMVSCFGAGHLICLYCYQTPFIQDMLPP-GNIWARLFGLK 294 610 ::** ** :**.: .*:: :*.: : * . * :: *:.:. 611 TM8 612 AtPIEZO TYIFNVAFSFLNTKVGKDMQIWEMVGLWHYTIPGFFLLAQFGLGMLVALGNLVNNSVFLY 446 613 mPiezo1 NFVDLPNYSSPNALVLNTKHAWP-----IYVSPGILLLLYYTATSLLKLHKSCPSE---- 345 614 .:: :* *: * : : * *. **::** : *: * : .. 615 TM9 616 AtPIEZO LSEESSRSSNERSY----VEADE----ETKVLVVATIAWGLRKCSRAIMLA--------- 489 617 mPiezo1 LRKETPREDEEHELELDHLEPEPQARDATQGEMPMTTEPDLDNCTVHVLTSQSPVRQRPV 405 618 * :*: *..:*:. :* : *: : * .* :*: :: : 619 TM10 620 AtPIEZO ---LIFLIAM-----------KPGFFHA------------VYVIFFLMYLLSHNINRKIR 523 621 mPiezo1 RPRLAELKEMSPLHGLGHLIMDQSYVCALIAMMVWSIMYHSWLTFVLLLWACLIWTVRSR 465 622 * * * . .:. * :: *.*: . . : * 623 624 AtPIEZO KSLILLCEVHFAL--------LYILEIDLVSNSLKQEGSASREVLFQLGLLRSESSWDFL 575 625 mPiezo1 HQLAMLCSPCILLYGLTLCCLRYVWAMELPELP----TTLGPVSLHQLGLEHTR--YPCL 519 626 :.* :**. : * *: ::* . : . *.**** ::. : * 627 Chimera6 TM11 Chimera7 628 AtPIEZO EIALL||ACFCAIHNHGFEVLFSFSAIVRHTPSPPIGFSILKAGLNK||SVLLSVYSSPSSSYS 635 629 mPiezo1 DLGAM||----------LLYLLTFWLLLR--------------QFVK||EKLLKKQKVPAALLE 555 630 ::. : : *::* ::* : *. **. . *:: . 631 TM12 TM13 632 AtPIEZO QD-NTTYERHIASFLSAIGQKFLSMYRSCGTYIAFITILISVYLVKPNYVSFGYIFLLLL 694 633 mPiezo1 VTVADTEPTQTQTLLRSLGELVTGIYVKYWIYVCAGMFIVVSFAGRLVVYKIVYMFLFLL 615 634 * : ::* ::*: . .:* . *:. ::: : : .: *:**:** 635 TM14 Myc2 636 AtPIEZO WI----TGRQLFEETKRRLWFPLKAYAVLVFMFIYCLSSFVSLQLW--LSGFIDLYFYLG 748 637 mPiezo1 CLTLFQVYYTLWRKLLRVFWWLVVAYTMLVLIAVYTFQFQDFPTYWRNLTGFTDEQL--G 673 638 : . *:.: * :*: : **::**:: :* :. * *:** * : * 639 TM15 640 AtPIEZO YNSKAPLLDNVWESLAVLIVMQLYSYERRQSGHYIPGQSSLLHPGVFGF--------FE- 799 641 mPiezo1 --------DLGLE---------QFSVSEL--------FSSILIPGFFLLACILQLHYFHR 708 642 * * :* .. **:* **.* : *. 643 TM16 644 AtPIEZO ----------------RFLAWHGQKIL-FAALFYASLSPISVFGF--VYLLGLVICTTFP 840 645 mPiezo1 PFMQLTDLEHVPPPGTRHPRWAHRQDAVSEAPLLEHQEEEEVFREDGQSMDGPHQATQVP 768 646 *. * :: * : . .** : * .* .* 647 TM17 648 AtPIEZO KSSSIPSKSFLIYTGFLVSAEYLFQLWGMQAQMFPGQK----------------YAELSF 884 649 mPiezo1 --------------------EGTASKWGLVADRLLDLAASFSAVLTRIQVFVRRLLELHV 808 650 * . **: *: : . ** . 651 TM18 TM19 652 AtPIEZO YLGLRVYEPGFWGIESGL--RG-KVLVVAACTLQYNVFRWLERTSGLTVIKGKYEEPCPL 941 653 mPiezo1 FKLVALY--TVWVALKEVSVMNLLLVVLWAFALPYPRFRPMAS--CLSTVWTCIIIVCKM 864 654 : : :* .* . : . ::*: * :* * ** : *:.: * : 655



