Review

Tansley review
Calcium transport across plant membranes:
mechanisms and functions

Author for correspondence: Vadim Demidchik1,2,3, Sergey Shabala1,4, Stanislav Isayenkov5,

Vadim Demidchik
Tel: +375 172 095934 Tracey A. Cuin4 and Igor Pottosin6
Email: dzemidchyk@bsu.by 1
Department of Horticulture, Foshan University, Foshan 528000, China; 2Department of Plant Cell Biology and Bioengineering,
Received: 26 September 2017 Biological Faculty, Belarusian State University, 4 Independence Avenue, Minsk 220030, Belarus; 3Komarov Botanical Institute,
Accepted: 21 April 2018 Russian Academy of Sciences, 2 Professora Popova Street, St Petersburg 197376, Russia; 4Tasmanian Institute of Agriculture, University
of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia; 5Institute of Food Biotechnology and Genomics, National Academy of
Science of Ukraine, 2a Osipovskogo Street, Kyiv 04123, Ukraine; 6Centro Universitario de Investigaciones Biom
edicas, Universidad de
Colima, Avenida 25 de julio 965, Colima 28045, Mexico

Contents

Summary 1 IV. Concluding remarks 16

I. Introduction 1 Acknowledgements 16

II. Physiological and structural characteristics of plant References 16

Ca2+-permeable ion channels 2

III. Ca2+ extrusion systems 13

New Phytologist (2018)
doi: 10.1111/nph.15266
Key words: calcium, calcium extrusion
systems, cyclic nucleotide-gated channels, ion
channels, ionotropic glutamate receptors,
reactive oxygen species, ROS-Ca2+ hub,
signalling.
Summary
Calcium is an essential structural, metabolic and signalling element. The physiological functions
of Ca2+ are enabled by its orchestrated transport across cell membranes, mediated by Ca2+-
permeable ion channels, Ca2+-ATPases and Ca2+/H+ exchangers. Bioinformatics analysis has not
determined any Ca2+-selective filters in plant ion channels, but electrophysiological tests do
reveal Ca2+ conductances in plant membranes. The biophysical characteristics of plant Ca2+
conductances have been studied in detail and were recently complemented by molecular genetic
approaches. Plant Ca2+ conductances are mediated by several families of ion channels, including
cyclic nucleotide-gated channels (CNGCs), ionotropic glutamate receptors, two-pore channel 1
(TPC1), annexins and several types of mechanosensitive channels. Key Ca2+-mediated reactions
(e.g. sensing of temperature, gravity, touch and hormones, and cell elongation and guard cell
closure) have now been associated with the activities of specific subunits from these families.
Structural studies have demonstrated a unique selectivity filter in TPC1, which is passable for
hydrated divalent cations. The hypothesis of a ROS-Ca2+ hub is discussed, linking Ca2+ transport
to ROS generation. CNGC inactivation by cytosolic Ca2+, leading to the termination of Ca2+
signals, is now mechanistically explained. The structure–function relationships of Ca2+-ATPases
and Ca2+/H+ exchangers, and their regulation and physiological roles are analysed.

I. Introduction
Calcium is an essential macronutrient in plants (Marschner, 2012).
In a broad sense, calcium influx in plant cells is required either for
‘nutritional’ or ‘signalling’ purposes. The nutritional mode
involves passive Ca2+ supply via ion channels for structural and
metabolic needs and relies on the operation of constitutive Ca2+
influx channels (Demidchik & Maathuis, 2007). By contrast, the
signalling mode depends on a rapid and transient ion channel-
mediated increase in cytosolic Ca2+ [Ca2+]cyt., referred to as a Ca2+

