SCIENCE SIGNALING | RESEARCH ARTICLE

PLANT BIOLOGY Copyright © 2020

Two glutamate- and pH-regulated Ca2+ channels are

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required for systemic wound signaling in Arabidopsis American Association
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Qiaolin Shao1,2*, Qifei Gao1,3,4*, Dhondup Lhamo1, Hongsheng Zhang2, Sheng Luan1† to original U.S.
Government Works
Plants defend against herbivores and nematodes by rapidly sending signals from the wounded sites to the whole
plant. We investigated how plants generate and transduce these rapidly moving, long-distance signals referred
to as systemic wound signals. We developed a system for measuring systemic responses to root wounding in
Arabidopsis thaliana. We found that root wounding or the application of glutamate to wounded roots was sufficient
to trigger root-to-shoot Ca2+ waves and slow wave potentials (SWPs). Both of these systemic signals were inhibited
by either disruption of both GLR3.3 and GLR3.6, which encode glutamate receptor–like proteins (GLRs), or constitutive
activation of the P-type H+-ATPase AHA1. We further showed that GLR3.3 and GLR3.6 displayed Ca2+-permeable
channel activities gated by both glutamate and extracellular pH. Together, these results support the hypothesis
that wounding inhibits P-type H+-ATPase activity, leading to apoplastic alkalization. This, together with glutamate
released from damaged phloem, activates GLRs, resulting in depolarization of membranes in the form of SWPs
and the generation of cytosolic Ca2+ increases to propagate systemic wound signaling.

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INTRODUCTION
Plants are constantly under mechanical stress caused by weather
conditions such as wind and by biotic factors, including pathogens
and herbivorous animals. Such factors can lead to tissue damage or
wounding in both underground and aboveground organs. Particularly
impactful to crop production is pest infestation by leaf-chewing
insects and root nematodes. In response to wounding, plants initiate
sophisticated mechanisms to repair the wounded tissues and defend
against pests. The plant hormone jasmonic acid (JA) is a critical
regulator of wound responses and is synthesized upon tissue damage
(1–6). Studies have revealed molecular pathways for JA signal trans-
duction that begins with the intracellular receptor COI1 (7–10),
followed by the degradation of JAZ family repressors to activate
JA-responsive genes (11–14). JA-based responses occur immediately
not only at the wound site but also in unwounded distal organs
linked to the wound site through the vasculature (15). JA biosynthesis
takes place within 1 min in leaves distal to the wounded leaf (16–18),
implicating a long-distance rapid wound signal moving from the
wounded leaf to distal leaves. Several candidates for this rapid signal
have been identified, including slow wave potentials (SWPs), glutamate,
Ca2+, and reactive oxygen species (ROS) (19–22).
SWPs are electrical signals generated upon wounding by the
depolarization and repolarization of the cell membrane in some cell
populations in plants. Although the precise origin of SWPs is still
under debate, SWPs remain a viable candidate for the systemic
wound signal (19–23). In particular, several studies have linked
SWPs to systemically induced JA biosynthesis and wound responses
through genetic analysis of SWP-defective mutants and artificial
manipulation of the leaf potential by current injection (24). The
Arabidopsis thaliana genetic mutants defective in SWP include those
1
Department of Plant and Microbial Biology, University of California, Berkeley,
Berkeley, CA 94720, USA. 2State Key Laboratory of Crop Genetics and Germplasm
Enhancement, Jiangsu Collaborative Innovation Center for Modern Crop Production,
College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.
3
School of Life Sciences, Northwest University, Xi’an 710069, China. 4School of
Life Sciences, Shaanxi Normal University, Xi’an 710119, China.
*These authors contributed equally to this work.
†Corresponding author. Email: sluan@berkeley.edu
with disrupted glutamate-receptor-like (GLR) genes, such as GLR3.3
and GLR3.6. The glr3.3glr3.6 double mutant is specifically compro-
mised in the capacity to propagate the wound SWP signal to the
distal leaves, which is accompanied by impaired JA biosynthesis in
undamaged leaves, supporting the hypothesis that electrical signals
can be a rapid systemic wound signal (24, 25). SWP signals in most
studies are recorded by tissue surface electrodes, making it difficult
to interpret the contribution of different cell types to these SWP
signals. To overcome this problem, the electrical penetration graph
(EPG) has been used to detect changes in the membrane potential
of Arabidopsis sieve elements during plant-herbivore interactions
(26). EPG recordings have shown that the glr3.3glr3.6 double mutant
fails to propagate wound-induced SWPs, consistent with the results
from conventional surface electrode recording and confirming the
importance of GLR3.3 and GLR3.6 in SWP propagation (27).
Work by Kumari et al. (28) suggests that the P-type adenosine
triphosphatase (ATPase), Arabidopsis H+-ATPase 1 (AHA1), plays
a role in modulating SWPs in response to wounding, suggesting
that SWPs may be a proxy of plasma membrane potential. Because
depolarization is critical for systemic wound signals, AHA1 activity
should inhibit depolarization through hyperpolarization or re-
polarization of the membrane. As a result, the repolarization phase
is extended in the aha1 mutant and shortened in the gain-of-function
AHA1 allele ost2-2D (28). These changes have altered the SWPs with
a consequence in systemic wound response. Thus, the exact pattern
of the SWPs is functionally relevant to wound signal propagation.
The systemic cytosolic Ca2+ wave has been shown to be another
early wound response event (29–34). Plant GLRs are putative
Ca2+-permeable channels and receptors for glutamate, and direct
testing of the involvement of glutamate in wound responses has
identified a connection between glutamate and wound-responsive
Ca2+ waves (34). The application of 50 to 100 mM glutamate at a
wound site triggers a long-distance Ca2+ wave typical of that induced
by mechanical or insect-inflicted wounding events, indicating that
glutamate may serve as a trigger of the long distance rapid wound
signal. The plant phloem contains 10 to 100 mM glutamate, and the
concentration of glutamate in the apoplast, the space between the
cell membrane and cell wall, may rise to 50 mM at damage sites (34).

