Baluska & Mancuso - Root Phonotropism

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Root phonotropism: Early signalling events following sound perception in
Arabidopsis roots
Ana Rodrigo-Morenoa,⁎, Nadia Bazihizinaa, Elisa Azzarelloa, Elisa Masia, Daniel Tranb,
François Bouteaub, Frantisek Baluskac, Stefano Mancusoa
a Department of Agrifood Production and Environmental Sciences − Università degli Studi di Firenze, Viale delle Idee 30, 50019 Sesto Fiorentino, Florence, Italy
b Université Paris Diderot, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de Demain, Paris, France
c University of Bonn, IZMB, Kirschallee 1, 53115, Bonn, Germany
A R T I C L E I N F O
Keywords:
Cytosolic calcium
Gravitropism
Mechanotransduction
Ion fluxes
Ion channels
Phonotropism
Reactive oxygen species
Sound vibrations
Voltage clamp
A B S T R A C T
Sound is a fundamental form of energy and it has been suggested that plants can make use of acoustic cues to
obtain information regarding their environments and alter and fine-tune their growth and development. Despite
an increasing body of evidence indicating that it can influence plant growth and physiology, many questions
concerning the effect of sound waves on plant growth and the underlying signalling mechanisms remains un-
known. Here we show that in Arabidopsis thaliana, exposure to sound waves (200 Hz) for 2 weeks induced
positive phonotropism in roots, which grew towards to sound source. We found that sound waves triggered very
quickly (within minutes) an increase in cytosolic Ca2+, possibly mediated by an influx through plasma mem-
brane and a release from internal stock. Sound waves likewise elicited rapid reactive oxygen species (ROS)
production and K+ efflux. Taken together these results suggest that changes in ion fluxes (Ca2+ and K+) and an
increase in superoxide production are involved in sound perception in plants, as previously established in ani-
mals.
1. Introduction
Sound is a fundamental form of energy and many organisms have
evolved to use it for a range of ecological processes [1]. In animals, it
has been shown that individuals can use sound vibrations, including
those produced by the behaviour of other individuals (i.e. public in-
formation), for social interactions as well as water and food recruit-
ments and enhance their fitness [2]. Recent advances in plant research
suggest that the same is true in plants. For instance, as demonstrated for
animals and their use of “public information”, it is now well established
that plants can use volatile organic compounds to provide community
members with information about their states [3]. These volatile signals
tailor plant defences towards biotic and abiotic stresses, ultimately in-
creasing their fitness [4–6]. As for the perception of the volatile organic
compounds, it is then logical to expect that plants can make use of
acoustic cues to obtain critical information regarding their environ-
ments to alter and fine-tune their growth and development based on
their external contingencies and stimuli [7].
Sound is ubiquitous in nature. It is, therefore, not surprising that
plants sense and respond to vibrating sound waves [8,9]. Sound waves
have been found to trigger a wide array of responses in plants at
different levels, from altering gene expression [10] and root growth [7]
at the single plant level; up to the ability of plants to discriminate be-
tween the vibrations caused by insect chewing and those caused by
wind or insect song [11]. Furthermore the recent finding that some
carnivorous plants have developed ultrasound reflectors in order to
attract bats, enabling them to improve their echolocation system to
easily locate and identify their pitchers, strongly suggests that acoustic
communication could also play an important role in plant-animal in-
teractions, as well as in the selection of their mutualistic partners
[1,9,12,13].
Given the physical properties of sound waves, sound is likely to have
a mechanical impact on plant cell membranes, suggesting that per-
ception and signalling of mechanical and sound stimuli are based on
many similarities [8]. Supporting further this view, mechanosensory
transduction has been found to underlie many processes in animals
hearing, and the mechanosensory transduction channels NompC and
Nan, both transient receptor potential (TRP) superfamily members, are
fundamental in sound perception in Drosophila [14–17]. While the
molecular basis for perception of a mechanical stimulus remains to be
identified in plants, there is a wealth of evidence linking transient in-
creases in cytosolic Ca2+ levels to mechanoresponses at the whole plant
http://dx.doi.org/10.1016/j.plantsci.2017.08.001
Received 20 April 2017; Received in revised form 31 July 2017; Accepted 1 August 2017
⁎ Corresponding author.
E-mail address: anarodrigomoreno@hotmail.com (A. Rodrigo-Moreno).