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signal. This signal is essential for decoding internal and external
stimuli, transducing them into physiological and gene expression
responses (Dodd et al., 2010; Hedrich, 2012; Swarbreck et al.,
2013; Edel et al., 2017; Demidchik & Shabala, 2018).
The chemical activity of Ca2+ in the cytosol is maintained by
living cells with amazing accuracy. Plants evolved Ca2+-
transporting and Ca2+-buffering systems that work in concert to
maintain [Ca2+]cyt at the 50–150 nM level, even though the
cytoplasm is surrounded by much higher Ca2+ activities (0.1–
10 mM) in the cell wall and certain organelles (endoplasmic
reticulum (ER) and vacuole; Stael et al., 2012). Moreover, the
negative electric potential of the plasma membrane (PM) creates
additional forces to attract Ca2+ into the cell (each
28 to 29 mV
provides the same energy for the Ca2+ influx as a 10-fold difference
in Ca2+ concentration across a membrane). The fixed charges in the
cell wall and the Donnan potential cause accumulation of
extracellular Ca2+ in close proximity to the PM and also contribute
to a steep Ca2+ gradient across the PM. Overall, this means that any
Ca2+-permeable pore in the PM will provide a very strong Ca2+
influx along the electrochemical gradient. Thus, very tight
regulation of Ca2+ transport is vital.
Constant [Ca2+]cyt is maintained by the orchestrated regulation
of the following processes: a passive influx of Ca2+ along the
electrochemical gradient through Ca2+-permeable cation channels
of the PM, tonoplast and other endomembranes; the removal of
Ca2+ against the electrochemical gradient by Ca2+-ATPases and
Ca2+/H+ exchangers; and [Ca2+]cyt buffering by intracellular
compounds, including free polyvalent inorganic and organic
anions (such as phosphates, ATP, ADP etc.), anionic heads of
lipids, Ca2+-binding proteins and carboxyl residues of biopolymers
and similar ligands. All these processes are modulated by internal
and external stimuli such as phytohormones, reactive oxygen
species (ROS), regulatory proteins and gene expression pro-
grammes.
Owing to a low energy barrier and no need for the formation of
covalent bonds, calcium passage through ion channels is one of the
most rapid ‘elementary’ physiological process, lasting for only
nanoseconds (Kuyucak et al., 2001). Pumping out or exchanging
Ca2+ against the electrochemical gradient is much slower (the
microsecond range). Buffering of excess Ca2+ is relatively fast, but
slower than diffusion via channels. However, the specifics and
physiological role of such buffering are poorly understood. Some
estimates suggest that c. 1700 of total Ca2+ can be bound per one
free Ca2+ in the cytosol (Fleet et al., 1998). The activity of Ca2+-
ATPases and Ca2+/H+ exchangers (jointly known as Ca2+-
extruding systems) is directly up-regulated by [Ca2+]cyt, increasing
upon [Ca2+]cyt elevation mediated by ion channels. Calcium-
extruding systems are energized by ATP or an ATP-dependent H+
gradient.
The molecular nature, biophysical and physiological properties
of Ca2+-transporting systems, including Ca2+-permeable ion
channels, Ca2+-ATPases and Ca2+/H+ exchangers, have been
addressed by a large number of studies. It has become increasingly
evident that Ca2+ transport across plant membranes is rather
complicated; it is mediated by highly redundant systems, which in
some cases show no similarity to animal, fungal or prokaryote
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counterparts. Here the Ca2+-transporting systems of the PM and
endomembranes are discussed in a physiological context, with
emphasis on recent developments in the field of ion channels.
II. Physiological and structural characteristics of plant
Ca2+-permeable ion channels
Plant genomes do not encode ion channel subunits with Ca2+-
selective filters as found in animals. Nonetheless, plant membranes
are permeable to Ca2+ and conduct Ca2+ currents, as shown by
electrophysiological measurements (Demidchik et al., 2002b).
Thus plants, despite not bearing canonical Ca2+ channels, still
have Ca2+ conductances that must be mediated by other types of
cation channels. These are collectively called the Ca2+-permeable
cation channels. The majority of animal Ca2+-permeable cation
channels, such as ionotropic purinoceptors, glutamate receptors
and ‘transient receptor potential’ channels, are also not highly
selective to Ca2+. Similar to animal Ca2+-permeable channels, plant
Ca2+ conductances are present in all cells and show a variety of
properties to suit different physiological requirements, with
sophisticated regulation and pharmacology. Plant Ca2+ conduc-
tances have been well explored physiologically, but a link to the
exact gene products is still largely missing.
The evolutionary analysis of plant Ca2+-permeable channel
families reveals a dramatic divergence between higher plants and
animals (Edel et al., 2017). Compared with animals and algae,
higher plants have lost the four-domain voltage-dependent cation
channels, inositol trisphosphate receptors, P2X purinoceptors,
‘cys-loop’ ligand-gated ion channels and ‘transient receptor
potential’ channels. Nevertheless, plant Ca2+-permeable channels
such as cyclic nucleotide-gated channels (CNGCs), ionotropic
glutamate receptors (GLRs) and reduced hyperosmolality-induced
[Ca2+] increase 1’ channel (OSCA1) demonstrate a greater variety
of phylogenetic clades, selectivity filters and potential regulatory
structures than their animal counterparts (see Supporting Infor-
mation Figs S1–S7).
The bulk of previously published phylogenetic analyses on Ca2+-
transporting proteins focused heavily on animal systems or model
plant species. Here we compare up to 600 sequences from most
important crop and model species from different families, with
diverse physiological and adaptive strategies (see Figs S1–S7). In
addition to reporting phylogenetic relationships, the aspects of
evolutional phylogeny are also covered. Finally, our cladistics
analyses include more detailed distribution into subclades not
reported by previous studies (see Section II).
As single-channel patch-clamp recordings are difficult to obtain,
most electrophysiological tests are conducted in the whole-cell
mode. In the latter case, the net membrane Ca2+ conductance,
which represents the activities of all contributing Ca2+-transporting
channels and transporters at given time in a given cell, is recorded.
The measured conductance is a result of a highly dynamic and
constantly changing assembly of different channel subunits, which
also potentially includes currents of active transporters. A number
of genes encoding channel subunits can potentially be responsible
for one type of Ca2+ conductance. As a result, Ca2+ conductances,
representing activities of different ion channels, can be recorded
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simultaneously (Miedema et al., 2008; Straltsova et al., 2015). Gao
et al. (2016) recently reported that up to eight different subunits of
putative Ca2+-permeable channels can be expressed at the same
time in the PM of an individual plant cell.
Genetic approaches aim to modify channel protein expression; if
a gene is made dysfunctional by T-DNA or retrotransposon inserts,
RNAi knockdown technologies or genome editing using TALEN
or CRISPR-Cas9, then conductance can be lost. However, a high
redundancy of Ca2+-permeable subunits, such as CNGCs and
GLRs, decreases the effectiveness of this reverse genetic approach.
Overexpression or conditional RNAi-based knockouts are used to
deal with this problem. The recent applications of these technolo-
gies to study Ca2+ conductances are summarized in Table 1.
Another approach is expressing plant channel subunits in high-
throughput animal expression systems such as HEK cells and
Xenopus oocytes for rapid patch-clamping and two-electrode
voltage clamp tests, respectively. However, this approach is
jeopardized by a potential lack of plant-native protein folding
during post-translational regulation when expressed in animal cells.
Thus, reports on expression of plant Ca2+-permeable channel
subunits in animal cells are useful, but they can be controversial. For
example, Tapken et al. (2013) found that the ionotropic glutamate
receptor AtGLR1.4 expressed in Xenopus oocytes is not activated by
either glutamate or glycine, but can be stimulated by some other
amino acids such as methionine and tryptophan, even though these
amino acids do not induce [Ca2+]cyt elevation in intact plants
(Davenport, 2002; Demidchik et al., 2004).
1. The electrophysiological properties and classification of
Ca2+ conductances studied in vivo
Calcium conductances have been recorded and analysed in
different plant preparations using electrophysiological techniques
since the 1980s (reviewed by Demidchik et al., 2002b). This has
allowed the determination of the key biophysical characteristics (as
described later) of Ca2+ conductances and created the basis for the
classification of Ca2+ conductances as well as further molecular
studies. Ideally, all these characteristics should be linked to genetics,
structure and physiology.
Membrane localization Based on target membranes, Ca2+ con-
ductances can be classified as PM, tonoplast, chloroplast, mito-
chondria and ER conductances. Unfortunately, mitochondrial,
chloroplast and ER Ca2+ conductances/transporters are insuffi-
ciently studied in vivo (Carraretto et al., 2016).
Voltage-dependence According to their voltage-dependence,
plant Ca2+ conductances are classified as: depolarization-activated
Ca2+ conductance (DACC); hyperpolarization-activated Ca2+
conductance (HACC); and voltage-independent Ca2+ conduc-
tance (VICC) (see typical I–V types in Fig. 1). Sometimes these
conductances are called ‘channels’, using the same abbreviations.
Theoretically, all Ca2+ conductances should be encoded by
specific genes. Some candidate genes have been found and are
discussed later (Fig. 2). Among the conductances measured
in vivo (Table 2), the DACCs are still not associated with any
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gene, although this type of conductance dominates animal Ca2+
influx.
Kinetics of activation Ca2+ conductances include the following
groups: rapidly activating (fully activated in milliseconds), slowly
activating (fully activated in seconds), and ‘spiky’ burst-like
conductances or single-channel events in the whole-cell mode.
Rapidly activated channels (Fig. 1) could be permanently open,
although this is still debated (Zhang et al., 2000; Demidchik,
2012). The third class is rare, but it is observed, for example, under
treatment with glutamate, nanoparticles, H2O2 or during
pathogen attack, potentially reflecting the current through GLRs
or mechanosensitive channels, respectively (Demidchik et al.,
2004; Sosan et al., 2016).
Pharmacological properties Lanthanides (Gd3+, La3+), dihy-
dropyridines and phenylalkylamines, quinine, mibefradil, bepridil
or flunarizine (10
6–10
3 M) are the most widely used blockers of
Ca2+ conductances in plants (Demidchik et al., 2002b; Demidchik,
2012). Unfortunately, screening and synthesis of pharmaceuticals
that specifically bind and block different classes of plant Ca2+-
permeable channels have not been carried out. In animal physi-
ology, this field of research is far better developed, because of its
direct medical impact (Arranz-Tagarro et al., 2014).
Constitutive presence vs ligand-induced opening Some Ca2+
conductances are considered as ‘constitutive’, meaning they do not
need any specific activators apart from voltage. By contrast, ligand-
gated Ca2+ conductances activate only after binding a ligand to the
specific ligand-binding moiety. The following extracellular ligands/
activators can trigger Ca2+ conductances in plants: amino acids such
as glutamate and glycine; ROS (e.g. H2O2 or hydroxyl radicals
(•OH)); and purines, such as ATP and ADP. There is also a group of
intracellular ligands that includes 30 ,50 -cyclic adenosine
monophosphate (cAMP), 30 ,50 -cyclic monophosphate (cGMP),
cADPR, phosphate, phosphorylated sugars, lipid derivatives and
H2O2. Some substances, such as polyamines, H+, cytosolic Ca2+ and
ROS, may act as channel modulators, causing inhibitory or
stimulatory effects, depending on circumstances. For example,
polyamines stimulate ROS-induced Ca2+ conductance (Zepeda-
Jazo et al., 2011) but they can block constitutive Ca2+ conductances
(Pottosin & Dobrovinskaya, 2014). A special case of ligand-like
activation involves ROS-activated Ca2+-permeable channels
(Demidchik, 2015). Based on results obtained in molecular tests
with animal ROS-activated cation channels, these channels are
directly, and in most cases reversibly, activated by ROS from both
the outside and the inside (G€orlach et al., 2015). Plants have very
similar activation (Demidchik et al., 2007), but the molecular
mechanisms of this activation are still unknown (Demidchik, 2015).
Selectivity While the hyperpolarization-activated Ca2+ channels
present in root hairs are extremely selective for Ca2+ (V
ery &
Davies, 2000), the majority of plant Ca2+ conductances show
significant permeability to both mono- and divalent cations,
including K+, Na+, NH4+, Cs+, Rb+, Ba2+ and Sr2+, and are
generally weakly permeable to Zn2+, Mn2+ and Mg2+ (reviewed by
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Table 1 Characterization of plant Ca2+-permeable channels using genetic approaches
No. Subunit Techniques
Cyclic nucleotide-gated channels (CNGCs)
1 AtCNGC1, AtCNGC2, Reverse genetics via T-DNA insertional
AtCNGC5, AtCNGC6, mutagenesis
AtCNGC20
2 AtCNGC2 Reverse genetics via T-DNA insertional
mutagenesis
3 AtCNGC2 Reverse genetics via T-DNA insertional
mutagenesis
4 AtCNGC3 Reverse genetics via T-DNA insertional
mutagenesis; expression in yeast
mutants deficient in Ca2+ uptake
5 AtCNGC4 Reverse genetics via T-DNA insertional
mutagenesis; heterologous expression
and electrophysiological
characterization in frog oocytes of
Xenopus laevis
6 AtCNGC7, AtCNGC8 Reverse genetics via T-DNA insertional
mutagenesis
7 AtCNGC7, AtCNGC8, Heterologous expression and
AtCNGC9, AtCNGC10, electrophysiological characterization
AtCNGC16 in animal cells (HEK293T)
8 AtCNGC10 Expression of antisense AtCNGC10
construct
9 AtCNGC12 Transient expression of AtCNGC12
lacking functional CaMBS in leaves of
Nicotiana benthamiana
10 AtCNGC14 Reverse genetics via T-DNA insertional
mutagenesis
11 AtCNGC16 Reverse genetics via T-DNA insertional
mutagenesis
12 AtCNGC18 Heterologous expression and
electrophysiological characterization
in animal cells (HEK293T)
13 AtCNGC18 Reverse genetics via T-DNA insertional
mutagenesis
14 AtCNGC18 Heterologous expression and
electrophysiological characterization
together with Ca2+-dependent
protein kinase CPK32 in in frog
oocytes of Xenopus laevis;
overexpression in plants with a Ca2+-
dependent protein kinase CPK32;
pollen tubes coexpressing CNGC18–
YFP were examined with FRET
15 MtCNGC15 RNA interference and Rhizobium
rhizogenes-mediated transformation
16 NtCBP4 Overexpression in tobacco plants
Ionotropic glutamate receptors (GLRs)
17 AtGLR1.1 Expression of antisense AtGLR1.1
construct
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Phenotype References
KO atcngc1, atcngc2 and atcngc20 Wang et al. (2013)
showed unmodified guard cell cGMP-
activated Ca2+ influx current
compared with the WT. Double KO
atcngc5/atcngc6 lacked this current.
KO atcngc2-1 and atcngc2-2 were Chan et al. (2003)
sensitive to high external Ca2+
KO atcngc2 (dnd1) lacked Ca2+ current Ali et al. (2007)
and did not generate NO upon stim-
ulation by bacterial elicitor
KO atcngc3 showed the same root Gobert et al. (2006)
growth as WT in high Ca2+ medium;
AtCNGC3 expression did not change
growth of yeast strains lacking Ca2+
uptake systems
KO atcngc4 (hlm1) showed differential Balague et al. (2003)
abilities to restrict bacterial growth,
depending on the avirulence gene
expressed by the pathogen; patch-
clamp technique in frog oocytes
showed that CNGC4 is permeable to
both K+ and Na+ and is activated by
both cGMP and cAMP.
Double KO lines Atcngc7-1/atcngc8-2 Tunc-Ozdemir et al. (2013a)
and atcngc7-3/atngc8-1 showed male
sterility
Expression of AtCNGC7-10 and Gao et al. (2016)
AtCNGC16 increased Ca2+ current,
but did not change K+ current
Lower basal Ca2+ influx in root Guo et al. (2010)
epidermis and decreased calcium
content
Expression of AtCNGC12 with a non- DeFalco et al. (2016a)
functional N-terminal CaMBS induced
programmed cell death
KO atcngc14 exhibited a loss of rapid Shih et al. (2015)
auxin-induced Ca2+ signalling
KO atcngc16-1 and atcngc16-2 Tunc-Ozdemir et al. (2013b)
decreased pollen fitness and seed set
AtCNGC18 expression increased Ca2+ Gao et al. (2014)
conductance and evoked its activation
by the cyclic nucleotides
KO atcngc18 exhibited male sterility Frietsch et al. (2007)
Expression of AtCNGC18 caused Ca2+ Zhou et al. (2014)
current increase (in Xenopus), which
was additionally stimulated by coex-
pression with CPK32; AtCNGC18 was
expressed in the tip of the pollen tube
and caused Ca2+ influx in CPK32-
dependent manner
KO cngc15 showed symbiotic defects Charpentier et al. (2016)
Increased tolerance to Ni2+ and Arazi et al. (1999)
hypersensitivity to Pb2+
Modification of carbon and nitrogen Kang & Turano (2003)
metabolism
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Table 1 (Continued)