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SCIENCE SIGNALING | RESEARCH ARTICLE
Thus, it is proposed that the glutamate released from the phloem
may serve as a damage-associated molecular pattern (DAMP), and
GLR3.3 and GLR3.6 could be activated in response, leading to the
increase of cytosolic Ca2+ concentration ([Ca2+]cyt) (34). Furthermore,
wound-induced cytosolic Ca2+ increase, together with calmodulin,
initiates disassociation of the JAV1-JAZ8-WRKY51 complex, lead-
ing to the activation of JA biosynthetic genes (35). Although these
studies support the importance of local and systemic Ca2+ signals in
the wound response, the puzzling question concerns the underlying
mechanism for the propagation of this [Ca2+]cyt increase from wound
sites to distal leaves. Specifically, despite evidence supporting the
essential function of GLR3.3 and GLR3.6 proteins for the propagation
of the systemic wound signal, it remains unknown whether they are
indeed glutamate-sensitive Ca2+-permeable channels.
ROS have also been proposed to act as a long-distance rapid
wound signal (36). Using a transgenic plant expressing luciferase driven
by the ROS-responsive AtZAT12 promoter (37, 38), researchers
have identified a wound-induced systemic ROS signal traveling at a
rate of 8.4 cm/min, which depends on the respiratory burst oxidase
homolog D (RbohD) in Arabidopsis (36). A newly developed method
(39) can directly measure ROS produced upon wounding in planta using
the oxidation of fluorescence probes such as 2′,7′-dichlorofluorescin
diacetate. In addition, heat or high light stress can also generate
systemic electrical signals that may be linked to ROS, because the
amplitude of SWPs is reduced in the rbohD mutant (40).
Although parasitic nematodes do not wound plants the same way
as chewing insects, nematode attacks also induce a systemic defense
response that involves the production of JA (41). In tomato, the
root-knot nematode induces the systemic transmission of electrical
and ROS signals from attacked roots to the leaves, leading to an
increased accumulation of JAs in the leaves (42). Tomato mutants
lacking GLR3.5 or RBOH1 are compromised in nematode-induced
electrical and ROS signals in the leaves, suggesting that ROS and
SWPs may serve as nematode-induced root-to-shoot systemic
signals in tomato plants (42).
Although several candidates for systemic wound signals have been
identified, little is known about the relationship of these candidates,
especially concerning SWPs and systemic Ca2+ waves. In particular,
GLR3.3, GLR3.6, and AHA1 are involved in both the SWPs and Ca2+
waves, but little is known about the mechanism underlying the func-
tion of GLRs and AHA1 in the wound response. Here, we established
a root-to-shoot long distance signaling system in Arabidopsis and
showed that glutamate activated both SWPs and systemic Ca2+
waves that depended on the actions of GLR3.3, GLR3.6, and AHA1.
The GLRs were functionally connected to AHA1 in systemic signal-
ing because they were glutamate-activated and proton-inhibited
Ca2+-permeable channels required for generating both the membrane
depolarization and Ca2+ increases responsible for electrical and
Ca2+ waves in plants.
RESULTS
Wounding and glutamate trigger systemic root-to-shoot
[Ca2+]cyt waves
As previously reported (34), systemic [Ca2+]cyt increase is a hallmark
of the leaf-to-leaf long distance signaling in response to wounding.
To establish a model for studying the root-to-shoot systemic wound
response, we used transgenic plants carrying a single copy of the
Ca2+-responsive fluorescent protein GCaMP6s (43) to monitor
Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020
[Ca2+]cyt changes in live plants while wounding the roots. When
mechanical wounding was applied to roots by severing the main root
at a site 2 cm away from the root-shoot junction, a strong vasculature-­
based [Ca2+]cyt increase was observed in the rosette leaves (Fig. 1, A and B,
and movie S1). Also, the [Ca2+]cyt increase was seen in all leaves,
unlike the leaf-to-leaf system in which the [Ca2+]cyt changes prefer-
entially propagated to leaves at positions n ± 3 and n ± 5 relative
to the wounded leaf (34). Such differences likely resulted from the
vascular connection of main roots to all the above-ground organs.
Video recording of the time course of the [Ca2+]cyt rise indicated a
rapid and uniform change in Ca2+ in all leaves within 1 min of
wounding (movie S1), similar to the time course of the leaf-to-leaf
systemic [Ca2+]cyt wave (34). We then tested whether glutamate at
the wound site in the root also elicited systemic [Ca2+]cyt changes, as
shown in the leaf-to-leaf signaling system (34). Fifteen minutes
after wounding, when the wound-responsive Ca2+ waves returned
to the resting level, application of a 10-l drop of 100 mM glutamate
to the wound site induced another strong and rapid systemic
[Ca2+]cyt increase in all the leaves of the rosette, with similar kinetics
as that caused by wounding (Fig. 1, C and D, and movie S2).
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We explored the effect of different wound sites along the roots
on the systemic response and found that the response weakened
with distance from the root-shoot junction. The maximum F/F0
induced by a cut 2 cm away from the junction was significantly larger
than that caused by a cut at 5 cm (Fig. 1E). We also found that
wounding a lateral root triggered a similar but weaker systemic
[Ca2+]cyt increase as compared to wounding the main root (fig. S1A).
To test the effect of different glutamate concentrations on systemic
Ca2+ waves, we divided plants into three groups (A, B, and C)
and then wounded the plant roots 2 cm away from the root-shoot
junction. The three groups showed similar [Ca2+]cyt increases in
response to wounding (Fig. 1F). After 15 min, we added 100, 50,
and 20 mM glutamate to the wound site of the group A, B, and C
plants, respectively, and found that 50 and 100 mM glutamate
triggered a significantly larger [Ca2+]cyt increase than did 20 mM
glutamate (Fig. 1G). These results demonstrated that wound-induced
root-to-shoot systemic [Ca2+]cyt rise was dependent on both the
concentration of apoplastic glutamate and the distance of wound
sites from the root-shoot junction.
Glutamate, like wounding, induces a root-to-shoot systemic
electrical wave
Wounding initiates leaf SWPs that propagate from the wounded leaf
to undamaged distal leaves at a speed of ~5 cm/min, which induces
systemic JA biosynthesis and qualifies SWPs as a systemic wound
signal (24). We performed leaf-to-leaf SWP recordings as described
in earlier studies (24) and obtained results similar to those published
(fig. S2). We then tested whether SWPs were also produced during
the root-to-shoot systemic wound response. When the main root
was wounded, the surface potential (SWP) measured at a rosette
leaf showed a quick hyperpolarization, followed by prolonged de-
polarization and repolarization phases (Fig. 2, A to D). Wounding
lateral roots also triggered a similar systemic SWP (fig. S1B). The
SWPs measured for the root-to-shoot wound response appeared
similar to SWPs in the leaf-to-leaf response, consistent with the
hypothesis that the root-to-shoot wound response also involves SWPs
as a long-distance signal. As one of the plant DAMPs, glutamate
triggers a systemic [Ca2+]cyt rise that, like SWPs, also causes JA
synthesis in distal leaves (34). To address the relationship between
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A C D
0.8 1.0

F/F ([Ca2+]cyt))
0s 0s **
0.8

F/F ([Ca2+]cyt)
0.6
0.4 0.6

0.2 0.4
Leaf 4 0.2
0.0 Leaf 5
Leaf 6 0.0
0 120 240 360 Leaf 4 Leaf 5 Leaf 6
Time (s)
60 s 60 s E
0.8 1.0

F/F ([Ca2+]cyt))
0.8

F/F ([Ca2+]cyt)
0.6
**
0.6
0.4
0.4
0.2
2 cm
0.2
0.0 5 cm 0.0
0 120 240 360 2 cm 5 cm
300 s 300 s
Time (s)
F 1.0
0.8

F/F ([Ca2+]cyt))
0.8

F/F ([Ca2+]cyt)
0.6
0.6

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0.4
0.4
0.2 Group A
0.2
Group B
Group C
0.0 0.0
[Ca2+]cyt Low High A B C
0 120 240 360
Time (s) Group
B G
0.8 1.0 0.8 1.0 **

F/F ([Ca2+]cyt))
Group A
F/F ([Ca2+]cyt))

Group B **
0.8 Group C 0.8
F/F ([Ca2+]cyt)

0.6
F/F ([Ca2+]cyt)

0.6
0.6 0.4 0.6
0.4 **
0.4 0.4
0.2
0.2 0.2 0.2
Leaf 4
Leaf 5 0.0
0.0 Leaf 6 0.0 0.0
0 120 240 360 Leaf 4 Leaf 5 Leaf 6 0 120 240 360 A B C
Time (s) Time (s) Group

Fig. 1. Wounding and glutamate trigger root-to-shoot transmission of [Ca2+]cyt waves. (A) Imaging of WT (Col-0) plants expressing the genetically encoded intracellular
Ca2+ indicator GCaMP6s at the indicated time points after cutting the main root. (B) Kinetics of [Ca2+]cyt increases in leaves 4, 5, and 6 quantified along the time course
after wounding. n = 10 plants. Boxplot shows max [Ca2+]cyt in the time frame. (C) Ca2+ imaging of WT plants expressing GCaMP6s at the indicated time points after adding
100 mM glutamate at the wound site. Scale bar, 1 cm. (D) Kinetics of [Ca2+]cyt increases in leaves 4, 5, and 6 quantified along the time course after application of glutamate.
n = 10 plants. Boxplot shows max [Ca2+]cyt in the time frame. (E) Comparison of the time courses of [Ca2+]cyt increase in leaf 6 when roots were cut at either 2 or 5 cm away
from the root-shoot junction. n = 10 plants (2 cm) and 13 plants (5 cm). Boxplot shows max [Ca2+]cyt in the time frame. (F and G) [Ca2+]cyt increases recorded in leaf 6 of
three groups of plants after wounding (F) and in the same leaves again after application of 100 mM (group A), 50 mM (group B), or 20 mM (group C) glutamate to the
wound sites (G). n = 8 plants (group A), 10 plants (group B), and 10 plants (group C). Error bars, means ± SEM. Boxplots show the minimum, 25th percentile, median, 75th
percentile, and maximum of the max [Ca2+]cyt data points in the time frame. Asterisks indicate statistically significant differences compared with the WT by two-tailed
Student’s t test. **P < 0.01.