Plant Science 264 (2017) 9–15
Available online 10 August 2017
0168-9452/ © 2017 Elsevier B.V. All rights reserved.
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level, placing the secondary messenger Ca2+ at the centre of these
mechanical signal transduction pathways [18–21]. Other processes,
such as proton fluxes and ROS generation, have also been shown to play
critical role in the downstream responses involved in plant mechan-
otransduction, modulating the activity of membrane transporters and
regulating gene transcription [19,21]. For instance, mechanical stress
has been show to induce a localized generation of ROS in intact root
tissue within few seconds from the stimulus, which was activated by the
touch-associated Ca2+ increase and was dependent on RBOH C, an
Arabidopsis homolog of the phagocyte NADPH oxidase subunit
gp91phox [19].
The majority of studies on plants responses to sound have focused
on its effect on plant growth and germination. Less is known, however,
about the early initial signalling events that occur within the first few
minutes and how plants sense sound vibrations in their environments
[8,9]. To address these questions, we monitored the initial cellular
events triggered by following sound exposition in the root of Arabidopsis
thaliana to define the rapid cellular processes induced by sound per-
ception. We show that sound reduced plant growth and lateral root
growth, which was likely associated with an increase K+ leakage, and
altered the gravitropic response of Arabidopsis thaliana roots. We also
provide evidence that increase in cytosolic Ca2+ is a key player in the
initial events of sound wave vibration perception and early transduc-
tion in plants. Changes in K+ fluxes and an increase in superoxide
production were also found to be involved in sound perception. These
data strongly suggest that changes in ion fluxes (Ca2+ and K+) and an
increase in superoxide production are involved in the early signalling
events in sound perception in plants, as previously established in ani-
mals.
2. Materials and methods
2.1. Seedling material and growth conditions
Arabidopsis (Arabidopsis thaliana L.) seeds ecotype Columbia-0 were
surface‐sterilized and placed on half-strength Murashige and Skoog
(MS) culture medium without vitamins and containing 1% sucrose,
solidified using 0.8% phytagel (Sigma-Aldrich). The plates were store at
4 °C for 48 h in darkness to break dormancy and synchronize germi-
nation. Subsequently plants were kept vertically under a long-day cycle
of 16 h light (78 mmol m−2 s−1 irradiance)/8 h dark at 22 °C, with the
exception of plants used for the gravistimulation experiment, which
were placed horizontally in darkness to avoid phototropism and the
angle of curvature measured after 5 days. 4-day old uniformseedlings
were subsequently transferred in square plates without sucrose for 1 or
2 weeks with or without unilateral sound in egg-box faced foam isolated
boxes.
2.2. Sound treatment
The single frequency signal was generated using the NCH Software
− Tone Generator v2.10. During the sound treatments, the 28 mm
speaker (RS components, Italy) and the plants were placed on different
shelves to prevent transfer of vibrations. We selected 200 Hz as pre-
vious work demonstrated that this frequency has a physiological re-
levance for plants; indeed, this frequency was previously found to in-
duce positive phonotropism both in maize roots growing in water/
solute [7] and in Arabidopsis roots growing in air/on agar surface in
Petri dishes.
2.3. Monitoring of ion changes with fluorescent dyes
The acetoxymethyl ester (AM) of Fluo-4, a Ca2+-sensitive fluor-
escent dye was purchased from Molecular Probes (F14201, Invitrogen,
Molecular Probes, Eugene, OR, USA). 7–10 days-old intact seedlings
were incubated in a solution containing 10 μM Fluo-4/AM, 100 mM
KCl, 5 mM Ca-EGTA, 10 mM MOPS pH 7.2 for 30 min at room tem-
perature in the dark, and then washed in a free Fluo-4/AM solution and
placed in 0.01% Poly-L-Lysine (P4832, SIGMA)-coated small Petri
dishes filled with Fluo-4/AM free solution. After 1 h, roots were vi-
sualized under a confocal microscope (LEICA Laser scanning confocal
microscope SP5, Leica Microsystems, Germany) with a 20 x dry objec-
tive. The excitation wavelength was set at 488 nm, and emission was
detected at 505 ± 20 nm. Short sound exposition was performed with
a 28 mm speaker (RS components, Italy) attached to the microscope, to
avoid direct contact with the samples and prevent transfer of vibrations.