No. Subunit Techniques Phenotype References

18 AtGLR1.1, AtGLR1.4 Heterologous expression and Increase of Ca2+ currents in AtGLR1.1- Tapken & Hollmann (2008)
electrophysiological characterization and AtGLR1.4-expressing oocytes
in frog oocytes of Xenopus laevis
19 AtGLR1.4 Heterologous expression and AtGLR1.4 expression yielded Ca2+ Tapken et al. (2013)
electrophysiological characterization influx currents stimulated by amino
in frog oocytes of Xenopus laevis acids; nine amino acids were
antagonists, and four, including
glutamate and glycine, had no effect
on Ca2+ conductance
20 AtGLR1.2, AtGLR3.7 Reverse genetics via T-DNA insertional AtGLR1.2 and AtGLR3.7 were Michard et al. (2011)
mutagenesis activated by serine and mediated Ca2+
influx inducing pollen tube elongation
21 AtGLR3.1 Reverse genetics via T-DNA insertional AtGLR3.1 showed involvement in a Cho et al. (2009)
mutagenesis; overexpression in generation of Ca2+ oscillations in
Arabidopsis thaliana guard cells needed for anion channel-
dependent stomata closure
22 AtGLR3.2 Overexpression in Arabidopsis thaliana Poor Ca2+ utilization Kim et al. (2001)
23 AtGLR3.3 Reverse genetics via T-DNA insertional Atglr3.3 was defective in [Ca2+]cyt. Qi et al. (2006)
mutagenesis elevation induced by amino acids
24 AtGLR3.3 Reverse genetics via T-DNA insertional Atglr3.3 showed an impaired gravit- Miller et al. (2010)
mutagenesis ropic response
25 AtGLR3.3, AtGLR3.4 Reverse genetics via T-DNA insertional Amino acid-induced Ca2+ signatures Stephens et al. (2008)
mutagenesis were modified in KO atglr3.3 and
atglr3.4.
26 AtGLR3.4 Transformation of Arabidopsis thaliana AtGLR3.4 was present in plasma Vincill et al. (2012)
with GFP-fused GLRL3.4; heterolo- membrane; its expression in animal
gous expression and electrophysio- cells resulted in voltage-independent
logical characterization in animal cells Ca2+ influx current activated by amino
(HEK293T) acids
27 AtGLR3.6 Reverse genetics via T-DNA insertional KO had smaller root meristem size and Singh et al. (2016)
mutagenesis decreased mitotic activity.
28 AtGLR3.7 Overexpression in Arabidopsis Lack of amino acid-induced activation; Roy et al. (2008)
thaliana; heterologous expression and increase of constitutive Ba2+, Ca2+ and
electrophysiological characterization Na+ conductance of oocytes
in frog oocytes of Xenopus laevis
29 OsGLR3.1 Reverse genetics via T-DNA insertional KO osglr3.1 showed a decreased size of Li et al. (2006)
mutagenesis quiescent centre and increased Evans
Blue staining of root tip, suggesting
enhanced cell death
30 PpGLR1, PpGLR2 Generation of gene knockouts by KO glr1-19, glr2-11 and a double Ortiz-Ram
ırez et al. (2017)
homologous recombination mutant glr1/2 showed defects in
chemotaxis and reproduction
Two-pore channel (TPC)
31 AtTPC1 Reverse genetics via T-DNA insertional Attpc1 lacked SV channel activity and Peiter et al. (2005)
mutagenesis; overexpression in was defective in ABA-induced
protoplasts isolated from Arabidopsis germination repression and in
thaliana guard cells stomatal closure caused by a high
extracellular Ca2+
32 AtTPC1 Reverse genetics via T-DNA insertional Attpc1 did not show changes in stress- Ranf et al. (2008)
mutagenesis induced Ca2+ signals
33 OsTPC1, TaTPC1 Cross-species complementation OsTPC1 and TaTPC1 showed presence Dadacz-Narloch et al. (2013)
expression in the background of the in vacuole and SV channel activity
Arabidopsis tpc1-2 mutant and in
HEK293 cells
34 AtTPC1 Reverse genetics via T-DNA insertional Systemic Ca2+ response to wounding Kiep et al. (2015)
mutagenesis and herbivory in leaf rosettes was
inhibited in Attpc1-2 mutant
35 AtTPC1 Reverse genetics via T-DNA insertional Attpc1-2 had slower NaCl-induced Choi et al. (2014)
mutagenesis propagation of Ca2+ signal along the
root

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Table 1 (Continued)

No. Subunit Techniques Phenotype References

Mechanosensitive-like channels (MSLs)

36 AtMSL2, AtMSL3 Reverse genetics via T-DNA insertional
mutagenesis
37 AtMSL2 Reverse genetics via T-DNA insertional
mutagenesis; expression in atmsl2
mutant and WT plants of Arabidopsis
thaliana
38 AtMSL4, AtMSL5, Reverse genetics via T-DNA insertional
AtMSL6, AtMSL9, mutagenesis; generation of double,
AtMSL10 triple and quintuple mutants;
overexpression in root protoplasts of
Arabidopsis thaliana combined with
patch-clamping
Mid1-complementing activity channels (MCAs)
39 AtMCA1 Reverse genetics via T-DNA insertional
mutagenesis; heterologous expression
in yeast deficient in Ca2+ uptake
systems; overexpression in mesophyll
protoplasts of Arabidopsis thaliana
combined with patch-clamping
40 AtMCA1 Overexpression in Arabidopsis thaliana
41 AtMCA1 Heterologous expression and
electrophysiological characterization
in frog oocytes of Xenopus laevis
42 AtMCA1, AtMCA2 Heterologous expression in Sf9 cells and
structural crystallographic
characterization
Hyperosmolality-induced [Ca2+]cyt. channel 1 (OSCA1)
43 AtOSCA1,1, AtOSCA1,2 Calcium imaging-based unbiased
forward genetic screens to isolate
mutants with low hyperosmolality
induced by Ca2+; heterologous
expression and electrophysiological
characterization in animal cells
(HEK293T; Chinese hamster ovary
cells)
Double KO atmsl2/atmsl3 showed Haswell & Meyerowitz (2006)
abnormalities in plastid size and shape
PN(X)9N motif is essential for MSL2 Jensen & Haswell (2012)
function and for its proper
intraplastidic localization; two of the
three conserved motifs analysed were
critical for MSL2 function
A presence of all tested subunits is Haswell et al. (2008)
required for full-strength Ca2+ influx
currents activated by stretching
Expression in yeast improved Ca2+ Nakagawa et al. (2007)
uptake; various tests of KO and
overexpression lines showed that
MCA1 is crucial for Ca2+ balance;
stretch-activated Ca2+ current was
bigger in the overexpression line
Increased transcription of touch- Lee et al. (2005)
induced calmodulin-like protein TCH3
MS cation currents were activated by Furuichi et al. (2012)
hypo-osmotic shock or by membrane
stretch produced by pipette suction
MCA2 revealed tetrameric structure Shigematsu et al. (2014)
with a small transmembrane domain
and a large cytoplasmic region
The Atosca1mutant exhibits a reduced Yuan et al. (2014);
hyperosmolality; plants also display Hou et al. (2014)
impaired osmotic Ca2+ signalling in
guard cells and root cells, and altered
transpiration and root growth in
response to osmotic stress; construct
expressing DUF221 led to formation
of rapidly activating VICC-like con-
ductance

KO, knockout lines; WT, wild type; cGMP, 30 ,50 -cyclic guanosine monophosphate; cAMP, 30 ,50 -cyclic adenosine monophosphate; CaMBS, calmodulin-binding
site; VICC, voltage-independent Ca2+ conductance.

Demidchik & Maathuis, 2007). This leads to the conclusion that ORAI proteins (similar to animal store-operated Ca2+-release
plant Ca2+ conductances are mainly mediated by nonselective channels from the ER). This group is not yet studied and will not be
cation channels (NSCCs). discussed here.

Ionotropic glutamate receptors ‘Glutamate receptor-like’ (GLR)

2. Putative channel subunits with potential Ca2+
permeability
Most of the knowledge in the field comes from the studies on
Arabidopsis thaliana channel subunits. Their transmembrane
topology and structural highlights are summarized in Fig. 2 and
Table 3 and were recently comprehensively discussed by Demid-
chik & Shabala (2018). The heterologous expression studies
(Table 1) and in vivo electrophysiological tests (Table 2) demon-
strated that some of these genes do indeed encode Ca2+-permeable
channels. Edel et al. (2017) recently showed that gymnosperms,
mosses and algae (but not angiosperms) contain a few genes of
genes encode proteins resembling animal ionotropic GLRs (Fig. 2;
Table 3). GLRs are abundant family of plant ion channels, with 20
genes in A. thaliana and typically over 10 in all other higher plant
genomes (Lam et al., 1998; Davenport, 2002; Aouini et al., 2012;
Ni et al., 2016). A. thaliana GLRs show three phylogenetic clades
(GLR1–3; Chiu et al., 1999; Davenport, 2002). According to our
analysis, three large clades of GLRs comprise 10 different subclades
(Fig. S3), which all demonstrate early evolutionary divergence from
animal GLRs (Fig. S4). GLRs are believed to be tetramers
consisting of different subunits, with preferential expression in
root tissues (Price et al., 2013). Some specific subunits are expressed

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Phytologist Tansley review Review 7

(a) Membrane potential (voltage), mV Glutamate is recognized as a predominant excitatory neuro-

transmitter in the central nervous system (Julio-Pieper et al., 2011).
–180 –150 –120 –90 –60 –30 0 30 60 90 120
Hyper- Resting
As the concentrations of glutamate, glycine and other amino acids
Depolarization
polarization potential in the apoplast are within the 0.1–1 mM range (Lohaus et al., 1995,
I–V curve shows relationship between current and membrane voltage. 2001), the presence of GLRs make the glutamatergic Ca2+
Different types of Ca2+ conductance are activated at physiological signalling in plants plausible. It was shown that submillimolar
voltage range exogenous glutamate and glycine induces lanthanide-sensitive

Current (I)

n
atio
[Ca2+]cyt transients and PM Ca2+ conductances in plant cells

re i t h
fic
d w
c ti
(Dennison & Spalding, 2000; Dubos et al., 2003; Demidchik

ar I–V
tw
et al., 2004; Michard et al., 2011).
r ou AtGLR3.4 expressed in HEK cells possessed properties of
Linea ve
ur
I–V c voltage-independent Ca2+-permeable ion channels (Vincill et al.,
Voltage (V) 2012). However, cloned plant GLRs might be gated by a broader
range of amino acids, including 12 of the 20 proteinogenic amino
acids and glutathione (Table 1). However, these findings need to be
n
confirmed by in planta experiments.
o
ati
rec ith
tific

Analysis of GLR knockouts showed that a lack of GLR3.3 and

I– V
w
inw I–V

Be

ll - l i k e
GLR3.6 stops the glutamate-induced [Ca2+]cyt elevation (Qi et al.,
ard