SWPs and glutamate, we used the root-to-shoot wound response
system developed here to test the possibility that glutamate released
at (or added to) the wound site triggers electrical signals represented
by SWPs. Fifteen minutes after wounding, we added glutamate to the
wound site and observed an SWP on a rosette leaf (Fig. 2, A to C and E),
reminiscent of the one generated by wounding. We also observed
glutamate-triggered leaf-to-leaf SWPs that were similar to those
induced by wounding (fig. S2, A to C). This finding made a critical
link between glutamate and electrical signals (SWPs), indicating that
glutamate release at the wound site was sufficient for the generation
of both SWPs and systemic Ca2+ waves.
To quantitatively analyze root-to-shoot SWPs, we divided the
SWP into the hyperpolarization phase and depolarization phase
(Fig. 2A) and collected data on the amplitude and duration for both
phases. We found that both the amplitude and duration for hyper-
polarization and depolarization were similar between the wound-
and glutamate-induced SWPs (Fig. 2, B and C), indicating that
glutamate-induced SWPs mimic the wound-induced SWPs.
To further compare leaf-to-leaf and root-to-shoot SWPs, we
analyzed in detail the depolarization phases of these SWPs. In
previous studies, the depolarization phase of wound-induced leaf-
to-leaf SWPs appears smooth with a single sustained phase (24, 27).
We found SWPs with monophasic patterns similar to those previously
described and, in addition, more complex leaf-to-leaf SWPs consist-
ing of an initial fast depolarization followed by a slower and sustained
depolarization (fig. S2). We found that 56% (44 of 78) of wound-­
induced root-to-shoot SWPs consisted of an initial fast depolarization,
followed by a slower depolarization, and 44% (34 of 78) were simpler,
showing a single sustained phase (Fig. 2D). For glutamate-induced
root-to-shoot SWPs, the depolarization phase of 61% (42 of 69) of

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SCIENCE SIGNALING | RESEARCH ARTICLE

A mate as the initial signal at the wound

site. Our results showed that glutamate
Amphyp
at the wound site induced both [Ca2+]cyt
Dur dep Glu increases and SWPs, consistent with the
Amplitude (mV)

W idea of glutamate being a trigger for sys-

Ampdep

temic wound signaling. We thus tested

the possibility that GLR3.3 and GLR3.6
may also be essential for the root-to-
Dur hyp
shoot wound response.
When roots were wounded, the
amplitude and duration of the depolariza-
Time (s)
tion phase of the systemic SWPs were
B D
significantly reduced in the glr3.3glr3.6
double mutant (Fig. 3, A to F), whereas
the duration of the hyperpolarization
Amplitude (mV)

phase was slightly longer than wild type

(Col-0) (Fig. 3D). We then added gluta-
mate to the wound sites of plant roots
and found that, compared with Col-0,
the SWPs in a glr3.3glr3.6 mutant

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rosette leaf were also significantly im-
W W Glu Glu paired in both the hyperpolarization
hyp dep hyp dep

C E and depolarization phases (Fig. 3, A,

B, G to J). These results showed that
GLR3.3 and GLR3.6 played essential roles
in the wound- and glutamate-­induced
systemic SWPs in root-to-shoot long-­
Duration (s)

distance signaling.
In parallel assays, we found that wound-
and glutamate-induced root-to-shoot
systemic [Ca2+]cyt increases were nearly
abolished in the glr3.3glr3.6 double mu-
W W
hyp
Glu
dep
Gluhyp dep
tant (fig. S3, A to D, and movies S3 and
Fig. 2. Wounding and glutamate trigger a root-to-shoot systemic electrical signal. (A) Typical wound- and S4). Furthermore, we measured the sys-
glutamate-induced SWPs recorded at leaf 6. The time points at which the root was wounded (W) and 100 mM temic JA response by checking the mRNA
glutamate was added to the wound site (Glu) are marked with arrowheads. Four parameters, including hyper- abundance for marker genes JAZ7, JAZ10,
polarization amplitude (Amphyp), hyperpolarization duration (Durhyp), depolarization amplitude (Ampdep), and and OPR3 in rosette leaves and found
depolarization duration (Durdep) were used to compare SWPs. (B and C) Comparison of amplitude (B) and dura- that wound-induced, JA-dependent tran-
tion (C) of wound- and glutamate-­induced SWPs. n = 78 plants for wound-induced SWPs, and 69 plants for glutamate-­ script abundances were much lower in
induced SWPs. Boxplots show the minimum, 25th percentile, median, 75th percentile, and maximum of the data the glr3.3glr3.6 double mutant compared
points. (D) Two typical wound-induced SWPs recorded at leaf 6. (E) Two typical glutamate-induced SWPs recorded to Col-0 (fig. S3, E to G). These results
at leaf 6.
confirmed the essential roles of GLR3.3
and GLR3.6 during the systemic wound
plants was monophasic, whereas 39% (27 of 69) of plants showed response not only in the leaf-to-leaf system but also in the root-to-
an initial fast and then slower depolarization phase (Fig. 2E). We shoot system.
concluded that root-to-shoot SWPs and leaf-to-leaf SWPs in our
study and those described in previous studies were comparable P-type H+-ATPase activity plays a role in the root-to-shoot
during the depolarization phase (24,  27). Together, these results systemic wound response
suggest that glutamate released at the wound site may be a trigger of SWPs in the root-to-shoot model involved both hyperpolarization
SWPs in the systemic wound response in both root-to-shoot and leaf- and depolarization phases (Fig. 2). Although it is not clear exactly
to-leaf models. how SWPs correspond to plasma membrane potentials, previous
studies have suggested that P-type H+-ATPases may be involved in
GLR3.3 and GLR3.6 are required for root-to-shoot systemic producing the SWPs (44–49). We examined the importance of P-type
wound signaling H+-ATPase in root-to-shoot systemic signaling by two approaches.
In the leaf-to-leaf wound response model, GLR3.3 and GLR3.6 are First, we studied the effect of the fungus-derived, H+-ATPase–­
necessary for the propagation of wound-induced SWPs and systemic activating compound fusicoccin (FC) on SWPs induced by wound-
[Ca2+]cyt rise (24, 25, 34). Because GLRs are putative glutamate ing or glutamate application in the root-to-shoot systemic response
receptors in plants, the involvement of GLR3.3 and GLR3.6 in the (fig. S4). The rosette leaves of plants divided into three groups (A, B,
systemic wound response further supports the importance of gluta- and C) and wounded on the roots 2 cm away from the root-shoot

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A B
glr3.3glr3.6
W

20 mV
Glu

C D E F
40 **** 400 **** 20 **** 800 ****
35 10

30 300 0
Amplitude (mV)

Amplitude (mV)
25 Duration (s)

Duration (s)
20 200 400

15

10 100 200

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0 0 0
glr3.3glr3.6 glr3.3glr3.6 glr3.3glr3.6 glr3.3glr3.6
G H I J
**** **** 20 **** 800 ****
25
10

20 0
200
Amplitude (mV)

Amplitude (mV)

Duration (s)
Duration (s)

15
400

10 100
200
5

0 0 0
glr3.3glr3.6 glr3.3glr3.6 glr3.3glr3.6 glr3.3glr3.6
Fig. 3. GLR3.3 and GLR3.6 participate in root-to-shoot wound signaling. (A and B) Typical recordings of wound-induced (W) and glutamate-induced (Glu) SWPs in
leaf 6 in Col-0 (A) and glr3.3glr3.6 (B) plants. The time points at which the root was wounded and glutamate was added to the wound site are marked with arrowheads.
(C to F) Wound-induced SWPs in leaf 6 of Col-0 and glr3.3glr3.6 plants are described by amplitude (C) and duration (D) of hyperpolarization and by amplitude (E) and
duration (F) of depolarization. (G to J) Glutamate-induced SWPs in leaf 6 of Col-0 and glr3.3glr3.6 plants are described by amplitude (G) and duration (H) of hyperpolariza-
tion and amplitude (I) and duration (J) of depolarization. The WT (Col-0) data from leaf 6 are the same as those shown in Fig. 2A; n = 78 plants for wound-induced SWPs
and n = 69 plants for glutamate-induced SWPs. For experiments in glr3.3glr3.6, n = 71 plants. Boxplots show the minimum, 25th percentile, median, 75th percentile,
and maximum of the data points. Asterisks indicate statistically significant differences compared with the WT by two-tailed Student’s t test. ****P < 0.0001.