The single frequency signal was generated using the NCH Software −
Tone Generator v2.10.
The K+‐sensing fluorescent probe potassium-binding benzofur-
anisophthalate (PBFI) was used to quantify K+ activity in living root
exposed to sound as described in [22]. After PBFI (P1267MP, Molecular
probes) incubation, root tips were visualized under a ZEISS OBSERVER
Z1 AX10 microscope with a Kubler codix HXP120 fluorescence lamp, a
DAPI filter and a 20X dry objective. In addition, roots were also in-
cubated in measuring solution with 10 mM tetraethylammonium
chloride (TEA+, K+ channel blocker) for 1 h prior to visualization.
2.4. Monitoring in vivo K+ fluxes in roots
Following 2 weeks of continuous sound exposure or growth in
control conditions, K+ fluxes were monitored by placing the seedlings
in a measuring chambers filled with 4 mL of basal salt media (BSM,
0.1 mM CaCl2, 0.2 mM KCl), where the primary root was immobilized
in the horizontal position for 40 min to adapt the plants to the new
conditions prior to the measurement. Net K+ fluxes were then mea-
sured on roots using the vibrating probe non-invasive system
(University of Florence, Italy), a non-invasive technique for measure-
ments of specific ion fluxes from plant tissues. Details on fabrication
and calibrations of microelectrodes were described previously in [23].
Basal K+ were measured for 10 min in control plants and in plants
exposed to 200 Hz for 2 weeks.
2.5. Aequorin luminescence measurements
The [Ca2+]cyt variations were recorded with A. thaliana seedlings
expressing the aequorin gene. Aequorin was reconstituted by overnight
incubation in MS medium supplemented with 30 g L−1 sucrose and
2.5 μM native coelenterazine. Four 7d-old seedlings were transferred
carefully into a luminometer tube, and the luminescence counts were
recorded continuously at 0.2 s intervals with a luminometer. Sound
exposition was performed with a 28 mm speaker (RS components, Italy)
placed inside the luminometer measurement room. The residual ae-
quorin was discharged by addition of 500 μL of a 1 M CaCl2 solution
dissolved in 100% methanol. The resulting luminescence was used to
estimate the total amount of aequorin in each experiment. Calibration
of calcium measurement was performed by using the equation:
pCa = 0.332588(-logk) + 5.5593, where k is a rate constant equal to
luminescence counts per second divided by total remaining counts [24].
The Ca2+ channel blocker Gd3+ (50 μM Gd3+) and the inhibitor of the
cADP-ribose/ryanodine receptor dantrolene (100 μM prepared by dis-
solving dantrolene in methanol, with the final concentration of me-
thanol in the experimental solution never exceeding 0.5% (v/v)) were
added 15 min before sound exposition.
2.6. Cell culture conditions
Arabidopsis thaliana suspension cultured cells were grown in MS
medium, pH 5.8 augmented with 30 g L−1 sucrose, 0.2 mg L−1 2,4-D
[26] under control conditions (i.e. no sound). Cells were maintained at
22 ± 2 °C, under continuous darkness and continuous shaking (gyra-
tory shaker) at 150 rpm. Cell suspensions were sub-cultured weekly
using a 1:8 dilution. All experiments were performed at 22 ± 2 °C
A. Rodrigo-Moreno et al. Plant Science 264 (2017) 9–15
10
using log-phase cells (4 days after sub-culture) maintained in their
culture medium to limit stress. Cell densities were about 3.105 cells
mL−1 for A. thaliana. Freshly obtained A. thaliana cells were then used
to monitor ROS production and for voltage clamp measurements.
2.7. Monitoring of ROS production
The production of ROS of A. thaliana cells was monitored by the
chemiluminescence of the Cypridina luciferin analog (CLA) as pre-
viously described [25,26]. Freshly obtained cells were carefully trans-
ferred into a luminometer tube, and the luminescence counts were re-
corded after a few minutes with a FB12-Berthold luminometer. CLA is
known to react mainly with O2•‐and 1O2 with light emission [27]. The
NADPH-oxidase inhibitor diphenyleneiodonium chloride (10 μM DPI)
or 500 μM Gd3+ were added 15 min before sound exposition.