2006; Singh et al., 2016). Michard et al. (2011) demonstrated that

GLR1.2 is expressed in pollen tubes and is potentially involved in
Depolarization-activated cation channels (conductances) the polar Ca2+ influx needed for pollen tube elongation. Knocking-
Hyperpolarization-activated cation channels (conductances) out GLRs affected metal homeostasis, pollen incompatibility,
Voltage-independent cation channels (conductances)
immunity and photosynthesis (see review by Weiland et al., 2015).
(b) Ortiz-Ram
ırez et al. (2017) have recently shown that in
Hyperpolarization Slowly
Physcomitrella patens protonema, GLR1 and GLR2 form consti-
activating tutive, rapidly activating Ca2+-permeable channels (sensitive to
channels
animal GLR inhibitors), which are responsible for targeting female
Current

Rapidly
activating
channels
Slowly
activating
channels
Depolarization
Fig. 1 Schematic representation of the current–voltage curves (I–V) and
kinetics of Ca2+ currents associated with the plant plasma membrane Ca2+
conductances. (a) Three types of I–V curves corresponding to the coexisting
Ca2+ conductances (e.g. in root epidermal protoplasts; Miedema et al.,
2008; Straltsova et al., 2015). The ‘hyperpolarization-activated Ca2+
conductances’ (blue line; HACCs) and ‘voltage-independent Ca2+
conductances’ (green line; VICCs) have been linked to the activity of specific
cloned genes both in vitro (Table 1) and in vivo (Table 2). The
‘depolarization-activated Ca2+ conductances’ (red line; DACCs) are not yet
associated with any specific gene product. A bell-like DACC I–V (observed in
plants) is also typical for the animal voltage-gated Ca2+-selective channels,
but are not found in any genome of the higher plants. VICCs dominate Ca2+
influx at resting potential (green part of the voltage range), which is critical
for nutritional and structural needs, while HACCs and DACCs most likely
provide signalling functions. (b) Ca2+ current (grey line) activated by
hyperpolarization (blue arrow) and depolarization (red arrow). Rapidly and
slowly activating Ca2+ currents (corresponding to different groups of Ca2+-
permeable channels) are found in the plasma membrane. Rapidly activating
Ca2+ conductances can be related to channels that are permanently open or
to channels with extremely rapid activation kinetics, such as those
characterized by Zhang et al. (2000). The Ca2+ current kinetics is not yet
linked to genetics.
in leaf mesophyll, guard cells and pollen tubes (Weiland et al.,
2015). Plant GLRs show great divergence from their animal
counterparts, particularly in the pore region, and could potentially
have different permeability profiles. The selectivities of plant GLR
remain largely unknown.
reproductive organs by sperm and regulation of transcription
Time factors essential for zygote development. Nevertheless, mechanistic
explanations for GLR knockout phenotypes is still largely missing,
and many of observed effects are probably indirect.
Cyclic nucleotide-gated channels The distinct feature of these
tetrameric ion channels is their regulation by binding cyclic
nucleotides to conserved cyclic nucleotide binding domain
(CNBD) (Fig. 2; Table 3). CNGCs are structurally similar to
members of the superfamily of six transmembrane ‘Shaker-like’
pore-loop ion channels (DeFalco et al., 2016b; Jha et al., 2016).
Our cladistics analysis shows that plant CNGCs form six clades
(Fig. S1), not five as reported elsewhere (Saand et al., 2015), and
are significantly divergent from their animal counterparts (Fig. S2).
It is presumed that plant CNGCs open after binding cGMP and
guanosine 50 -triphosphate (GTP), although confirmation for this
activation mechanism from in vivo tests is missing. Most of the
CNGCs are expressed at the PM. They are abundant in the root and
leaf epidermis where they probably act for perceiving and encoding
environmental stimuli. CNGCs are found in guard cells and the
mesophyll, where they take part in the control of stomata closure
and photosynthesis (Gobert et al., 2006; Jammes et al., 2011).
CNGCs are also responsible for the nuclear Ca2+ oscillations in the
symbiotic signalling pathway in legume roots; they mediate a
targeted nuclear release of the ER Ca2+ store (Charpentier et al.,
2016).
Crystallography data reveal the presence of six major putative
plant CNGC selective filters: GQN (AtCNGC1, 3, 10–15, 17 and
18), GQG (AtCNGC5–9), AGN (AtCNGC19 and 20), GQS

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Cyclic nucleotide-gated Ionotropic glutamate Two-pore channel 1

channels (CNGCs) receptors (GLRs) (TPC1)
N
Apoplast
ATD
Apoplast Vacuole
LBD

P P P
M2
1 2 3 4 5 6 M1 M3 M4 1 2 3 4 5 6 7 8 9 10 11 12
P

CNB Kd: 950 nM C EF EF

NT D 14-3-3
cGM
Cytosol cAM P IQ IQ of Cytosol
Kd P C
:7 CT another N
M

N nM 4n Cytosol
o r8 subunit
K d: 18
nM
K d: 3 4 C
CaM–Ca2+
Po
te

ti a a
n

lN –C
T–
CT C aM
i n t e r a c ti o n v i a

Mechanosensitive-like channels (MSLs) Mid1-complementing Reduced ‘hyperosmolarity-induced [Ca2+]cyt.

MSLs groups I and II MSLs group III activity channels (MCA) increase’ channels (OSCA1)

Intermembrane space Apoplast Apoplast Vacuole

N N
N

1 2 3 4 5 1 2 3 4 5 6 P 1 2 3 4 5 6 7 8 9
P P

N PHM7_cyt
C Protein CMBS
kinase C
Stroma of chloroplasts
or mitochondrial matrix N Cytosol C C Cytosol Cytosol
2+
Fig. 2 Membrane topology of Ca -permeable channel subunits (adapted from Demidchik & Shabala, 2018, with modifications). NT and CT, N- and C-
terminal calmodulin-binding sites (CaMBS), respectively; IQ, ‘isoleucine-glutamine’ domain, which functions as calmodulin-binding site; CNBD, cyclic
nucleotide-binding domain; P, pore domain; EF, EF-hand motif, which binds Ca2+; ATD, amino terminal domain; LBD, ligand-binding domain; OSCA1
transmembrane domain (grey colour) could be a cleavable signal peptide. See Table 3 for details on the structure of these subunits. The mechanism for
calmodulin (CaM) and Ca2+ regulation of cyclic nucleotide gated channels (CNGS) is depicted in more detail in the top left panel. According to DeFalco et al.
(2016a) CNGC12 has three CaMBSs – one in the N-terminus (NT domain), and two in the C-terminus (CT and IQ domains). The NT and CT domains did not bind
free CaM but they do show high affinity for CaM–Ca2+ complexes (Kd of 7 and 34 nM, respectively). The IQ domain can bind free CaM (Kd of 950 nM) as well as
CaM–Ca2+ complexes (two sites for this interaction were predicted: Kd1 of 18 nM and Kd2 of 84 nM). Theoretically, these sites should be occupied at basal
[Ca2+]cyt, which ranges between 70 and 120 nM. As Kd2 (84 nM) is within the basal [Ca2+]cyt., the second binding to IQ can have a regulatory effect on CNGCs.

(AtCNGC16), AND (AtCNGC2), and GN-L (AtCNGC4) The ability of CNGC to permeate Ca2+ was recently demon-
(Jammes et al., 2011). The electrophysiological properties of strated using the patch-clamp and Ca2+ imaging technologies in
CNGCs have been tested using heterologous expression systems various CNGC mutants (Table 2), providing genetic-based evi-
(Table 1). These studies revealed that CNGCs that harbour the dence for Ca2+ currents measured in intact cells. For example,
GQN, GQG and GQS triplets are selective for Ca2+ and promote CNGC2 demonstrated hyperpolarization-activated inwardly rec-
Ca2+ uptake (Gao et al., 2012, 2014, 2016; Wang et al., 2013). tifying Ca2+ currents and corresponding [Ca2+]cyt., which was
Some heterologously expressed CNGCs are activated when stimulated by cyclic nucleotides and regulated by phytohormones
coexpressed with a Ca2+-dependent protein kinase, CPK32, (Ali et al., 2007; Lu et al., 2016). The pollen tube AtCNGC18
implying regulation of their activity by phosphorylation (Zhou mediated voltage-independent currents in response to cyclic
et al., 2014). Recent studies carried out on intact plants also nucleotides. Wang et al. (2013) found that CNGC5 and CNGC6
demonstrated that the AND (Ali et al., 2007; Lu et al., 2016) of A. thaliana guard cell PM are ABA-insensitive, cGMP-
triplet is potentially selective for Ca2+. These selective CNGC dependent Ca2+-permeable channels.
filters discovered in plants are absent in animal counterparts. Calcium entry through CNGCs evolved self-inactivation
Thus, an important evolutionary role of this divergence may be machinery to prevent Ca2+ overload and shape calcium signals.
suggested. The classical Ca2+-dependent inactivation (CDI) of animal