junction showed similar SWPs in response to wounding (fig. S4, A
to C). Fifteen minutes after wounding, we added 50 mM glutamate,
2.5 M FC, or 2.5 M FC combined with 50 mM glutamate to the
wound sites of the group A, B, and C plants, respectively. Whereas
glutamate treatment elicited a typical SWP and FC treatment alone
had no effect (fig. S4, B and C, right panel), the combination of
glutamate plus FC slightly increased the amplitude of the hyper-
polarization phase but, notably, eliminated both the amplitude and
duration of the depolarization phase (fig. S4, B and C, right panel),
demonstrating that the activation of H+-ATPases by FC blocked the
depolarization phase of glutamate-induced SWPs.
Second, we used a genetic approach to test the effect of ost2-2D,
a gain-of-function allele of AHA1, on the wound- and glutamate-­
induced SWPs (Fig. 4, A and B). When the roots of ost2-2D were
wounded, the SWPs of the rosette leaf showed higher hyper-
polarization amplitude and normal duration as compared with
Col-0 (Fig. 4, C and D). In the depolarization phase, ost2-2D
showed lower amplitude and shorter duration than did Col-0
(Fig. 4, E and F). We then added glutamate to the wound sites
and found that ost2-2D showed higher amplitude and normal
duration in the hyperpolarization phase as compared with Col-0
(Fig. 4, G and H). In the depolarization phase, the duration of SWPs
on the ost2-2D rosette leaf was significantly reduced as compared
to those of Col-0 (Fig. 4, I and J), showing again that constitutively
active AHA1 inhibited the depolarization phase of both wound-
and glutamate-induced SWPs. Together, the results from both
pharmacological and genetic approaches indicated that the inhibi-
tion of P-type H+-ATPase was required for plants to generate and/
or propagate SWPs in the root-to-shoot model, which is consistent
with published findings on the leaf-to-leaf wound response (28).

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A B ost2-2D

W
W Glu

20 mV
Glu

C D E F
40 *** 400 20 ** 800 ****
35 10

30 300 0
Amplitude (mV)

Amplitude (mV)
25

Duration (s)
Duration (s)
20 200 400

15

10 100 200

0 0 0

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ost2-2D ost2-2D ost2-2D ost2-2D
G H I J
*** 20 800 ****
25
10

20 0
200
Amplitude (mV)

Amplitude (mV)

Duration (s)
Duration (s)

15
400

10 100
200
5

0 0 0
ost2-2D ost2-2D ost2-2D ost2-2D
Fig. 4. Constitutive activation of P-type H+-ATPase inhibits wound- and glutamate-induced SWPs. (A and B) Typical recording of wound- and glutamate-induced
SWPs in leaf 6 in Col-0 (A) and ost2-2D (B) plants. The time points when the root was wounded (W) and glutamate was added (Glu) to the wound site are marked with ar-
rowheads. (C to F) Wound-induced SWPs in leaf 6 of Col-0 and ost2-2D plants as described by amplitude (C) and duration (D) of hyperpolarization and amplitude (E) and
duration (F) of depolarization. (G to J) Glutamate-induced SWPs in leaf 6 of Col-0 and ost2-2D plants as presented by amplitude (G) and duration (H) of hyperpolarization
and amplitude (I) and duration (J) of depolarization. n = 27 plants for Col-0 and n = 39 plants for ost2-2D. Boxplots show the minimum, 25th percentile, median, 75th
percentile, and maximum of the data points. Asterisks indicate statistically significant differences compared with the WT by two-tailed Student’s t test. **P < 0.01,
***P < 0.001, and ****P < 0.0001.

The Ca2+-permeable channel activities of GLR3.3 and GLR3.6
are gated by extracellular glutamate and pH
Data presented here and in earlier studies support the essential role
of GLR3.3 and GLR3.6 in the systemic wound response for both leaf-­
to-leaf and root-to-shoot models (24, 25, 34). These GLRs appear to
couple the SWPs and Ca2+ waves in some way so that these systemic
signals become interdependent. One hypothesis is that GLR3.3 and
GLR3.6 may function as glutamate-activated Ca2+-permeable channels,
stimulating Ca2+ influx that alters plasma membrane potentials.
However, it remains to be tested whether GLR3.3 and GLR3.6 are
indeed glutamate-gated Ca2+ channels. Our data herein (Fig. 4 and
fig. S4) and a previous report (28) both identified P-type H+-ATPase
as a critical component in the systemic wound response, possibly as
a result of its contribution to the plasma membrane potential and
thus to SWPs. We explored the interplay of the P-type H+-ATPase
and GLR3.3 and GLR3.6 by examining the channel properties of
Arabidopsis GLR3.3 and GLR3.6, which is key to further under-
standing systemic wound signaling.
The Arabidopsis GLR family consists of 20 members that are
implicated in various functions [reviewed in (50)]. The channel
properties, however, are largely unexplored because only a few of
the 20 members have been studied using patch-clamp approaches.
For example, AtGLR3.4 expressed in human embryonic kidney
(HEK) cells shows Ca2+-permeable channel activity that is enhanced
by the presence of 1 to 2 mM asparagine, serine, or glycine (51).
AtGLR1.4 appears to function as a nonselective cation channel when
expressed in frog oocytes and is sensitive to 1 mM of a broad
range of amino acids (52). AtGLR3.2 and AtGLR3.3 can conduct
Ca2+-permeable current in the absence of any ligand when co-
expressed in monkey kidney COS-7 cells with AtCORNICHON1
and AtCORNICHON4, Arabidopsis homologs of sorting proteins that
control the trafficking of ionotropic glutamate receptors (iGluRs)

Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020 6 of 13

SCIENCE SIGNALING | RESEARCH ARTICLE

in animals (53). Also, the biochemically reconstituted GLR3.3 ligand sensitivity between plant and animal GLRs resulted from
ligand-binding domain (LBD) appears to show a preference for problems in our Ca2+ imaging system, we used a rat iGLuR, gluta-
l-glutamate and the sulfur-containing amino acids l-cysteine and mate receptor ionotropic, kainate 2 (Grik2), as a control. We found
l-methionine (54). Together, the studies so far indicate that some that 100 M glutamate triggered a large [Ca2+]cyt increase in HEK293T
plant GLRs can be gated by amino acids at low millimolar or sub- cells expressing Grik2 and GCaMP6s under pH 7.5, whereas no
millimolar concentrations, and some are constitutively open without response was observed for Arabidopsis GLR3.3 or GLR3.6 under the
any ligands. However, Toyota et al. (34) suggest that glutamate, but same conditions (Fig. 5C).
not other amino acids, is specifically effective in eliciting Ca2+ waves Animal iGluRs are gated by multiple amino acids, including
in systemic wound response that requires putative glutamate receptors glutamate, glycine, serine, and alanine (57), and it has been shown
GLR3.3 and GLR3.6. Furthermore, the concentration of glutamate that several plant GLRs are also gated by multiple amino acids
must be 50 mM or higher to have a significant effect (34). These (51, 52, 58). We thus tested the effect of each of the 20 amino acids
findings are not consistent with the known channel properties of on the activity of GLR3.6 and found that only glutamate (at 100 mM),
GLRs reported so far, whether GLR3.3 and GLR3.6 indeed serve but not other amino acids, triggered significant [Ca2+]cyt increases
as the glutamate receptors responsible for the systemic wound in HEK293T cells coexpressing GLR3.6 and GCaMP6s (Fig. 5D and
response. We decided to bridge this critical gap between channel movies S5, S6, and S7). This result is consistent with the report
activities and in planta function of the GLRs in the context of showing that glutamate is the only amino acid that specifically elicits
systemic wound response by two approaches to delineating GLR3.3 a systemic Ca2+ wave in plants (34).
and GLR3.6 functional properties. To further investigate the channel properties of GLR3.3 and
First, we examined the activity of GLR3.3 and GLR3.6 in mediating GLR3.6, we conducted patch-clamp experiments on HEK292T cells
Ca2+ influx using single-cell Ca2+ imaging experiments in HEK293T expressing the individual GLRs. Using the standard bath solution