2.8. Voltage clamp measurements
Plasma membrane potential (Vm) were measured on 4-day-old cul-
tured cells maintained in their culture medium with the main ions in
MS medium being 28 mM NO3− and 16 mM K+ [26,28]. Individual
cells were immobilized by a microfunnel (approximately 50–80 μm
outer diameter) and impaled with microelectrode controlled by mi-
cromanipulators (WR6-1, Narishige, Japan) in a 500 μL chamber.
Sound exposition was performed with a 28 mm speaker (RS compo-
nents, Italy) attached to the microscope, avoiding direct contact with
the samples. Microelectrodes were made from borosilicate capillary
glass (Clark GC 150F, Clark Electromedical, Pangbourne Reading, UK)
pulled on a vertical puller (Narishige PEII, Japan). Their tips were less
than 1 μm diameter; they were filled with 600 mM KCl, and had elec-
trical resistances between 20 and 50 MΩ with the culture medium.
Voltage-clamp measurements of whole-cell currents from intact cul-
tured cells presenting stable running membrane potential (Vm) were
carried out using the technique of the discontinuous single voltage-
clamp microelectrode (dSEVC, Finkel and Redman, 1984) adapted to
plant cells as previously described [26]. Specific software (pCLAMP 8)
drives the voltage clamp amplifier (Axoclamp 2A, Molecular Devices,
Sunnyvale, USA). Voltage and current were simultaneously displayed
on a dual input oscilloscope (Gould 1425, Gould Instruments Ltd,
Hainault, UK), digitalised with a Digidata 1322A (Molecular Devices,
Sunnyvale, USA). In whole-cell current measurements the membrane
potential was held to the value of the resting membrane potential.
Current recordings were obtained by hyper- and depolarizing pulses
from −200 to +80 mV (20 mV, 2 s steps of current injection, 6 s of
settling time). Pre-treatment of cells with TEA-Cl (10 mM) was don
15 min before sound exposure.
2.9. Statistical analysis
All statistical analyses were performed with the software Graph
Path Prism 5.00. Normal distribution was tested with a Shapiro–Wilk
test and to identify any statistical differences between treatments, a
one-way ANOVA was performed. When significant differences (p-
value< 0.05) were found, a Tukey post hoc test was applied to further
discriminatebetween significantly different groups. Student’s unpaired
two-tailed t-test was used for single comparisons.
3. Results
3.1. Sound affects gravitropism and induces phonotropism of Arabidopsis
roots
We observed that sound reduced plant growth and modulated the
gravitropism of the primary roots of young Arabidopsis seedlings (Fig. 1
and S1). When grown vertically, the roots of the plants exposed for 2
weeks to 200 Hz showed positive phonotropism as they curved towards
to sound source (Fig. 1a,b). To confirm the effect of sound on root
growth direction, we repeated this experiment by growing the seedlings
horizontally in darkness to exclude any possible influence of light. As
shown in Fig. 1c, the dark-grown roots of control plants curved and
grew straight downwards. On the other hand, plants grown under
sound exposition lacked the ability to respond to the vector gravity, and
roots curved towards to sound source (Fig. 1c). Furthermore, sound-
treated plants always showed shorter lateral roots, which was likely
associated with an increase K+ leakage to the external medium as the
addition of 10 mM KNO3 to the growing medium restored wild type
phenotype under sound conditions (Fig. 1d).
3.2. Sound triggered rapid elevations in cytosolic Ca2+and increased O2−
production
There was a calcium increase at the pericycle level of intact sound-
treated seedlings, measured with FLUO-4, just after 5 min of sound
exposition (Fig. 2a,b). This calcium increase was inhibited by pre-
incubation with 50 μM Gd3+, an inhibitor of MCA1 channels ([29],
Fig. 2b). Subsequently cytoplasmic Ca2+ variations were also recorded
as described previously [28] in whole A. thaliana seedlings expressing
the apoaequorin gene [24], where a significant cytoplasmic Ca2+ in-
crease was detected within the first 2 min after 2 min of treatment.
These sound-induced Ca2+ increase could be inhibited by 500 μM
Gd3+, an inhibitor of plasma membrane Ca2+ channels, and by 100 μM
dantrolene, inhibitor of the cADP-ribose/ryanodine channel-receptor
involved in internal Ca2+ stock release ([30]; Fig. 2c).