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Table 2 Plasma membrane Ca2+ conductances in different tissues and corresponding subunits of Ca2+-permeable ion channels that were confirmed by patch-
clamp tests with protoplasts
Tissues, cell type and
proposed functions Major characteristics of Ca2+ conductances
Root epidermis and cortex: Co-existence of HACC, DACC and VICC
Growth Rapidly and slowly activating
Stress response currents in same cell
ROS sensing Stimulation by cyclic nucleotides, ROS,
Hormone action purines and amino acids
Gravitropic response Pressure-induced single-channel events
Heat perception in outside-out patches
Sensing of mechanical stimuli
Guard cells; HACC, VICC; activation by ABA
Induction of closure or cyclic nucleotides
Pollen tube; VICC; activation by ABA, cyclic
Stimulating of polar growth nucleotides or D-serine
ROS, reactive oxygen species; HACC, hyperpolarization-activated Ca2+ conductance; DACC, depolarization-activated Ca2+ conductance; VICC, voltage-
independent Ca2+ conductance; ABA, abscisic acid.
CNGCs, which includes interaction between CNGC-embedded
calmodulin (CaM) and Ca2+ (Ben-Johny et al., 2015), was
suggested to operate in higher plants (K€ohler et al., 1999; K€ohler
& Neuhaus, 2000). Binding CaM in the presence of elevated
[Ca2+]cyt. may displace cAMP or cGMP in the CNBD (overlapping
with calmodulin-binding site (CaMBS)), locking plant CNGC. In
animal cells, [Ca2+]cyt. increase results in a sequential Ca2+ binding
to so-called C- and N-lobes of CaM embedded into CNGC (Linse
et al., 1991). Occupation of both lobes triggers CDI, while binding
only to the C-lobe induces so-called ‘Ca2+-dependent facilitation’
(CDF), prompting further Ca2+ current (Mori et al., 2008; Ben-
Johny et al., 2015). Time-dependent behaviour of plant voltage-
dependent Ca2+ conductances (Demidchik et al., 2002a; Miedema
et al., 2008) can be explained by the CDI and CDF mechanism.
Recent data reveal that plant CNGC subunits possess more
CaMBS than initially shown (Fischer et al., 2013, 2017; DeFalco
et al., 2016a,b). CNGC20 and potentially some other CNGCs do
not show CNBD overlapping with CaMBSs. ‘Isoleucine-glutamine’
(IQ) domains adjacent to the aC-helix play a role of CaMBSs in 11
CNGCs (1–3, 5, 8, 9, 11, 12, 15, 17 and 20), while a complete C-
terminus, possibly including more CaMBSs, is required for CaM
binding in remaining CNGCs (Fischer et al., 2017). Using
CNGC12 as a model, DeFalco et al. (2016a) found that this
channel has three CaMBSs, allowing [Ca2+]cyt. to be maintained at
the basal concentration (c. 50–100 nM) and also allowing the
termination of Ca2+ signals of high amplitude (0.5–1 lM). The
schematic overview of these interactions is shown in Fig. 2.
‘Two-pore cation channel’ (TPC) The two-pore channel 1
(TPC1) has a single gene in A. thaliana (Peiter et al., 2005),
functioning as a dimer and encoding the conductance of the so-
called ‘slow vacuolar’ (SV) channel, which has been electrophys-
iologically characterized in detail (Hedrich & Marten, 2011;
Pottosin & Dobrovinskaya, 2017). This homologous channel
exists in plants, animals and protists (Hedrich & Marten, 2011;
Dadacz-Narloch et al., 2013; Patel & Cai, 2015). TPC1 channels
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Functioning of the channel’s subunit
confirmed by electrophysiological
tests in intact plant cells References
AtCNGC3, AtCNGC6, AtMSL9, Gobert et al. (2006);
AtMSL10, AtANN1 Peyronnet et al. (2008);
Gao et al. (2012);
Laohavisit et al. (2013)
AtCNGC2, AtCNGC5, AtCNGC6 Ali et al. (2007);
Wang et al. (2013)
AtCNGC18, AtGLR1.2 Michard et al. (2011);
Gao et al. (2016)
dominate vacuolar Ca2+ conductance (Pottosin et al., 2009) and its
relative expression determines the vacuolar Ca2+ storage capacity
(Gilliham et al., 2011). The transport of vacuolar Ca2+ to the
cytoplasm is tightly controlled to avoid a massive Ca2+ release that
would perturb metabolism and induce cell death.
Plant TPC1 is strictly selective for cations over anions and
conducts alkaline cations at a high rate (100–200 pS), but with little
preference: Na+ ~ Li+ ~ K+ > Rb+ > Cs+ (Pottosin & Dobrovin-
skaya, 2014; Guo et al., 2017). TPC1 also conducts divalent
cations as Ca2+ ~ Sr2+ ~ Ba2+ > Mg2+, with a relative permeability
higher than that for K+, but with a conductance an order of
magnitude lower (Johannes & Sanders, 1995; Pottosin et al., 2001;
Guo et al., 2017). The fact that SV channels generate large (several
pA) Mg2+ currents implies that Mg2+ is conducted through the
entire pore in a fully hydrated state (Pottosin & Sch€onknecht,
2007). Estimates of the cutoff diameter for the sugar beet SV
channel based on the permeability for Mg2+ and a block by different
quaternary ammonium ions yield a value between 7 and 8  A for the
SV channel diameter (Pottosin & Sch€onknecht, 2007). This is
consistent with the recently established crystal structure of
AtTPC1, which revealed an unusually short and wide (atom-to-
atom cross-distances of 8–9  A) selectivity filter (Guo et al., 2016;
Kintzer & Stroud, 2016). Human TPCs possess an Asn at a
position equivalent to 630 in Arabidopsis TPC1. This gives a rigid
constriction of 4.8 
A within the selectivity filter, resulting in a high
Na+ selectivity of the hTPC (cf. 2.4  A for an average centre-to-
centre Na-O distance in crystals; Guo et al., 2017). The introduc-
tion of Asn630 into AtTPC1 converts it to a channel with a high
selectivity for Na+ over K+ and decreases the relative Ca2+
permeability 10-fold (Guo et al., 2017). Animal TPCs, depending
on their mode of activation (by either an indirect effect of NAADP
or binding of PI(3,5)P2), appear either as a Ca2+-permeable NSCC
(Morgan et al., 2015; Pitt et al., 2016) or as Na+-selective channels
with low Ca2+ permeability (Guo et al., 2017; Lagostena et al.,
2017), respectively. AtTPC1 is not responsive to either PI(3,5)P2
or NAADP (Boccaccio et al., 2014).
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Table 3 Structural characteristics of Ca2+-transporting systems in Arabidopsis thaliana
Family of ion channels or
active transporters
Ca2+ influx systems
Cyclic nucleotide-gated
channels (CNGCs)
Ionotropic glutamate
receptors (GLRs)
Two-pore channel (TPC)
Mechanosensitive-like channels
(MSL channels)
Mid1-complementing activity
channels (MCA channels)
Reduced hyperosmolality-
induced [Ca2+] increase
1 channel (OSCA1)
Piezo channel
Annexins
Ca2+ extrusion systems
Ca2+-ATPases
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Number of Location in cell
genes in family membranes Structure of transmembrane domains Sites for binding of ligands and Ca2+
20 PM, VM Tetramers Up to three calmodulin-binding sites
Each subunit includes six transmembrane (CMBS)
domains Cyclic nucleotide binding site in
Each subunit has one pore region (between C-terminus
transmembrane domains 5 and 6); four
such regions form pore
Selectivity filters are specific to plants and
not examined
Voltage sensor
20 PM Tetramers Extracellular site of activation by
Three transmembrane domains (M1, M3 amino acids (LBD)
anmd M4) in each subunit Extracellular site for subunit inter
Each subunit has one pore region M2 connection (amino terminal
between transmembrane domains M1 domains: ATD)
and M3; four such regions form pore
Conservative NSCC-type selectivity filter
‘SYTANLAA’
Voltage sensor is not tested
1 VM Dimers Calmodulin-binding sites at
Twelve transmembrane domains in each cytosolic side;
subunit Requires Ca2+ binding to cytosolic
Each subunit has two pore regions between EF-hand for opening;
transmembrane domains 5 and 6, and 11 Modulation of voltage sensor by
and 12, respectively; pore regions from Ca2+, Mg2+, pH and other cations
each subunit form pore
Wide pore allowing movement of Ca2+ and
other cations
10 PM, EM (?) heptamers or pentamers (?)
Five (Group 1 and 2: MSL 1–8) or six (Group
3: MSL 9 and 10) transmembrane
domains
The pore is formed by fifth or sixth trans
membrane domains
Potential permeability to anions and large
organic molecules (permeability to Ca2+
is also proposed)
2 PM, VM (?) One transmembrane domain forming (?)
Ca2+-permeable pore – tetramer
Potential permeability to anions and Ca2+
Activation by membrane stretch
15 PM, EM (?) Eight or nine transmembrane domains (?)
Putative pore region between
transmembrane domains 8 and 9
1 PM Oligomer (?)
20–40 transmembrane domains
8 PM (?), EM (?) Peripheral plasma membrane and vesicle (?)
membrane proteins
Hypothetically insert to plasma membrane
to form a Ca2+-permeable water pore
Destabilize membrane bilayer
Regulate trafficking of Ca2+-permeable
channels
15 PM, EM Ten transmembrane domains Three cytosolic domains: actuator
Two classes: P2A-ATPase/ECA (four genes) (A), nucleotide-binding (N) and
and P2B-ATPase/ACA (11 genes) phosphorylation (P) domains,
1 : 1 Ca2+ to ATP stoichiometry respectively
N-terminus plays the role of Ca2+-
binding autoinhibitory domain
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Table 3 (Continued)
Family of ion channels or Number of Location in cell
active transporters genes in family membranes
Ca2+/H+ exchangers 6 PM, EM
PM, plasma membrane; VM, vacuole membrane; EM, endomembranes; (?), not established or require further research; NSCC, nonselective cation channels.
In the two-repeat six-transmembrane domain structure of A.
thaliana TPC1 (Fig. 2; Table 3), voltage-sensing domains (VSDs)
bearing the arrays of positively charged arginines are located in S4
(VSD1) and S10 helices (VSD2). However, the VSD1 is rigid in
space and only VSD2 moves in response to voltage change,
opening channels at cytosol-positive electric potentials (Guo
et al., 2016). In the animal TPC1, both VSDs are functional; the
neutralization of positive charges in any of them abolishes the
voltage dependence (Cang et al., 2014). A movement of the VSD2
in plant TPC1 is handicapped by vacuolar Ca2+ or Mg2+ binding
to the EF-like sites in the loop between S7 and S8, causing an
increase in the activation voltage (Dadacz-Narloch et al., 2011;
Kintzer & Stroud, 2016). Binding of luminal Ca2+ and/or
interaction between neighbouring binding sites are pH-
dependent and modulated by monovalent cations. The latter
‘buffers’ the TPC1 voltage-dependence against free luminal Ca2+
changes within a physiological range of concentrations (Pottosin
& Dobrovinskaya, 2017). Collectively, vacuolar cations strongly
impede opening of TPC1 at physiological potentials, so that only
a few of the thousands of TPC1s per vacuole may be open at
resting conditions (Pottosin et al., 2009).
A prerequisite for TPC1 opening is Ca2+ binding to the cytosolic
EF-hand (Fig. 2; Guo et al., 2016; Kintzer & Stroud, 2016). Thus,
successful opening of the TPC1 channel requires a concerted
movement of the cytosolic gates, coupled to VSD2 and EF2 in both
subunits (Kintzer & Stroud, 2016). The kinetic model explaining
the modulation of TPC1 gating by cytosolic Ca2+ and voltage in
detail is illustrated in Fig. 3. The fact that SV channels are activated
by cytosolic Ca2+ (Hedrich & Neher, 1987), in combination with
their high permeability to Ca2+, prompted the hypothesis that these
channels mediate Ca2+-induced Ca2+-release from the vacuole for
signalling needs (Ward & Schroeder, 1994). However, TPC1
channels cannot mediate a significant global Ca2+ release (Ranf
et al., 2008). TPC1 channels probably open locally within high
cytosolic Ca2+ microdomains where free Ca2+ reaches tens of lM
(Pottosin & Dobrovinskaya, 2017). Intriguingly, TPC1 channels
are essential components in long-distance Ca2+ signalling. To date,
their crucial role has been shown for systemic Ca2+ response to
herbivory in leaves (Kiep et al., 2015) and for rapid Ca2+ wave
propagation along the root in response to local NaCl application
(Choi et al., 2014). A rapid Ca2+ wave in the latter case also requires
a ROS-Ca2+ hub in the PM, in addition to TPC1 (Evans et al.,
2016). TPC1 channels have also recently been named as the most
likely oxygen sensors operating in plants under conditions of soil
flooding (Wang et al., 2017).
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Structure of transmembrane domains Sites for binding of ligands and Ca2+
Ten transmembrane domains
Cation-binding regions
Homomeric or heteromeric
dimers or trimers
Annexins These are cytosolic proteins that are capable of Ca2+-
dependent or Ca2+-independent association with membrane
phospholipids (Lizarbe et al., 2013; Konopka-Postupolska &
Clark, 2017) and are traditionally associated with the onset of
Ca2+-dependent apoptosis in animal cells. Eight putative genes
encoding annexins are found in Arabidopsis, and 25 and 11 genes
were detected in wheat and barley, respectively (Xu et al., 2016).
Plant annexins differ structurally from their animal counterparts,
consisting of repeated annexin domains with conserved endonexin
fold binding Ca2+ (Konopka-Postupolska & Clark, 2017). The
function of plant annexins is still far from understood, but some
studies show that they are involved in Ca2+ influx across the PM via
formation of redox-regulated Ca2+-permeable pores (Laohavisit
et al., 2009, 2013; Baucher et al., 2012).
Mechanosensitive channels Membrane tension response and
mechanical osmotic stimuli in plants can trigger Ca2+ signals via
mechanosensitive-like channels (MSLs; 10 genes in A. thaliana),
mechanosensitive ‘Mid1-complementing activity’ channels
(MCAs; two genes in A. thaliana) and mechanosensitive piezo
channels (one gene in A. thaliana) (Hamilton et al., 2015). The
cladistic analysis of plant MSL genes reveals the existence of four
clades (Fig. S5). Mitochondria- and chloroplast-based MSLs have five
transmembrane domains and hypothetically assemble in homohep-
tamers (similar to crystallized bacterial ‘mechanosensitive channel of
small conductance’). The fifth transmembrane domain plays the role
of a pore region, while the region encoding a voltage sensor is not
found in MSLs. Group 3 MSLs (MSL8, MSL9 and MSL10) are
localized to the PM and have six transmembrane domains, assembled
as tetramers (or multimers), with the sixth transmembrane domain
playing the role of the pore region (Hamilton & Haswell, 2017).
Compared with other Ca2+-permeable channels, MSLs demonstrate
higher unitary conductances, from 45 to 140 pS (Haswell et al., 2008)
and potentially higher preference to anions (Haswell et al., 2008;
Maksaev & Haswell, 2012). Calcium permeability of these channels
in vivo is debated.
Functions of MSLs include regulation of programmed cell death
(MSL10; Veley et al., 2014), water and ion balance in pollen tubes
(MSL8; Hamilton & Haswell, 2017), regulation of electrical
potential difference across the inner membrane of mitochondria
(MSL1; Lee et al., 2016), mechanical sensing in roots (MSL9 and
MSL10; Peyronnet et al., 2008) and chloroplast volume under
normal and stress conditions (MSL2 and MSL3; Veley et al., 2014),
along with other possible roles (Hamilton et al., 2015) (Table 1).
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(a) Plant TPC1 Subunits of MCA channels (MCA1 and MCA2) include one
transmembrane domain and assemble into a tetrameric complex
Vacuole
EF-like
to form a Ca2+-permeable pore near their N-terminus, oriented
P P to the apoplastic space (Shigematsu et al., 2014; Kamano et al.,
+
1 2 3
+
4 5 6 7 8 9
+
10 11 12
2015) (Fig. 2; Table 3). Similar to CNGCs, their activity could
+ +
+ be regulated by an increase in cytosolic Ca2+ via cytosolic EF
EF1 EF2 hand-like motifs. MCA1 and MCA2, when activated by
VSD1 VSD2 stretch, function as voltage-independent channels with unitary
(inactive) (active)
N C conductance resembling those found in NSCCs or K+-selective
Cytosol
Shakers (Furuichi et al., 2012; Kurusu et al., 2012; Hamilton
(b) (c) et al., 2015).
Ca2+ (µM)
1.0 2000
Closed Open
Relative open