Downloaded from http://stke.sciencemag.org/ on July 14, 2020

cells coexpressing the Ca2+ indicator GCaMP6s and plant GLRs. containing 10 mM Ca 2+ and 100 mM glutamate, we observed
However, we initially failed to observe any activity in plant GLR3.3 cation-carried inward currents in HEK293T cells expressing GLR3.3
and GLR3.6 using this assay. Further investigation of the literature or GLR3.6 under pH 7.5 (Fig. 6, A to D). Because several previous
on animal iGluRs indicated that a subset of these receptors, specifically reports showed that some plant GLRs can be activated by lower
the N-methyl-d-aspartate receptors (NMDARs), are not only gated amounts (<1 mM) of amino acids (51, 52, 58), we included rat Grik2
by their ligands but are also highly sensitive to protons. The open as a control. We consistently recorded large inward currents in
probability of NMDARs is reduced with increasing proton concen- HEK293T cells expressing Grik2 in the standard bath solution
tration (or lowering the pH) (55), and a structural study described the containing 10 mM Ca2+ and 100 M glutamate, whereas no inward
mechanism of this pH sensitivity (56). If
Arabidopsis GLR3.3 and GLR3.6 are also A B C 7
8
inhibited by low pH, then that would Mock 10 10 mM * 100 µM glutamate
* **
explain why we did not observe any ac- GLR3.3 25 mM
*
6 pH 7.5
6 GLR3.6 8 50 mM
tivity when we used typical acidic pH *
(F–F0)/F0

(F–F0)/F0
(F–F0)/F0

* 100 mM * 5
*
conditions to apply glutamate and other 6 Glutamate
*
4
amino acids to the HEK cells in the 4
3
attempt to activate GLR3.3 and GLR3.6. 4
We found that, under higher pH (7.5 and 2 2
2
8.5), the external application of 100 mM 1
glutamate induced a large [Ca2+]cyt in- 0 0 0
crease in the HEK293T cells transiently pH 5.5 pH 6.5 pH 7.5 pH 8.5 Mock GLR3.3 GLR3.6 ck 3.3 3.6 r i k 2
M o G LR G LR G
coexpressing GLR3.3 or GLR3.6 and
GCaMP6s (Fig. 5A and movies S5 and S6). D
10
Under lower pH (5.5 and 6.5), however, Mock *
2+ GLR3.6
glutamate-induced [Ca ]cyt increases were 8
negligible (Fig. 5A). These data indicated
(F–F0)/F0

that GLR3.3 and GLR3.6, like animal 6

NMDARs, were proton-sensitive. 4

We then applied different concen-
trations of glutamate (10, 25, 50, and 2
100 mM) to the HEK293T cells co-
expressing GCaMP6s plus either GLR3.3 0
e
et
n

ys

u
ln

lu

r
s

o
g

ly

is

l
a

r
Se

Va
Ph
As

As

Th

Ty
Le

or GLR3.6 under pH 8.5 conditions,

Ly
Ile

Pr
Ar

Tr
M
Al

G
C

2+ 2+
and found that only 50 and 100 mM Fig. 5. Ca imaging of HEK293T cells expressing GLR3.3 or GLR3.6. (A) Single-cell imaging of [Ca ]cyt increases
in HEK293T cells expressing GLR3.3 or GLR3.6, as induced by 100 mM glutamate at the indicated pH values. (B) [Ca2+]cyt
glutamate induced significant [Ca2+]cyt
increases induced by the indicated concentrations of glutamate at pH 8.5 in HEK293T cells expressing GLR3.3 or
increases (Fig. 5B). This was a surpris- GLR3.6. (C) [Ca2+] changes induced by 100 M glutamate in cells expressing GLR3.3, GLR3.6, or Grik2. (D) [Ca2+] in
cyt cyt
ing finding given that animal iGluRs can HEK293T cells expressing GLR3.6 and stimulated with the indicated amino acids at pH 8.5. Amino acids were added
be activated by micromolar concentra- at 100 mM, except for tyrosine and tryptophan, which were used at lower concentrations because of lower solubility.
tions of glutamate (57). To rule out the For relative max (F − F0)/F0, the mock control was set to 1. For all experiments, n = 3 independent experiments, with
possibility that the marked difference in ~60 cells imaged in each experiment. Error bars depict means ± SEM. Student’s t test. *P < 0.05, **P < 0.01.

Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020 7 of 13

SCIENCE SIGNALING | RESEARCH ARTICLE

A Voltage (mV) B Voltage (mV)

Because the standard bath solution
50
−160 −120 −80 −40 0 contained not only Ca2+ but also Na+ and
Mock 0 −160 −120 −80 −40 0
K+, we characterized the permeability
0
pH 5.5 of GLR3.3 and GLR3.6 to these mono­
pH 6.5 valent and divalent cations using patch-­
−50 −50
Mock pH 5.5 clamping. We added 140 mM Na+, K+,

Current (pA)
Mock pH 6.5
or Cs+, and 112 mM Ca2+ or Ba2+, re-

Current (pA)
pH 7.5 Mock pH 7.5 −100
GLR3.3 −100 Mock pH 8.5 spectively, to the bath solution contain-
pH 8.5 * GLR3.3 pH 5.5
−150
ing 100 mM glutamate with pH adjusted
GLR3.3 pH 6.5
to pH 8.5 and performed patch-clamping
−150 GLR3.3 pH 7.5
GLR3.3 pH 8.5 −200
in the whole-cell configuration (figs. S5, A
to J, and S6, A to J). We found that GLR3.3
or GLR3.6 conducted inward currents with
C Voltage (mV) D Voltage (mV) both monovalent and divalent cations
50
Mock −160 −120 −80 −40 0 as carriers with a preference for Ca2+ and
pH 5.5 0 −160 −120 −80 −40 0
Ba2+. The permeability sequence of GLR3.3
0
pH 6.5 was Ba2+ > Ca2+ > Na+ = K+ > Cs+, and for
−50 GLR3.6, the sequence was Ba2+ > Ca2+ >
−50
pH 7.5 Mock pH 5.5 K+  >  Na+  >  Cs+, showing that GLR3.3

Current (pA)
Mock pH 6.5 and GLR3.6 are nonselective cation chan-

Downloaded from http://stke.sciencemag.org/ on July 14, 2020

Current (pA)

−100 −100
Mock pH 7.5
Mock pH 8.5
nels that prefer Ba2+ and Ca2+ (table S1).
GLR3.6
−150
* GLR3.6 pH 5.5 −150
Animal iGluRs can assemble as homo-
GLR3.6 pH 6.5 and heterotetrameric channels, and func-
GLR3.6 pH 7.5
pH 8.5
GLR3.6 pH 8.5 −200
tional channels are formed exclusively
−200
by the assembly of subunits of the same
receptor class (57). Arabidopsis GLR sub-
E Voltage (mV) F Voltage (mV) 100
units interact with multiple other GLRs
50
−160 −120 −80 −40 0 −160 −120 −80 −40 0
in a yeast mating-based split ubiquitin
Mock
0 0 system (59), indicating that Arabidopsis
GLR3.3
GLRs might also form heteromeric com-
GLR3.6 −50
−100 plexes. However, a fusion of GLR3.3 to
−100 the reporter -glucuronidase (GLR3.3-
Current (pA)

Current (pA)