After a few minutes of treatment, sound induced an increase in
superoxide production in Arabidopsis cells, which was inhibited by
both Gd3+ (500 μM) and the NADPH oxidase inhibitor diphenyleneio-
donium chloride (DPI, 10 μM) (Fig. 3).
3.3. Sound activated K+ channels and trigger K+ efflux from Arabidopsis
roots
As above-mentioned the phenotype of sound-treated plants shows
shorter lateral roots (Fig. 1d), similar to control plants grown in po-
tassium deficiency [31]. We therefore measured K+ content in sound
treated plants and observed that after 9–12 days, sound treatment re-
sulted in a decline in total seedling K+ content (Fig. S2) and, compared
to control plants, there was a significant K+ decrease in lateral roots of
sound-treated plants (Fig. 4a). This was confirmed with electro-
physiological measurements with the VIP system which showed a sig-
nificant increase in K+ efflux in sound-treated plants (Fig. 4b).
Using the K+-sensitive fluorescent dye PBFI-AM in a time-course
experiment (0–10 min after sound exposure), we observed that six
minutes after exposing the plants to 200 Hz, sound induced a potassium
decrease in intact roots that was inhibited by the pre-incubation with
the K+ channel blocker TEA+ (Fig. 4c). These decrease in K+ content
observed in seedling exposed to sound could be related to the increase
in outward rectifying K+ currents sensitive to TEA+ as observed for
Arabidopsis cultured cells exposed to sound for 5 min (Fig. 5).
4. Discussion
In this paper, we present insights into the nature of the cellular
processes involved in sound perception and positive phonotropism of in
Arabidopsis roots. In particular it emerges that changes in ion (Ca2+
and K+) fluxes are involved in sound perception in plants. Importantly
in this respect, it is well demonstrated that these two ions play a key
role in the regulation of sound perception and mechanotransduction in
animals [32–35]. These results support the hypothesis that, regardless
of different structures involved in sound perception in different species
across kingdoms, local processing and response of sound stimuli are
more-or-less similar [7–9].
Results here show the involvement of Ca2+ ion in sound perception
A. Rodrigo-Moreno et al. Plant Science 264 (2017) 9–15
11
in Arabidopsis roots. In plants, intracellular Ca2+ is considered as one
of the most important players in the plants’ signal transduction path-
ways and elevation in cytosolic Ca2+ has been shown to be at the core
of a sophisticated network of signalling pathways that shape gene ex-
pression and cell physiology [36]. The calcium data suggests that this
cytosolic Ca2+ increase could be due to an influx through plasma
membrane and a release from internal stock, thus possibly involving a
Ca2+ induced Ca2+ release process as observed in inner hair cells of the
mice auditory system [37]. Interestingly, MCA1 and MCA2 (from mid1-
complementing activity 1 and 2), which are specifically Ca2+ channels
[38], have been found to mediate calcium influx in response to me-
chanical stimulation [29,39], favouring the hypothesis that these
channels might be involved in the sound-induced Ca2+ elevations. Al-
though we analysed the expression of both channels with RT-PCR, the
variability in MCA1 and MCA2 expression in control plants do not allow
us to conclude whether there was or not an increase in the expression of
the genes encoding these channels in sound-treated plants (data not
shown).
In vertebrates, hearing rely on tightly controlled ionic environ-
ments, and K+ fluxes play a fundamental role in sensory transduction
[33,35,40]. In plants, the early increase in outward K+ channel activity
concomitant to the early decrease in K+ content suggests a similar in-
volvement of K+ flux regulation in the signalization process induced by
sound. However, it is important to note that the delayed decrease in K+
content and observed K+ could be related to the sound-induced growth
reduction (Fig. S1, Supplementary data). Electrolyte leakage, mainly
related to K+ efflux, leads to irreversible K+ loss in stressed plants and
is considered a hallmark of response in intact plant cells in response to
most abiotic and biotic stresses that stops growth, possibly saving plants
resources for adaptation and reparation needs [41]. Indeed, confirming
the view that the declines in K+ concentrations in sound treated plants
were associated with the sound-induced growth reduction, the pheno-
type of sound-treated seedlings shows shorter lateral roots
(10.1 ± 2.8 mm in control plants, n = 8; and 2.8 ± 0.4 in sound-
treated plants, n = 10, T-test, P< 0.001), as observed in K+ deficient
plants [31], and following the addition in K+, root growth in sound
treated plants recovered to control values.