Piezo channels In animal cells, the so-called ‘piezo channels’

probability

500
provide the function of sensing touch and light mechanical stimuli
0.5
100
through activation of Ca2+ currents. Piezo channels have 2000–
50 4000 amino acids assembled in 20–40 transmembrane domains
12.5 (Hamilton et al., 2015). The Arabidopsis genome contains just one
–100 –50 0 50 100
piezo channel gene. This might function as a Ca2+-transporting
V (mV)
(d) system, although experimental data supporting a function or ion
1.0 selectivity for plant piezo proteins are still missing.
Limit of voltage
activation

0.5 Other mechanosensitive channels: OSCA Yuan et al. (2014)

recently identified a unique hyperosmolality-induced [Ca2+]cyt.
0 channel 1 (OSCA1) which mediates elevation of [Ca2+]cyt. via
10 100 1000
activation of Ca2+ influx conductance. This PM Ca2+-transporting
(e) 100
protein contains one pore region between the eight and nine
Midpoint potential

transmembrane domains (Fig. 2). OSCA1-mediated Ca2+ currents

(tested in HEK293 cells) are very similar to those mediated by
(mV)

50
Depolarization VSD2
Cytosolic Ca2+ EF2 0
Cytosolic Ca2+ or Mg2+ EF1
10
Cytosolic Ca2+ (µM)
Fig. 3 Gating of two-pore channel 1 (TPC1) (‘slow vacuolar’, SV) channel by
membrane voltage and Ca2+. (a) A two-repeat six-transmembrane domain
subunit of the dimeric plant TPC1 channel. Pore regions (P) are indicated in
each domain, and channel opening is a result of the dilation of the cytosolic
part of the pore. This is governed by the movement of the voltage sensor
VSD2 in the second repeat and Ca2+ binding to the cytosolic EF-hand, which
is transmitted to the pore in the first repeat. Luminal Ca2+ binding to the
EF-like site handicaps the movement of the VSD2, increasing the voltage
activation threshold. (b) Kinetic model of the channel activation by cytosolic
Ca2+. TPC1 opening requires a simultaneous opening of two distinct gates in
each subunit (depicted here for a single subunit). Gate 2 is coupled to the
movement of VSD2 and is opened by a positive voltage. Gate 1 opening
requires Ca2+ binding to the cytosolic EF (EF2?) site. Binding of Ca2+ (or
Mg2+) to the cytosolic EF1 domain potentiates Ca2+ binding to the EF2
receptor site, decreasing the threshold for voltage activation and facilitating
overall channel opening. In the present scheme, this event corresponds to a
transition between four upper and four lower kinetic states. (c) Voltage
activation of the SV channel at different levels of a free cytosolic Ca2+. The
curve parameter values, obtained for SV channels from barley mesophyll
vacuoles (Pottosin et al., 1997), were used to construct the plots in (d) and
(e). (d) The model predicts that, at saturating positive voltages, the maximal
fraction of open channels is limited by cytosolic Ca2+ binding (with an
apparent Kd = 55 lM). (e) A negative shift of the voltage dependence upon
an increase in cytosolic Ca2+. The solid line is the best fit of midpoint potential
values as a function of cytosolic Ca2+, highlighting that Ca2+ binding to the
modulatory site (EF1) cooperatively (15-fold) increases Ca2+ binding affinity
for the receptor site (EF2). Error bars are  SD.
constitutive voltage-independent NSCCs. A closely related A.
thaliana calcium-permeable stress-gated cation channel 1
(AtCSC1) probably forms hyperosmolality-activated Ca2+-
100 1000
permeable channels in the PM, which is self-regulated (closed) by
inflowing Ca2+ (Hou et al., 2014). OSCAs are a gene family
comprising 15 genes in A. thaliana with homologues existing in all
eukaryote kingdoms.
3. The hypothesis of a ROS-Ca2+ hub
An elevation in the [Ca2+]cyt induces an increase in the NADPH
oxidase-mediated production of ROS (O2 + e ? O2•
). Vice versa,
extracellular ROS (O2•
+ e ? H2O2 + e ? HO•) activate Ca2+
influx through Ca2+-permeable ion channels (Demidchik et al.,
2003, 2009, 2015; Foreman et al., 2003; Choi et al., 2017) (Fig. 4).
This supports the self-amplification mechanism, which increases the
duration of both Ca2+ and ROS signals and amplifies an initially
weak signal into a dramatic O2•–-Ca2+ increase (Demidchik et al.,
2007, 2009). Hypothetically, this ‘ROS-Ca2+ hub’ can be stimu-
lated by the CDF of CNGCs and phosphorylation of both the
channels and NADPH oxidase (Demidchik & Shabala, 2018).
Down-regulation of the ROS-Ca2+ hub may involve the CDI of
CNGCs (Ben-Johny et al., 2015; DeFalco et al., 2016b), induction
of Ca2+-ATPase through Ca2+-CaM binding to CaMBS1 and
CaMBS2 (Tidow et al., 2012), suppression of Rac/Rop GTPases
(Baxter-Burrell et al., 2002) and CDPK-catalysed phosphorylation
of ion channels (Zhou et al., 2014) or BIK1-catalysed phosphory-
lation of NADPH oxidase (Kadota et al., 2015).