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Myc5 656 AtPIEZO FVSAEDTTASVSSSNGEN--PSSTDHASIS-----MKQGEATSNSWPFFSPRGNQGAGFL 994 657 mPiezo1 LYQLKIVNPHEYSSNCTEPFPNNTNLQPLEINQSLLYRGPVDPANWFG-VRKGYPNLGYI 923 658 : . : .. *** : *..*: :. : :* . .* :* . *:: 659 TM20 660 AtPIEZO HPKTGGSESGSSRKFSFGHFWGSI-KESHRWNRRR---I-----LALKKERFETQKNLLK 1045 661 mPiezo1 QNHLQI--------LLLLVFEAVVYRRQEHYRRQHQQAPLPAQAVCADGTRQRLD-QDLL 974 662 : : : : * . : :...::.*:: :. . * . : : * 663 TM21 TM22 664 AtPIEZO IYLKFWIENMFNLYGLEINMIALLLASFALLNAISMVYIALLAACV------LLRRRVIQ 1099 665 mPiezo1 SCLKYFINFFFYKFGLEICFLMAVNV-----IGQRMNFMVILHGCWLVAILTRRRREAIA 1029 666 **::*: :* :**** :: : . . * ::.:* .* **..* 667 TM23 668 AtPIEZO KLWPVVVFLFASIL--------------AIEYVATWNSFLPSDQAPSETSVHCHDCWSIA 1145 669 mPiezo1 RLWPNYCLFLTLFLLYQYLLCLGMPPALCIDYPWRWSKAIPMNSALI--------KWLYL 1081 670 :*** :::: :* .*:* *.. :* :.* * 671 TM24 672 AtPIEZO ALYFKFCRECWLGVRVDDPRTLISYFVVFMLACFKLRADHISSFSESSTYHQMKSQRKNS 1205 673 mPiezo1 PDFF----------RAPNSTNLISDFLLLL--CASQQWQVF-SAERTEEWQRMAGINTDH 1128 674 :* *. : .*** *:::: * . : : : * ..:. :::* . ..: 675 Tm25 Chimera1(mPiezo1/AtPIEZO) 676 AtPIEZO FVWRDLSFETK------SMWTVLDYLRLYCYVHLLDVVLILILITGTLEYDILHLGYL||AF 1259 677 mPiezo1 L--EPLRGEPNPIPNFIHCRSYLDMLKVAVFRYLFWLVLVVVFVAGATRISIFGLGYL||LA 1186 678 : . * * : : ** *:: : :*: :**::::::*: . .*: **** 679 TM26 TM27 680 AtPIEZO ALVFARMRLEILKKKNK----IFRFLRVYNFVLIIFSL---AYQSPFVGNFNDGKCETVD 1312 681 mPiezo1 CFYLLLFGTTLLQKDTRAQLVLWDCLILYNVTVIISKNMLSLLSCVFVEQMQSNFCWVIQ 1246 682 .: : : :*:*..: :: * :**..:** . .. ** :::.. * .:: 683 TM28 684 AtPIEZO ---YIYEVIGFYKYDYG--------FRITARSALVEIIIFMLVSLQSYMFSSQEFDYVSR 1361 685 mPiezo1 LFSLVCTVKGYYDPKEMMTRDRDCLLPVEEAGIIWDSICFFFLLLQRRIFLSHYFLHVSA 1306 686 : * *:*. . : : . : : * *::: ** :* *: * :** 687 688 AtPIEZO YLEAEQIGAI-----------------VREQEKKAARKTEQLQQIREAEEKKRQRNLQVE 1404 689 mPiezo1 DLKATALQASRGFALYNAANLKSINFHRQIEEKSLAQLKRQMKRIRAKQEKYRQSQASRG 1366 690 *:* : * : :**. *: ..