Mock −200 GUS) is expressed in the phloem re-

−150
** GLR3.3 gion, whereas GLR3.6-GUS is expressed
Grik2 100 µM glutamate −200 GLR3.6 in the xylem contact cells (25), suggest-
−300
pH 7.5
−250
Grik2 ing that GLR3.3 and GLR3.6 are unlikely
−400 to form heterotetrameric channels in
Fig. 6. Patch-clamp recordings of the channel activities of GLR3.3 and GLR3.6. (A) Typical whole-cell recordings plants because they are present in dis-
of inward currents in HEK293T cells expressing GLR3.3, as induced by 100 mM glutamate at the indicated pH values. tinct cell populations.
(B) Average current–voltage curves for GLR3.3 at different pH values, n = 4 to 10 cells per condition. (C) Typical whole-
cell recordings of inward currents in HEK293T cells expressing GLR3.6, as induced by 100 mM glutamate at the indi-
cated pH values. (D) Average current–voltage curves for GLR3.6 at different pH values. n = 4 to 10 cells per condition. DISCUSSION
(E) Typical whole-cell recordings of inward currents in cells expressing GLR3.3, GLR3.6, or Grik2 as induced by 100 M The root-to-shoot systemic
glutamate at pH 7.5. (F) Average current–voltage curves for GLR3.3, GLR3.6, and Grik2. n = 4 to 6 cells per condition.
wound response is a robust
Error bars depict means ± SEM. Student’s t test. *P < 0.05, **P < 0.01.
model for studying plant long-
distance signaling
Most studies on systemic wound signaling
current was recorded for GLR3.3 or GLR3.6 beyond that observed have focused on the leaf-to-leaf model in Arabidopsis (24, 25, 28, 34).
in the mock control (Fig. 6, E and F). We thus concluded that Although it is established that the amounts of 12-oxo-phytodienoic
GLR3.3 and GLR3.6 were not activated by low millimolar or sub- acid, a JA precursor, increase and JA-responsive genes are induced
millimolar concentrations of glutamate, consistent with the earlier in the shoot when the root is wounded (60, 61), root-to-shoot
results using single-cell Ca2+ imaging (Fig. 5, A to C). This conclu- systemic wound signaling remains largely unexplored. During the
sion supports the finding that only high millimolar concentrations preparation of this study, it was reported that electrical signals and
of glutamate (above 50 mM) can elicit a systemic Ca2+ wave in ROS are involved in the systemic root-to-shoot response when
plants (34). Moreover, we found that the inward currents mediated tomato plants are damaged by the root-knot nematode, leading to
by GLR3.3 or GLR3.6 were significantly larger when bath pH was an increased accumulation of JAs in the leaves (42). The authors
adjusted to 7.5 and 8.5, as compared to lower pH (5.5 and 6.5) describe the importance of tomato GLR3.5 (a homolog of Arabidopsis
(Fig. 6, A to D), confirming that GLR3.3 and GLR3.6 were inhibited GLR3.3 and GLR3.6) and RBOH1 in the nematode-induced root-
by the extracellular protons. to-shoot systemic signaling process (42). Although it is difficult to

Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020 8 of 13

SCIENCE SIGNALING | RESEARCH ARTICLE
compare this study using nematode in tomato with earlier studies
using mechanical wounding in Arabidopsis, the results suggest that
root-to-shoot signaling and leaf-to-leaf signaling may share some
common components.
Here, we established a root-to-shoot systemic wound signaling
system in Arabidopsis and compared it with the leaf-to-leaf model
in the same species. In addition to showing the participation of the
same signals, including glutamate, SWPs, and Ca2+ waves, in the
root-to-shoot systemic wound response, we also made progress in
understanding the functional relationship of these signals in the
context of the channel properties of plant GLRs.
The glutamate-dependent Ca2+ channel activity of GLR3.3
and GLR3.6 couples systemic Ca2+ and electrical waves
in the wound response
Although systemic signaling through nerve action potentials has been
well established in animals, electrical signaling mechanisms in plants
had been controversial until a pioneering study (24) identified the
genes encoding GLR3.3 and GLR3.6 as essential genetic determinants
for propagation of electrical signals from a wounded leaf to distal
leaves, leading to a systemic response to wounding. An important
advance has linked wound- and glutamate-induced Ca2+ waves to
these same GLRs (25, 34), further emphasizing the central role of
GLRs in long-distance signaling during the plant wound response.
What are plant GLRs, and why are they required for systemic
wound signaling? At the center of this question lie the functional
properties of plant GLRs.
Animal iGluRs are nonselective cation channels, and some of
them, especially those of the NMDAR family, are Ca2+-permeable
(57). Although plant GLRs contain similar domains to those present
in the NMDARs, including the N-terminal domain, the receptor or
LBD, the pore domain, and the cytosolic C-terminal domain, it is
difficult to predict channel activity and ionic selectivity of plant
GLRs by comparing with animal NMDARs because of the low con-
servation of the pore sequence, and particularly the residues dictating
the Ca2+ permeability of NMDAR channels (50, 62). Previous studies
support the hypothesis that plant GLRs, like animal NMDARs,
function as Ca2+-permeable channels that may be activated by amino
acids (51–53, 63). Therefore, it makes sense that GLRs are important
in wound- and glutamate-induced long-distance Ca2+ signaling.
However, there exists a major gap between studies using electro-
physiological approaches and those measuring systemic wound
responses in plants. The data collected by patch-clamping animal
cells expressing plant GLRs indicate that at least two plant GLRs,
including AtGLR3.4 and AtGLR1.4, are Ca2+-permeable channels
gated by 1 mM amino acids including Asn, Ser, Gly, Met, Trp, Phe,
Leu, Tyr, and Thr (51, 52, 58). In contrast, 50 mM or higher concen-
trations of glutamate, but not other amino acids, specifically induce
GLR3.3- and GLR3.6-dependent Ca2+ waves in the leaf-to-leaf
wound response (34). The concentrations of glutamate required for
systemic signaling are a couple of magnitudes higher than those
shown to induce channel activity in the electrophysiological studies,
although different members of the GLR family have been analyzed
in different studies. To connect the channel activity and the role of
these GLRs in the systemic wound response, our work here has
addressed both the electrophysiological properties of GLR3.3 and
GLR3.6 and their function in systemic wound responses.
We confirmed that high concentrations of glutamate (50 mM or
higher) were essential to elicit a robust systemic Ca2+ wave and elec-
Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020
trical signal in the root-to-shoot system. Further, we showed that
GLR3.3 and GLR3.6 expressed in HEK293T cells are activated by
the same concentration of glutamate (50 to 100 mM), bridging the
gap between channel properties of these GLRs and their function in
systemic wound response. Our finding that Arabidopsis GLR3.3
and GLR3.6 were activated by 50 to 100 mM glutamate (Figs. 5 and
6, figs. S5 and S6, and table S1) is in stark contrast to the ligand
gating of animal iGluRs that are activated by a micromolar range of
external glutamate (57). This finding, however, is consistent with
the special physiology of plants in that apoplastic concentrations of
glutamate are normally in the low millimolar range, whereas 20 to
100 mM glutamate is found in the phloem sap (34, 64, 65), making
it feasible for plants to use a glutamate gradient as a DAMP. Under
resting conditions, apoplastic glutamate should not be sufficient to
activate GLRs, consistent with our finding that GLR3.3 and GLR3.6
were not active in the presence of low millimolar glutamate. How-
ever, upon wounding, the damaged phloem cells would release glu-
tamate to the apoplastic space, where it reaches 50 to 100 mM and
activates GLRs in the plasma membranes of adjacent cells required
for systemic wound signaling. This low ligand sensitivity may be
Downloaded from http://stke.sciencemag.org/ on July 14, 2020
unique to plant GLRs, and the structural basis for such a property
deserves investigation.
The pH-sensitive activity of GLR3.3 and GLR3.6 links
the function of P-type H+-ATPase to the systemic
wound response
Protons profoundly inhibit animal NMDAR activity, and alkali pH
enhances the open probability of the channels (55). Here, we found
that Arabidopsis GLR3.3 and GLR3.6 channels are also highly sensitive
to apoplastic pH, with alkali pH as an essential condition for channel
opening. This finding provides further insight into the function of
P-type H+-ATPase in the systemic root-to-shoot wound response
documented herein and in the leaf-to-leaf long-distance signaling
previously described (28). We propose that upon wounding, inhibition
of P-type H+-ATPase activity causes apoplastic alkalinization, which
cooperates with glutamate released into the apoplastic space to activate
GLRs, leading to the influx of Ca2+ and other cations through GLRs,
thereby depolarizing the membrane. As a result, constant activation
of P-type H+-ATPase through a gain-of-function mutation in ost2-2D
mutant or by FC treatment might block the alkalization of apoplastic
space (Fig. 4 and fig. S4) and therefore the activation of GLRs
(Figs. 5 and 6), inhibiting membrane depolarization and Ca2+ influx.
This does not conflict with the AHA1 function in the report in
which AHA1 may be activated after the Ca2+ influx to repolarize the
membrane (28). In other words, inhibition and activation of AHA1
may act as a critical cycle for changes in membrane potential and
apoplastic pH that hold the key for Ca2+ entry and signal propaga-
tion through GLRs.
To support the hypothesis that AHA1 is involved in the wound
systemic response, the measurement of apoplastic pH changes upon
wounding will be important. However, because of technical limita-
tions, we did not have any success in the rapid and accurate record-
ing of apoplastic pH changes in response to wounding. One previous
report (66) shows that wounding a leaf induces cytosolic acidification,
suggesting that proton pumps are inhibited upon wounding, which
supports our hypothesis.
The finding that GLR3.3 and GLR3.6 were only active when pH
was higher than 6.5 raises another key question: Could plant ap-
oplastic pH reach the threshold high enough to activate GLR3.3 and
9 of 13
SCIENCE SIGNALING | RESEARCH ARTICLE
GLR3.6? In one study (67), the authors fused pHluorin, a well-­
established pH sensor, to a transmembrane domain so that the
fusion protein would be anchored in the plasma membrane (PM)
with the pH-sensing moiety facing the apoplast. Using this PM-­
anchored pHluorin, membrane-associated apoplastic pH (around
6.5) is found to be much more alkaline than that of the overall cell
wall (67). Thus, inhibition of P-type H+-ATPase would increase the
pH further to a range higher than the resting level of 6.5, which
would be sufficient for the activation of GLR3.3 and GLR3.6. Fu-
ture studies should directly analyze the apoplastic pH changes in
response to wounding and glutamate application in the root-to-shoot
and leaf-to-leaf models.
In addition, the data presented herein and results from other
studies (28) may also provide a possible mechanism for how plants
terminate the SWPs and Ca2+ waves: When the [Ca2+]cyt waves pass
by, the cells would re-activate the P-type H+-ATPase that would pump
protons out, repolarize the cells, and acidify the apoplast, which
would inhibit GLR channel activity and terminate the systemic
SWP and Ca2+ signal. In summary, our results reported here have
uncovered critical regulatory properties of GLR3.3 and GLR3.6
that connect glutamate, H+-ATPase, SWP, and Ca2+ waves in plant
systemic wound signaling. Some key questions remain to be answered
regarding the mechanisms underlying both the initiation and prop-
agation of the signals. What triggers the inhibition of H+-ATPase
immediately after wounding to increase apoplastic pH to enable GLR
activation by glutamate? How does a signal propagate along the vas-
cular tissues? Future studies will be directed toward answering these
questions on plant long-distance signaling that, to some extent, mimics
nerve transmission of signals in animals in that glutamate, elec-
trical, and Ca2+ signals are intertwined into a complex network.
MATERIALS AND METHODS
Plant material and growth conditions
Seeds were sterilized with 10% (v/v) bleach and sown on agar plates
containing 1/2 Murashige and Skoog (MS) medium [1/2 MS, 0.8% (w/v)
Phyto agar, and 1% (w/v) sucrose, pH adjusted to 5.8 with KOH].
Plates were incubated at 4°C for 3 days for stratification and then
transferred to 22°C growth room with a 12-hour light/12-hour dark
cycle (100 mol m−2 s−1) for 6 days. Seedlings were then transferred
onto new plates (2 plants per plate) and grown for additional 15 days
before electrical recording or calcium imaging upon wounding. The
ost2-2D seeds were kindly provided by Jian-Min Zhou (68). The glr3.3
(SALK_099757C) and glr3.6 (SALK_091801C) were ordered from
Arabidopsis Biological Resource Center (ABRC) and crossed to ob-
tain the glr3.3glr3.6 double mutant. The glr3.3glr3.6 mutant was crossed
with a transgenic line containing UBQ10 promoter-driven GCaMP6s
and further brought to homozygosity with both GCaMP6s and
glr3.3glr3.6 background.
Surface potential recordings and current injection
The leaf surface potential recording method was modified from the
published method (24). An Axopatch 200B patch-clamp amplifier
(Axon Instruments) with a Digidata 1550 digitizer (Axon Instru-
ments), was applied to record the surface potential. The pClamp
10.7 software (Axon Instruments) was used for data acquisition and
analysis. The amplifier was turned to I-Clamp Normal mode, and
the sampling rate is 1000 Hz. The recording silver electrode was
connected to the leaf surface using 10 l of 0.5% (w/v) agar contain-
Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020
ing 10 mM KCl, and the reference silver electrode was inserted into
the agar medium.
Vector construction, cell culture, and transfection
The cDNA encoding GCaMP6s was amplified from HBT-GCaMP6-
HA43 (43), and the cDNAs encoding GLR3.3 and GLR3.6 were
kind gifts from José A. Feijó (53) and Edward E. Farmer (24). The
GLR3.3 and GLR3.6 and GCaMP6s were cloned into a single vector,
pBudCE4.1 (Invitrogen), for coexpression in HEK293T cells.
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum in a 5% CO2 incubator
at 37°C in a moist atmosphere. HEK293T cells were transfected
using the Lipofectamine 3000 Transfection Reagent Kit (Invitrogen).
Plasmids for transfection were extracted from Escherichia coli (DH5)
using QIAGEN Plasmid Mini Kit (Qiagen), and 2 g of plasmids in
total was added into each well of six-well plates (Nunc). HEK293T
cells showing bright green fluorescence were used for patch-clamp
experiments and Ca2+ imaging experiments 48 hours after transfection.
Whole-cell patch-clamp recording
Downloaded from http://stke.sciencemag.org/ on July 14, 2020
The whole-cell patch-clamp experiments were performed using
an Axopatch 200B patch-clamp setup (Axon Instruments) with a
Digidata 1550 digitizer (Axon Instruments) as previously reported
(69, 70). pClamp10.7 software (Axon Instruments) was used for
data acquisition and analysis.
The standard pipette solution for all experiments contained 140 mM
CsCl, 5 mM EGTA, and 10 mM Hepes (pH 7.5; adjusted with CsOH)
as previously described (71). The standard bath solution contained
100 mM Na-glutamate, 40 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM
CaCl2, 10 mM Hepes, and 10 mM glucose (pH 7.5) (adjusted with
NaOH). Different pH values were indicated in the figures.
For monovalent-cation substitution experiments, the bath solution
was changed as follows: (i) 100 mM Na-glutamate, 40 mM NaCl,
10 mM glucose, and 10 mM Hepes (adjusted to pH 8.5 with NaOH);
(ii) 100 mM K-glutamate, 40 mM KCl, 10 mM glucose, and 10 mM
Hepes (adjusted to pH 8.5 with KOH); and (iii) 100 mM Cs-­
glutamate, 40 mM CsCl, 10 mM glucose, and 10 mM Hepes (adjusted
to pH 8.5 with CsOH).
For divalent-cation substitution experiments, the bath solution
was changed as follows: (i) 50 mM Ca-(glutamate)2, 62 mM CaCl2,
10 mM glucose, and 10 mM Hepes [adjusted to pH 8.5 with Ca(OH)2];
and (ii) 50 mM Ba-(glutamate)2, 62 mM BaCl2, 10 mM glucose, and
10 mM Hepes [adjusted to pH 8.5 with Ba(OH)2]. A ramp voltage
protocol of 2-s duration from −160 to +30 mV (holding potential
0 mV) was applied 1 min after accessing a whole-cell configuration,
and currents were recorded every 20 s for 40 times in total for
each cell. The 40 current traces were used for statistical analysis for
average current-voltage curves. Permeability ratios for monovalent
and divalent cations against Cs+ were calculated as previously de-
scribed (71).
Single-cell calcium imaging in HEK293T cells
HEK293T cells expressing GCaMP6s were monitored by a Zeiss
Axio Observer Z1 Inverted Microscope using a 20× objective as
previously reported (72). The interval of data acquisition was 5 s.
The software iVision 4.5 (BioVision Technologies) was used for
data acquisition and analysis. The standard solution for Ca2+ imaging
contained 120 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1.2 mM NaHCO3,
10 mM glucose, and 10 mM Hepes (pH 7.5). About 60 s after initiation
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SCIENCE SIGNALING | RESEARCH ARTICLE
of imaging procedure, the bath was perfused using a peristaltic
pump with a solution containing 100 mM Na-glutamate, 20 mM NaCl,
3 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 1.2 mM NaHCO3, 10 mM
glucose, and 10 mM Hepes (pH 7.5) to elicit Ca2+ entry through
active channels. Different pH values were indicated in the figures.
Whole-plant calcium imaging
Transgenic Arabidopsis plants stably expressing GCaMP6s were
imaged with a Zeiss Lumar v12 epifluorescence stereoscope equipped
with a 6× objective lens and Retiga 12-bit camera. The green fluo-
rescent protein–based Ca2+ indicator, GCaMP6s, was excited using
a mercury lamp (Intensilight Hg Illuminator, Zeiss), and a 440/470-nm
excitation filter. The green fluorescent signal passing through a
535/550-nm filter was acquired every 2 s with the Retiga 12-bit camera
using iVision software (BioVision Technologies).
To elicit wound responses, Arabidopsis roots were severed with
a pair of scissors. For glutamate treatment, after a 15-min recovery
period after cutting, 100 mM glutamate (solved in ddH2O and
adjusted pH to 5.5 using tris base) was added to the cut site.
Using the ImageJ software, GCaMP6s signals were analyzed
overtime at several regions of interest. To calculate the fractional
fluorescence changes (F/F), the equation F/F = (F − F0)/F0 was
used, where F0 denotes the average baseline fluorescence determined
by the average of F over the first 10 frames of the recording before
the wound/treatment (72).
Total RNA isolation, cDNA synthesis, and qPCR
For quantitative polymerase chain reaction (qPCR) analysis, the main
roots of Col-0 and the glr3.3 and glr3.6 mutant were severed with
scissors at 2 cm away from the root-shoot junction. After the wound,
shoot samples were harvested at 0, 0.5, 1, and 2 hours, and rapidly
frozen in liquid nitrogen. Total RNA was extracted from plant sam-
ples using TRIzol reagent (Invitrogen), followed by synthesis of Poly
(dT) cDNA using the M-MLV Reverse Transcriptase (Promega).
Real-time quantitative reverse transcription polymerase chain reaction
(qRT-PCR) was performed using the SYBR Green Supermix (Bio-Rad)
according to the manufacturer’s instructions on a CFX96 Connect
Real-Time System (Bio-Rad). All individual reactions were done
in triplicate. The primers used for qRT-PCR are listed in table S2.
SUPPLEMENTARY MATERIALS
stke.sciencemag.org/cgi/content/full/13/640/eaba1453/DC1
Fig. S1. Systemic [Ca2+]cyt waves and SWPs induced by cutting a lateral root.
Fig. S2. Leaf-to-leaf SWPs induced by wounding and glutamate.
Fig. S3. Participation of GLR3.3 and GLR3.6 in wound- and glutamate-triggered root-to-shoot
[Ca2+]cyt waves.
Fig. S4. Inhibition of glutamate-induced SWPs by FC.
Fig. S5. Conductivity of GLR3.3 to divalent and monovalent cations.
Fig. S6. Conductivity of GLR3.6 to divalent and monovalent cations.
Table S1. Relative ion permeability of GLR3.3 and GLR3.6.
Table S2. Primers.
Movie S1. Systemic [Ca2+]cyt waves in response to cutting the main root.
Movie S2. Root-to-shoot [Ca2+]cyt waves induced by application of glutamate at the wound site.
Movie S3. Loss of root-to-shoot [Ca2+]cyt waves in a glr3.3glr3.6 mutant upon root wounding.
Movie S4. Loss of root-to-shoot [Ca2+]cyt waves in a glr3.3glr3.6 mutant upon application of
glutamate at the wound site.
Movie S5. [Ca2+]cyt imaging of control HEK293T cells upon application of glutamate at pH 8.5.
Movie S6. [Ca2+]cyt imaging of GLR3.6-expressing HEK293T cells upon application of glutamate
at pH 8.5.
Movie S7. [Ca2+]cyt imaging of GLR3.6-expressing HEK293T cells upon application of histidine
at pH 8.5.
View/request a protocol for this paper from Bio-protocol.
Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020
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Acknowledgments: We thank J.-M. Zhou for providing ost2-2D seeds, and J. A. Feijó and
E. E. Farmer for providing cDNAs encoding GLR3.3 and GLR3.6. Funding: Q.S. was
supported by a scholarship from the China Scholarship Council and Q.G. was supported, in
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SCIENCE SIGNALING | RESEARCH ARTICLE