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Class III Reducing agents

peroxidases (ascorbic acid, etc.)
Reduced transition
metals (Cu+, Fe2+)
Apoplast H2O2
Developmental e–
and stress stimuli HO•
Ca2+ Temperature Ca2+ O2• –

Flu
id NADPH
it y oxidase 10–3 M
CN
EF EF P
P EF 10–7 M
Receptor kinases Ca2+-influx channel
(some act as CNGC, GLR, MSL
CDPK
guanylate cyclases) MCA, OSCA, Piezo
Activation of channels via Ca2+ e–
?
receptor kinase-mediated
ic a ti o n
pathway A m p lif
Annexin
TPC1 Cytosol
Insertion to membrane, Ca2+-dependent
EF EF
membrane destabilization activation?
or increase of vesicle traffic

Vacuole Ca2+ 10–3 M

Fig. 4 The hypothetical scheme of the reactive oxygen species (ROS)-Ca2+ hub (‘ROS-Ca2+’) (adapted with changes from Demidchik & Shabala, 2018).
Calcium-permeable channels, which could be encoded by different genes, are stimulated by ROS produced by NADPH oxidases, activated by cytosolic Ca2+.
This system forms a positive feedback mechanism with a hypothetical self-amplification loop that can intensify weak Ca2+ or ROS signals. Class III peroxidases,
other ROS-generating systems and transition metal-reducing agents can also contribute to the functioning of the ROS-Ca2+ hub. Calcium can also be released
from the vacuole via TPC1. A multitude of stimuli act on Ca2+-permeable channels and NADPH oxidases via receptor kinase- and Ca2+-dependent protein
kinases pathways. See text for further details. CNGC, cyclic nucleotide-gated channel; GLR, glutamate receptor; MSL, mechanosensitive-like channel; MCA,
mechanosensitive ‘Mid1-complementing activity’ channel; OSCA, hyperosmolality-induced [Ca2+]cyt. channel.

The first established physiological role of the ROS-Ca2+ hub was
Ca2+ loading for the polar elongation of root hairs and root
elongation zone (Demidchik et al., 2003; Foreman et al., 2003).
Later, a similar mechanism was detected in growing pollen tubes
(Potock
y et al., 2007). In this case, the [Ca2+]cyt. in the growing part
of the cell, such as the tips of root hairs or pollen tubes, is
maintained by the ‘ROS-Ca2+ hub’ at a constantly high value (up to
1 lM), stimulating formation of longitudinal cytoskeleton bundles
and tip-directed exocytosis (Rounds & Bezanilla, 2013). Other
pairs of ion channel – NADPH oxidase (RBOHs – ‘respiratory
burst oxidases’) and their potential roles are summarized in Table 4.
Evans et al. (2016) recently found that, as well as PM Ca2+-
permeable channels, vacuolar TPC1-mediated Ca2+ release can
additionally stimulate the NADPH oxidase-mediated ROS-Ca2+
hubs (Fig. 4). Recent data demonstrate that ROS generation and
Ca2+ influx are also involved in hormonal signal transduction,
responses to stresses, osmoregulation, programmed cell death,
mineral uptake and long-distance signalling (summarized by Choi
et al., 2017; Shabala et al., 2016; Demidchik & Shabala, 2018;
Demidchik et al., 2018).
III. Ca2+ extrusion systems
When external Ca2+ activity increases by several orders of
magnitude, the cytosolic basal [Ca2+]cyt increases several-fold
(Demidchik et al., 2002a). Elevations of [Ca2+]cyt up to 0.5–10 lM
are typical for plant cell responses to phytohormones and
environmental cues. However, the basal [Ca2+]cyt concentration
recovers within a few minutes, even in the presence of these stimuli.
This recovery is mediated by Ca2+ extrusion and sequestration
mechanisms and requires energy-consuming Ca2+ transporters that
function against the electrochemical gradient.
Two types of active Ca2+ transport systems are found in plants:
the P-type Ca2+-ATPases, which rely on the hydrolysis of ATP and
use the energy released in this process (Bonza & De Michelis, 2011;
Huda et al., 2013a,b; Fig. 5); and the Ca2+/H+ exchangers of the
CAX family (calcium/cation exchangers), which derive energy
from the electrochemical gradient of protons across membranes
facing the cytosol (Emery et al., 2012; Pittman & Hirschi, 2016).
The ATPase-mediated mechanism has low capacity but high
affinity for Ca2+ (Km = 0.01–2 lM), while the exchanger-mediated
mechanism has low affinity and high capacity (Km = 2–10 lM)
(Tidow et al., 2012; Yadav et al., 2015). Together, they function
over a wide range of possible physiological cytosolic Ca2+ activities
(from 10
8 to 10
4 M).
1. Ca2+-ATPases
Classification and structure of plant Ca2+-ATPases Two types of
Ca2+-ATPase operate in plants (Geisler et al., 2000; Sze et al.,
2000) and belong to either the P2A- or P2B- group (Fig. S6). The
P2A-type Ca2+
ATPases are also known as ER-type Ca2+-ATPase
(ECA) and are located mostly on the endomembranes (Huda et al.,
2013a,b). In addition, according to our cladistic analysis, ECAs can
be further divided into three different subclades (Fig. S6). The
P2B-ATPases are known as autoinhibited Ca2+-ATPase (ACA type)
and are located at both the endomembranes and the PM (Bonza &
De Michelis, 2011; Huda et al., 2013a,b). It should be noted that

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Table 4 Physiological functions that involve plasma membrane Ca2+-permeable cation channels and NADPH oxidases and potentially rely on self-amplifying
reactive oxygen species (ROS) and Ca2+ signals mediated by ROS-Ca2+ hubs

Function Plasma membrane Ca2+-permeable channel NADPH oxidase References

Root cell growth AtCNGC3 AtRBOHC Foreman et al. (2003); Gobert et al.
(2006)
Pollen tube growth AtCNGC18, AtGLR1.2, AtGLR3.7 AtRBOHH, AtRBOHJ Michard et al. (2011); Kaya et al.
(2014); Guo et al. (2016)
ABA signalling AtCNGC5, AtCNGC6 AtRBOHD, AtRBOHF Mori & Schroeder (2004); Wang
et al. (2013)
Brassinosteroid signalling DACC NbRBOHB Kolupaev et al. (2014); Straltsova
et al. (2015); Deng et al. (2016)
Auxin signalling AtCNGC14 AtRBOHD Peer et al. (2013); Shih et al. (2015);
Nguyen et al. (2016)
Methyl jasmonate signalling AtCNGC2 AtRBOHD, AtRBOHF Maruta et al. (2011); Lu et al.
(2016)
Salicylic acid AtGLR3.3 AtRBOHD Kawano et al. (1998); Manzoor
et al. (2013); Jayakannan et al.
(2015)
Signalling by exogenous ATP and ADP VICCs, HACCs AtRBOHC Demidchik et al. (2009, 2011)
Hypersensitive response (massive PCD AtCNGC2, AtCNGC4, AtCNGC11, AtCNGC12 AtRBOHD, AtRBOHF Torres et al. (2002); Moeder et al.
around the spot of infection, preventing (2011); Dubiella et al. (2013)
spread of the disease)
Drought-induced stomata closure AtCNGC5, AtCNGC6 AtRBOHD, AtRBOHF Wang et al. (2013)
Response to salt stress (NaCl) AtCNGC10, Mechanosensitive channels NtRBOHD, NtRBOHF Guo et al. (2008, 2010); G
emes
et al. (2016)
Response to extreme temperatures AtCNGC6 AtRBOHD, AtRBOHB Larkindale et al. (2005); Gao et al.
(2012)

PCD, programmed cell death.

this classification is slightly vague; there is evidence that ECAs are
also localized to the tonoplast and PM (Wimmers et al., 1992;
Ferrol & Bennett, 1996). In this context, it is probably prudent to
focus on the dominant location.
Ca2+-ATPases are single polypeptides with 10 transmembrane
domains (1000–1100 amino acids; 110–125 kDa; Fig. 5; Table 3).
The first six segments (TM1–TM6) form the transport
domain (T-domain) while the remaining four form a support
domain (S-domain), which provides structural support to the
T-domain and coordinates ion binding (Kabala & Klobus, 2005;
Bonza & De Michelis, 2011). Located in the cytosol are the actuator
domain (A), nucleotide-binding domain (N) and phosphorylation
domain (P). The N-domain has a highly conserved KGAPE
sequence and interacts with ATPase via its adenosine part. The N-
domain resides within the P-domain, which catalyses the pump
operation by phosphorylation of the Asp residue within the
DKTGT motif. The A-domain has a highly conserved TGES motif
that accounts for an intrinsic protein phosphatase involved in
dephosphorylation of the P-domain during each catalytic cycle. The
major structural difference between the ECA and ACA pumps is the
presence of the autoinhibitory domain at the N-terminus in ACA.
Operation and regulation of plant Ca2+-ATPases Plant Ca2+
pumps belong to the P-type superfamily of ATPases and are thus
energized by ATP hydrolysis by binding the c-phosphate of ATP to
the aspartate residue within the DKTGT motif of the P-domain
(Palmgren & Harper, 1999). This enzyme has two conformations
known as the E1 and E2 states. The former has high affinity to Ca2+
and binds it at the cytosolic side of the membrane. Following ATP
hydrolysis and phosphorylation, the pump changes its confor-
mation to the E2 state, with has a much lower affinity to Ca2+ and
has an ion binding site located on the opposite (to cytosol) side
(Kabala & Klobus, 2005). As a result, Ca2+ dissociates from the
protein on the outer side of membrane. In general, the ATPase-
mediated mechanism is considered to be of a low capacity, with a
high affinity for Ca2+ (Km = 0.01–2 lM; Huda et al., 2013a,b),
and acts with a 1 : 1 Ca2+ to ATP stoichiometry (Lopreiato et al.,
2014).
Autoinhibited Ca2+-ATPase and ECA have different affinities
for Ca2+ (micromolar and submicromolar ranges, respectively;
Bonza et al., 2001; Meneghelli et al., 2008) and different specifici-
ties for divalent cations (Bonza & De Michelis, 2011). ACA is
highly selective and transports only Ca2+, whereas ECA may
additionally mediate uphill transport of Mn2+, Cd2+ and Zn2+
(Huda et al., 2013a,b).
The characteristic feature of the P2B ACA-type Ca2+ ATPase is
the presence of a CaM-binding domain at the N-terminal. This
domain contains two nonoverlapping CaM-binding sites (termed
CaMBS 1 and 2) that partially overlap with the autoinhibitory
domain (Baekgaard et al., 2006). In the absence of bound Ca2+, the
interaction between the autoinhibitory domain and A, N and P
domains keeps the enzyme in what is known as an ‘autoinhibited
state’. Binding the Ca2+–CaM complex to CaMBS1 partially
activates the A and N domains and enables Ca2+ transport, albeit at
a relatively low rate. This state is referred as the ‘basal activated state’
(Tidow et al., 2012). When both CaMBS1 and CaMBS2 are
occupied, a full displacement of the autoinhibitory domain from
the A, N and P domains occurs. This results in maximal Ca2+-

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New
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pollination and compatible pollen coat treatment (Iwano et al.,