*:::** :** ** : . 691 692 AtPIEZO KMKSEMLNLRVQLHRMNSDSNFGVASPRTEGLRRRKSPYLIPDSGAASPEIDGVVHRKEE 1464 693 mPiezo1 QLQSKDP--QDPSQEPGPDSPGGSSPPRRQWW----RPWLDHA---------TVIHSGDY 1411 694 :::*: : :. . ** * : ** : *:* *:* : 695 696 AtPIEZO QPIDEDSQYPFEAHEFPVSTTPEALDSPEYSFGASPCEI-TEVQQDLDVMSMERERKQKS 1523 697 mPiezo1 FLFESDSE--EEEEALPEDPRPAAQSAFQMAYQAWVTNAQTVLRQRRERARQERAEQLAS 1469 698 ::.**: * . :* . * * .: : :: * : * ::* : ** .: * 699 700 AtPIEZO EGKENPLISAVQL----------------------------------------------- 1536 701 mPiezo1 GGDLNPDVEPVDVPEDEMAGRSHMMQRVLSTMQFLWVLGQATVDGLTRWLRAFTKHHRTM 1529 702 *. ** :. *:: 703 704 AtPIEZO --------------------IGDGVSQVQFIGNQAVNN---------------------- 1554 705 mPiezo1 SDVLCAERYLLTQELLRVGEVRRGVLDQLYVGEDEATLSGPMETRDGPSTASSGLGAEEP 1589 706 : ** : ::*:: .. 707 708 AtPIEZO LVNFL--NISPENSDTNEQSSVDDEVYDEME------------------SQKRKHT--PF 1592 709 mPiezo1 LSSMTDDTSSPLSTGYNTRSGSEEIVTDAGDLQAGTSLHGSQELLANARTRMRTASELLL 1649 710 * .: . ** .:. * :*. :: * * : :: *. : : 711 TM29 712 AtPIEZO ERSTSLQSDRSSDGTSFQIGR---IFRHIWSRMQSNNDIVCYCCFIIAFLWNFSLLSMVY 1649 713 mPiezo1 DRRLHIPELEEAERFEAQQGRTLRLLRAGYQCVAAHSELLCYFIIILNHMVTASAASLVL 1709 714 :* : . ..:: . * ** ::* :. : ::.:::** :*: .: . * *:* 715 TM30 Chimera2 TM31 716 AtPIEZO LAALFLYALCVHTGPTHIFW||VIMLMYTEIYILLQYLYQIIIQHCGLSIDAPLL-HELGFP 1708 717 mPiezo1 PVLVFLWAMLTIPRPSKRFW||MTAIVFTEVMVVTKYLFQFGFFPWNSYVVLRRYENKPYFP 1769 718 . :**:*: . *:: **: :::**: :: :**:*: : . : :: ** 719 TM32 720 AtPIEZO TQRIK----SSFVVSSLPLFLIYIFTLIQSSITVKDGDWVPSADFTSRRNARGSQKDLTR 1764 721 mPiezo1 PRILGLEKTDSYIKYDLVQLMAL---FFHRSQLLCYGLWDHEEDRYPKDHCRSSVKDREA 1826 722 : : .*:: .* :: ::: * : * * . * : :.*.* ** 723 724 AtPIEZO IRLSQRILDVFKKLRDSAKLVIRSIYRYWISLTRGAESPPYFVQVTMDVHMWPEDGIQPE 1824 725 mPiezo1 KEEPEA------------KLESQS------ETGTGHPKEPVLAGT-------PRDHIQGK 1861 726 . : ** :* . * . * :. . *.* ** : 727