Author contributions: S.L. and Q.G. designed the research. Q.G. performed electrophysiology Submitted 8 November 2019
analysis; Q.S. performed whole-plant calcium imaging and q-PCR; Q.S. and D.L. performed Accepted 30 April 2020
SWP recordings; Q.S., Q.G., and H.Z. analyzed the data; and Q.G. and S.L. wrote the paper. Published 14 July 2020
Competing interests: The authors declare that they have no competing financial or 10.1126/scisignal.aba1453
nonfinancial interests. Data and materials availability: All data needed to evaluate the
conclusions in the paper are present in the paper or the Supplementary Materials. Plasmids Citation: Q. Shao, Q. Gao, D. Lhamo, H. Zhang, S. Luan, Two glutamate- and pH-regulated Ca2+
and Arabidopsis mutants are available upon request. channels are required for systemic wound signaling in Arabidopsis. Sci. Signal. 13, eaba1453 (2020).

Downloaded from http://stke.sciencemag.org/ on July 14, 2020

Shao et al., Sci. Signal. 13, eaba1453 (2020) 14 July 2020 13 of 13

Two glutamate- and pH-regulated Ca2+ channels are required for systemic wound signaling in
Arabidopsis
Qiaolin Shao, Qifei Gao, Dhondup Lhamo, Hongsheng Zhang and Sheng Luan

Sci. Signal. 13 (640), eaba1453.

DOI: 10.1126/scisignal.aba1453

Glutamate-dependent systemic signaling in plants

Upon wounding, plants generate systemic Ca2+ waves and electrical signals that propagate from the wound site
to distal tissues (see the Focus by Fichman et al.). The glutamate receptor−like proteins GLR3.3 and GLR3.6 are
required for leaf-to-leaf systemic wound signals in Arabidopsis thaliana. Shao et al. found that wounding or the
application of glutamate induced root-to-shoot Ca 2+ and electrical signaling in Arabidopsis, which required GLR3.3,

Downloaded from http://stke.sciencemag.org/ on July 14, 2020

GLR3.6, and inhibition of the proton pump AHA1. In cultured mammalian cells, GLR3.3 and GLR3.6 functioned as
pH-sensitive, glutamate-gated Ca2+ channels. These findings suggest that wounding induces both the leakage of
glutamate from the phloem into the apoplastic space and an increase in the apoplastic pH, leading to the activation of
GLRs and the generation of systemic Ca2+ waves and electrical signals.

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