Sound exposures induced ROS generation that was inhibited by DPI,
inhibitor of NADPH-oxidase, and Gd3+, inhibitor of Ca2+ channels.
These findings suggest that NADPH-oxidase could be the source of ROS
and that cytosolic Ca2+ variations in response to sound occurs upstream
of ROS generation. Supporting further the importance of ROS in sound
signalling, the NADPHoxidase NOX3, a superoxide (O2−) generating
enzyme, is highly expressed in the inner ear of animals, although its
physiological role in vivo in hearing has yet to be determined [42].
Plants homologues to these respiratory burst oxidases, Rboh, can
mediate an essential role in the initiation and amplification of different
signals related to stress processes but also growth [43]. It is further
noticeable that, mechanical stimulation elicits in Arabidopsis Ca2+-
dependent activation of RBOH C, resulting in ROS production [19].
Sound vibrations, like gravity, touch or sound waves; are pressure
waves that have a mechanical influences on cellular and subcellular
structures [44]. Although the activation of mechanosensitive channels
in plants is still discussed, two models have been proposed: one isthe
Fig. 1. Sound waves affect Arabidopsis root gravitropism and reduced lateral root length. (a) Control plants after 2 weeks; (b) Arabidopsis plants grown under a constant unilateral
frequency of 200 Hz. The images in (a) and (b) are representative images for each treatment and the red arrow shows the direction of the unilateral sound. (c) The gravitropic response of
sound-treated roots is altered in plants exposed to 200 Hz (n = 47) compared with control plants (n = 41). In the graphs in (c) the length of each single line indicates the number of
seedling, with the length of the black line indicating 10 seedlings. (d) Lateral root length (mm) of control and 2 weeks sound-exposed Arabidopsis seedlings grown in half-strength
Murashige and Skoog (MS) culture medium or with supplemental 10 mM KNO3 (for a final KNO3 concentration of 20 mM). Data are mean ± SE; n = 8-19. * α = 0.05. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Sound waves altered Ca2+ fluxes in the primary root of Arabidopsis. (a) Overlay images (bright field and FLUO-4 fluorescence) showing the increase in fluorescence intensity at
the pericycle level in the primary root of Arabidopsis plants exposed to 200 Hz alone, or (b) after pre-treatment with 50 μM Gd3+. (c) Mean increase in fluorescence intensity at the
pericycle after 300 s of 200 Hz exposition in control and gadolinium (50 μM Gd3+) pre-treated plants for 1 h; n = 15. (d) Changes in [Ca2+]cyt of Arabidopsis whole seedlings expressing
the aequorin gene upon 200 Hz exposition, alone or after a 15 min pre-treatment with channel inhibitors (500 μM Gd3+, or 100 μM dantrolene). Data are mean ± SE; n = 15, number of
independent measurements; Asterisks mark significant differences from 200 Hz; * α = 0.05; **α = 0.01; ***α = 0.001.
A. Rodrigo-Moreno et al. Plant Science 264 (2017) 9–15
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direct activation by tension at the plasma membrane and the other
involves the cytoskeleton. In mammalian cells, mechanical stimulation
of actin filaments can directly activate mechanosensitive channels [45]
and the same model has been hypothesized for plant gravitropism,
causing the initial calcium increase [46,47]. Based on this last hy-
pothesis, here we propose a novel model in plant communication in-
itiated by sound waves (Fig. 6). Sound activates mechanosensitive Ca2+
channels, as occurs in animal hearing. The Ca2+ dependent generation
of reactive oxygen species by NADPH oxidases would amplify the signal
and activates outward rectifying K+ channels [48].
What could be the ecological significance and benefits of sound
perception in plants? At the shoot level, the study by Appef and Cocroft
[11] suggests that vibrations may represent a new long distance sig-
nalling mechanism in plant–insect interactions that contribute to
systemic induction of chemical defences. Therefore might be ecological
benefits of sound perception in roots. For example, the regular clicking
sounds generated by tips of growing maize root apices has previously
been hypothesized to have a role for root echolocation, allowing roots
growing in soil to locate in advance water and obstacles deep in soil [7].