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728 AtPIEZO RVECRMNQLLRLVHNERCEKGNPDLCPYSSRVHVQSIERSTETPNEA-LVVLEVEYASPT 1883 729 mPiezo1 GS-IR--------SKDVIQDPPEDLKPRHTRHISIRFRRRKETPGPKGTAVMETEHEE-G 1911 730 * :: :. ** * :* :.* .***. .*:*.*: . 731 732 AtPIEZO NGCSSAEWYKSL--TPASDVAKEI----RKAQHSGLGEGTGFPYPILSV-----IGGGKR 1932 733 mPiezo1 EGKETTERKRPRHTQEKSKFRERMKAAGRRLQSFCVSLAQSFYQPLQRFFHDILHTKYRA 1971 734 :* .::* : *.. :.: *: * :. . .* *: . : 735 Chimera3 TM33 TM34 736 AtPIEZO D||TDLYAYIFGADLIVFFLVAI-FYQSVIKNKSEFIDVYQLEDQFPFDFVIILMVIFFLIV 1991 737 mPiezo1 A||TDVYALMFLADIVDIIIIIFGFWAFGKHSAATDIASSLSDDQVPQAFLFMLLVQFGTMV 2031 738 **:** :* **:: :::: : *: :. : * :**.* *:::*:* * :* 739 M35 Myc4 M36 740 AtPIEZO VDRVIYLCSFATGKVVYYLFSLILFTYAVTEYAWSIYPTQ---QHAAGLALRIIFLAKAM 2048 741 mPiezo1 IDRALYLRKTVLGKLAFQVVLVVA----IHIWMFFILPAVTERMFSQNAVAQLWYFVKCI 2087 742 :**.:** . . **:.: :. :: : : : * *: .: . . :: ::.*.: 743 744 AtPIEZO SLALQAIQIRYGLPHKSTLYRQFLTSEVSRINYYGYRLYRALPFLYELRCVLDWSCTATS 2108 745 mPiezo1 YFALSAYQIRCGYPTR--ILGNFLTKKYNHLNLFLFQGFRLVPFLVELRAVMDWVWTDTT 2145 746 :**.* *** * * : : :***.: .::* : :: :* :*** ***.*:** * *: 747 TM37 748 AtPIEZO LTMYDWLKLEDVNASLYLVKCDTVLNRA-THKHGEKQTKMTKCCNGICLFFILLCVIWAP 2167 749 mPiezo1 LSLSNWMCVEDIYANIFIIKCSRETEKKYPQPKGQKKKKIVKYGMGGLIILFLIAIIWFP 2205 750 *:: :*: :**: *.::::**. :: : :*:*:.*:.* * ::::*:.:** * 751 752 AtPIEZO MLMYSSGNP-TNIANPIKDASVQIDLKTVGGKLTLYQTT------------LCERISGDN 2214 753 mPiezo1 LLFMSLIRSVVGVVNQPIDVTVTLK---LGGYEPLFTMSAQQPSIVPFTPQAYEELSQQ- 2261 754 :*: * . ..:.* *.:* :. :** *: : *.:* : 755 756 AtPIEZO IDLGLDLGSQSFLPTYNKNDIQLICCQADASVLWLVPDTVVTRFIQS-LDWDTDMDITFT 2273 757 mPiezo1 --FDPYPLAMQFISQYSPEDIVTAQIEGSSGALWRISPPSRAQMKQELYNGTADITLRFT 2319 758 :. : .*: *. :** :..:..** : ::: *. : :*: : ** 759 Myc3 760 AtPIEZO WVLNRDRPKGKETVKYERSVDPLDLPKRSD----IQMVLNG-SMDGFRVHNLYPKFFRVT 2328 761 mPiezo1 WNFQRDLAKGGT-VEYTNEKHTLELAPNSTARRQLAQLLEGRPDQSVVIPHLFPKYIRAP 2378 762 * ::** ** *:* .. . *:* .* : :*:* :.. : :*:**::*. 763 764 AtPIEZO GSGDVRSFEDQTDEV---------------------SADILINHANFKWWWSFHNLKASE 2367 765 mPiezo1 NGPEANPVKQLQPDEEEDYLGVRIQLRREQVGTGASGEQAGTKASDFLEWWVIELQDCK- 2437 766 .. :.. .:: : . : : ::* ** :. ... 767 TM38 768 AtPIEZO NISACEGMDGPVAIIMSEET-PPQGFLGDTLSKFSIWGLYITFVLAVGRFIRLQCSDLRM 2426 769 mPiezo1 --ADCNL--LPMVIFSDKVSPPSLGF----LAGYGIVGLYVSIVLVVGKFVRGFFSEISH 2489 770 : *: *:.*: .: : * ** *: :.* ***:::**.**:*:* *:: 771 772 AtPIEZO RIPYENLPSCDRLIAICEDLYAARAEGELGVEEVLYWTLVKIYRSPHMLLEYTKLDYDA 2485 773 mPiezo1 SIMFEELPCVDRILKLCQDIFLVRETRELELEEELYAKLIFLYRSPETMIKWTRERE-- 2546 774 * :*:**. **:: :*:*:: .* ** :** ** .*: :****. ::::*: 775