Furthermore, as sound travels readily and far in dense environments
such as soil, a plant’s ability to detect vibrations might enable plants to
acoustic cues to locate distant water sources [49]; indeed the ambient
noise spectra for water streams and rivers, despite being variable de-
pending on turbulence, the presence of water breaking the surface or
waterfalls, can be measured in the range 0–500 Hz [50], which falls
within the range of frequencies that affect plant root growth rates and
directions [7]. In support of physiologically relevant root phonotropism
scenario, apices of growing maize roots produce regular sound waves.
Fig. 3. Sound waves altered reactive oxygen species production in Arabidopsis cells. ROS production in Arabidopsis cells after 200 Hz exposure, detected with the Cypridina luciferin
analog (CLA): (a) Kinetics of ROS production under control conditions, (b) after sound exposure and after sound exposure with pre-treatment (15 min before sound exposure) with (c) the
NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI, 10 μM) or (d) the MCA1 blocker gadolinium (Gd3+, 500 μM). (e) Mean ROS generation in Arabidopsis cells 3 min after
200 Hz exposure, with or without pre-treatment with 10 μM DPI or 500 μM Gd3+. In (a-d) values are mean ± SE; n = 3-5. In (e) values are mean ± SE; n = 6-10. Asterisks mark
significant differences from control or 200 Hz; ***α = 0.001.
Fig. 4. Sound waves altered K+ fluxes in the primary root Arabidopsis (a) Average PBFI fluorescence change in control and tetraethylammonium chloride (10 mM TEA+) pre-treated
Arabidopsis lateral roots for 1 h and then exposed for 6 min to 200 Hz; Values are mean ± SE, n = 12 (b) Average steady stake K+ fluxes of primary roots in control Arabidopsis
seedlings or in seedlings exposed to sound (200 Hz) for 2 weeks; Values are mean ± SE, n = 8-10. (c) Average fluorescence intensity due to PBFI in control and 9–12 days sound-exposed
Arabidopsis lateral roots; Values are mean ± SE, n = 21.
A. Rodrigo-Moreno et al. Plant Science 264 (2017) 9–15
13
These acoustic emissions generate relatively loud (ca. 2 cm/s) and
frequent clicks, which can be measured at some distance within the
fluid medium (Fig. 1c in [7]). Albeit being at this stage speculative and
considering that environmental vibrations rarely come in single fre-
quencies of steady pattern and are usually complex in both frequency
and amplitude, these finding highlights further the importance of un-
derstanding sound perception in plants.
Acknowledgments
The authors would like to thank Bernadette Biligui for technical
support. This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.plantsci.2017.08.001.
References
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Fig. 5. Sound waves increased the outward recti-
fying K+ current sensitive to TEA+ in Arabidopsis
thaliana cultured cells. Sound-induced increase in
outward-rectifying K+ currents in Arabidopsis
thaliana cultured cells. The different current traces
correspond to the time course experiment: (a)
Typical currents recorded for cultured cells about
one min before applying sound, (b) 5 min after
sound exposure and (c) 1 min after adding 5 mM
tetraethylammonium chloride (TEA+) and thus
6 min after sound application. The protocol is as il-
lustrated in (d) with eight steps ranging from −200
to +80 mV, and the holding potential (Vh) was Vm.
Mean increase in time and voltage dependent out-
ward K+ currents at + 80 mV was 1.24 +/−
0.35 nA, n = 5.
Fig. 6. Proposed model summarizing the events in-
itiated by sound waves in a plant cell. Sound acti-
vates mechanosensitive Ca2+ channels, and subse-
quently the Ca2+ dependent generation of reactive
oxygen species by NADPH oxidases amplifies the
signal and activate outward rectifying K+ channels.
A. Rodrigo-Moreno et al. Plant Science 264 (2017) 9–15
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	Root phonotropism: Early signalling events following sound perception in Arabidopsis roots
	Introduction
	Materials and methods
	Seedling material and growth conditions
	Sound treatment
	Monitoring of ion changes with fluorescent dyes
	Monitoring in vivo K+ fluxes in roots
	Aequorin luminescence measurements
	Cell culture conditions
	Monitoring of ROS production
	Voltage clamp measurements
	Statistical analysis
	Results
	Sound affects gravitropism and induces phonotropism of Arabidopsis roots
	Sound triggered rapid elevations in cytosolic Ca2+and increased O2− production
	Sound activated K+ channels and trigger K+ efflux from Arabidopsis roots
	Discussion
	Acknowledgments
	Supplementary data
	References