776

Extended Data Figure 2. Alignment between Arabidopsis PIEZO and mouse Piezo1 777

highlights the residues of interest. A multiple sequence alignment between mPiezo1 and 778

AtPIEZO was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The 779

transmembrane topology prediction for TM1- TM14 was obtained using on TOPCONS software 780

(http://topcons.cbr.su.se/) and the topology from TM15 to TM38 was derived from the structure 781

of mPiezo123. Residues highlighted in grey indicate the transmembrane domain. Residues in pink 782

are transmembrane domains predicted to be only in AtPIEZO, but not mPiezo1. Note that there 783

is higher homology between mPiezo1 and AtPIEZO in the regions where the structure of 784

mPiezo1 is resolved. Residues highlighted in green indicate the junction between mPiezo1/ and 785

AtPIEZO in the chimeras. Residues highlighted in red indicate the position of the Myc tag on 786

mPiezo122. Residues highlighted in yellow, indicate the position of the Myc tag on AtPIEZO. 787

The PFEW motif highlighted in blue conserved among plants, mammals and protozoa40. 788



http://topcons.cbr.su.se/

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789

Extended Data Figure 3. AtPIEZO transcript level in roots of WT and mutant plants. qRT-790

PCR was performed on samples harvested from root and leaf from 4 different plants. 791

***P < 0.001, N=4 (mean ± s.d.). 792



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793

Extended Data Figure 4. Root length of piezo mutants. a, Representative image indicating the 794

root lengths of piezo mutants when grown on top of MS media in plates tilted at a 900 angle. b, 795

Root length of WT and both piezo mutants piezo-FL and piezo-CT (n=30). c, Root length of 796

piezo mutants when grown inside the MS media containing the indicated agar concentrations in 797

plates positioned at a 600 angle. Data shown are for roots growing inside the MS media. 798

***P < 0.001 (N=23, mean±s.d.). 799



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800

Extended Data Figure 5. piezo mutants are defective at penetrating a hard barrier. a, 801

Original image of the plant root that was challenged by barriers from Fig. 2e. b, Same image 802

with adjusted light exposure for better visibility of roots grown inside the MS media. Black 803

arrowheads indicate roots within the MS media or at the barrier interface, white arrowheads 804

indicate root growth on the surface of MS media. c and d, The barrier is imaged at an angle that 805

enables visualization of the root tips at the 5 mm wide barrier (brown; same data shown in panel 806

A). c, Usually WT roots are observed to grow fairly straight through the harder barrier. d, Some 807

of piezo-CT roots are observed to also form swirls at the barrier. 808

809



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810

Extended Data Figure 6. Myc tag staining of AtPIEZO and chimera. Representative images 811

of non-permeabilized staining using an anti-Myc antibody (red) in AtPIEZO-myc -ires- GFP 812

transfected cells, mPIEZO1- 508-Myc (Myc tag located after 508 amino acid) and 813

mPIEZO1/AtPIEZO chimera containing 508-Myc. mPIEZO1-508-myc used as positive 814

control22. Scale bar is 20 µm. 815



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Extended Data Figure 7. Electrophysiological characterization of mPiezo1 and mPiezo1/ 816

AtPIEZO chimera. 817

818

SAC Parameter mPiezo1 mPiezo1/AtPIEZO

Imax (pA) -28 ± 8 (n=16) -25 ± 14 (n=8)

Threshold (mmHg) -31 ± 6 (n=16) -31 ± 7 (n=8)

P50 (mmHg) -47 ± 7 (n=15) -61 ± 6 (n=8)

Vrev of current in cell-attached patch

(mV)a

0.4 ± 2.3 (n=6) 0.7 ± 1.3 (n=2)

Inactivation rate, tau (ms) 67 ± 13 (n=15) >250 ms (n=8) ***

Percent of peak current at 250 ms (%) 23 ± 6 (n=16) 92 ± 4 (n=8) ***

a Vpipette at which SAC reversed under cell-attached patch recording conditions used here to

increase intracellular K+; the pipette solution (extracellular) contained high Na+ (see

Methods).

819

820



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Supplementary Video 1. piezo mutant poorly penetrate into hard MS media. Seeds of WT and 821

piezo-CT mutant plated on the surface of agar 2 cm above the barrier (12 g/l agar in MS media). 822

Supplementary Video 2. Mechanical indentation causes Ca2+ responses in the lateral root cap 823

cells and columella cells in the WT expressing GCaMP3. Four mechanical stimuli were applied 824

to the root cap beginning at 30s and followed in increasing increments of 20µm at 15s intervals. 825

Supplementary Video 3. Extensive mechanical stimulation (100 µm) lead to wound/systemic 826

Ca2+ fluxes that travel in both directions from the stimulation site. Five mechanical stimuli were 827

applied to the upper root beginning of 25s followed in increasing increments of 20µm at 15s 828

intervals. At the 100 µm of mechanical stimulation, Ca2+ responses travel bidirectionally. 829

Supplementary Video 4. Mechanical indentation causes Ca2+ responses only in the lateral root 830

cap cells in piezo knockdown (PIN3::amiRNA-PIEZO) mutant expressing GCaMP3. Four 831

mechanical stimuli were applied to the root cap beginning at 30s and followed in increasing 832

increments at 15s intervals. 833

834



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PIEZO ion channel is required for root mechanotransduction in … · 2020. 8. 27. · 1 1 PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana 2 Seyed

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