(a) Ca2+–ATPase (ACA)
2014), suggesting that stigmatic ACA13 functions in the export of
Ca2+ to the compatible pollen tube, promoting successful fertil-
ization. Another P2B-type isoform, ACA7, is expressed exclusively
in developing pollen grains and is required for normal pollen
development (Lucca & Leon, 2012). Indeed, significant negative
1 2 3 4 5 6 7 8 9 10
correlations between Ca2+-ATPase activity and pollen germination
N exist (An et al., 2016). AtACA10 regulates a vegetative phase
KGAPE
AD development and inflorescence architecture in Arabidopsis (Huda
et al., 2013a,b), while ACA8 plays a role in sucrose signalling
DTKGT during early seedling development (Zhang et al., 2014). CrACA1
N TGES Cytosol C
P was shown to be an important component of the gravity-sensing
A
mechanism in spores of the fern Ceratopteris richardii (Bushart
et al., 2014).
(b) Ca2+/H+ exchangers (CAX) P2A-type Ca2+ ATPases also contribute to pathogen-induced cell
C death (Zhu et al., 2010). A double knockout mutation of the
CaD vacuolar Ca2+ pumps ACA4 and ACA11 in A. thaliana results in a
high frequency of hypersensitive response-like lesions, suggesting
that these pumps are an essential component of the salicylic acid-
1 2 3 4 5 6 7 8 9 10 11 dependent programmed cell death pathway (Boursiac et al., 2010).
Another study by Frei dit Frey et al. (2012) revealed that ACA8 and
ACA10 interact with membrane-resident receptor kinases and
AD MnD represent an important component of receptor-mediated signalling
SOS2 Acidic motif in plant immune responses and development. These findings were
Cytosol
N recently confirmed by Yang et al. (2017), who showed that the
evolutionarily conserved protein BONZAI1 interacts with ACA10
and ACA8 to function in the generation of cytosolic calcium
signatures that are critical for stomatal movement and associated
Fig. 5 A schematic representation of the membrane topology of Ca2+ efflux
systems. (a) Ca2+-ATPases (ACA). The difference between endoplasmic
plant immunity. Finally, various ACA and ACA isoforms were
reticulum (ER)-type Ca2+-ATPase (ECA) and ACA lies in the presence of an shown to be central to plant adaptive responses to a broad range of
autoinhibitory domain (a red box labelled AD) at the N-terminus in the latter. abiotic stresses (see Table S1).
TM1–TM6 form the transport domain (T-domain), while the remaining four
form a support domain (S-domain). Located in the cytosol are the actuator
domain (A), nucleotide-binding domain (N) and phosphorylation domain 2. Ca2+/H+ exchangers (CAXs)
(P), each bearing a highly conserved motif. Based on Huda et al. (2013a,b),
with modifications. (b) Typical structure of a Ca2+/H+ exchanger (CAX). CAX Classification and structure of CAXs These transport proteins
possess an N-terminal autoinhibitory domain (AD) and several specific belong to the multigene family of cation/H+ exchangers (CAXs)
cation-binding regions that determine CAX selectivity. CaD, Ca-specific (Shigaki et al., 2006; Martinoia et al., 2007; Pittman & Hirschi,
binding domain; MnD, Mn-specific binding domain. Based on Shigaki & 2016). The CAXs have much lower transport affinities for
Hirschi (2006) and Manohar et al. (2011), with modifications.
Ca2+ than do Ca2+-ATPase pumps. Typically, each plant species
contains between four and 14 CAX genes (Manohar et al., 2011;
Pittman & Hirschi, 2016). In the Arabidopsis genome, six CAX
ATPase activity (‘fully activated state’) with a high rate of Ca2+ genes (AtCAX1–AtCAX6) have been identified (Manohar et al.,
extrusion (Tidow et al., 2012). 2011). In addition, five further transporters belonging to two
In stark contrast to ACA, very little is known about ECA phylogenetic clades and termed the Ca2+/cation antiporter super-
regulation. The observation that a peptide corresponding to an family, mediate active Ca2+ efflux from the cytosol in Arabidopsis
autoinhibitory region of ACA blocked the Ca2+ pumping activity (Manohar et al., 2011) (Fig. S7). These transporters were previ-
of ECA (Hwang et al., 2000) led to suggestions that ECA activity ously defined as AtCAX7–AtCAX11 and encode K+-dependent
may be regulated by a reverse interaction with some regulatory Na+/Ca2+ exchangers (Maser et al., 2001; Shigaki et al., 2006). The
protein (Sze et al., 2000). Explicit proof for this hypothesis is as yet list is growing; recently a novel chloroplast-localized potential
unavailable. Ca2+/H+ antiporter (CCHA1) from the UPF0016 calcium-proton
exchanger family was discovered (Wang et al., 2016a,b).
Physiological roles of plant Ca2+-ATPases A large number of Cation/H+ exchangers have 11 transmembrane helices (Fig. 5).
studies have implemented Ca2+-ATPases in a broad range of The subunits assemble into either homomeric or heteromeric
developmental and adaptive responses. A decline in Ca2+-ATPase dimers or trimers (Pittman & Hirschi, 2016). CAXs possess an
activity was shown to be causally linked to leaf senescence (Yang N-terminal autoinhibitory domain and a highly conserved cation-
et al., 2013). In Brassica, ACA13 was induced after both compatible binding region, with their transporter specificity being controlled

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16 Review Tansley review
by the nine-amino-acid region between transmembrane 1 (TM1)
and TM2 (Shigaki & Hirschi, 2006; Manohar et al., 2011). The
primary amino acid sequences in the transmembrane regions are
highly conserved in all plant CAXs, and the majority of the
sequence diversity of CAXs exists in the loop and tail regions, which
most likely explains their different gating properties (Manohar
et al., 2011).
Operation and regulation of CAXs These proteins possess two
cation-binding sites, termed a1- and a2-repeat regions, which are
located within transmembrane helixes 2–3 and 7–8, respectively
(Fig. 4; Table 3). It was shown that Ca2+ and H+ binding within
these repeat regions are mutually exclusive (Nishizawa et al., 2013;
Waight et al., 2013), suggesting the existence of a ‘one-H+-in, one-
Ca2+-out’ exchange mechanism (Pittman & Hirschi, 2016). The
protein structure is reset by H+ binding. Autoinhibition of CAX
proteins is caused by the N-terminus physically interacting with a
neighbouring N-terminal region (Manohar et al., 2011). Modu-
lation of CAX activities could occur by phosphorylation, changes of
pH and reaction to regulatory proteins such as the serine/threonine
kinase SOS2 with the CAX N-terminal domain (Demidchik &
Shabala, 2018).
Physiological roles of CAXs Similar to Ca2+-ATPase, CAXs are
critical for Ca2+-mediated phenomena in plants (Pittman &
Hirschi, 2016). Owing to their important role in regulating both
intracellular and apoplastic pH (Cho et al., 2012), CAXs are
instrumental in a broad range of developmental processes. Both
CAX expression levels and activity are highly cell type-specific
(Punshon et al., 2012; Wang et al., 2016a,b) and modulated by
various stresses, particularly by heavy metals. Some selected
examples depicting the role of specific CAX isoforms in stress-
induced adaptive responses are given in Table S2. Of specific
importance is the role of CAXs in cell-specific calcium storage and
defence responses (Conn et al., 2011; Hocking et al., 2017).
IV. Concluding remarks
Significant progress in our understanding of membrane calcium
transport has been achieved over the last decade. Most Ca2+-
permeable channels have been cloned and electrophysiologically
tested in heterologous expression systems. A putative pore sequence
harbouring Ca2+ selectivity has been proposed. Some of the
CNGCs demonstrate high Ca2+ selectivity and thus are likely to act
as plant ‘calcium channels’, although this remains to be confirmed
by experimentation on intact plants.
Significant progress has also been achieved in understanding the
structure and operation of the TPC1 channel. This channel has a
uniquely wide pore, allowing the movement of hydrated divalent
cations. It is likely that TPC1 is an Mg2+/Ca2+-permeable channel
mediating Mg2+ redistribution within a cell and Ca2+ flux to the
cytosol. Further roles of TPC1 in Mg2+ balance and nutrition need
to be elucidated.
ANNEXIN1, MCA1, MCA2 and OSCA1 have also been
reported as Ca2+-permeable channels. Plant annexins appear to be
different from their animal counterparts; the plant type can
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probably be inserted into the PM, thus forming a channel that
conducts Ca2+ currents. Advances in understanding the
mechanosensitive channels have allowed the elucidation of their
substantial roles not only in perception of mechanical stimuli, but
also in programmed cell death, responses to salt stress, regulation of
organelle shape and ROS sensing. It also appears that more genes of
Ca2+-permeable mechanosensitive channels, such as OSCA1, exist
in plants, prompting further studies in this field.
Plasma membrane Ca2+-permeable channels interact with Ca2+-
activated NADPH oxidase to form a self-amplifying system: a
ROS-Ca2+ hub. This system could provide the transduction and
amplification of the initial Ca2+ or ROS stimuli into a more
sustainable response, with implications for cell growth, hormonal
signalling and stress responses.
Ca2+-extruding systems, the Ca2+-ATPases and Ca2+/H+ exchang-
ers, operate in concert with Ca2+-permeable channels to form a finely
tuned mechanism for Ca2+ removal from the cytosol. The crystal
structure of the Ca2+-ATPase autoinhibitory domain demonstrates a
biphasic activity of this protein in Ca2+ extrusion, which increases
with [Ca2+]cyt., thus explaining its action in signalling cascades. The
role of other putative calcium exchangers, such as the calcium-
sodium like exchanger (NCL; Wang et al., 2012; Li et al., 2016), is
also emerging but requires more detailed research.
A large number of studies have been conducted using KO
mutants and overexpressing lines of Ca2+-transporting systems,
revealing critical roles of calcium transport systems in intracellular
signalling, Ca2+ and Mg2+ nutrition, elongation growth, cytoskele-
ton regulation, biotic and abiotic stress responses, programmed cell
death, gravity sensing, ROS, hormones, temperature changes,
mechanical stimuli, control of stomatal closure and photosynthesis.
All this makes the Ca2+-transporting machinery a highly attractive
target for genetic improvement of plants for environmental fitness.
However, the large number of Ca2+ transporting systems and the
complexity of their regulation make the practical task of repro-
gramming stress resilience and control over plant development and
productivity extremely challenging. Further progress in this
direction may therefore only be achieved by comprehensive
functional studies. This will reveal the role of a particular Ca2+-
transporter encoding gene(s) in specific developmental and/or
stress responses. When complemented by the full knowledge of the
molecular structure of such a transporter, this will enable it to be
edited for optimal performance.
Acknowledgements
This study was supported by the Russian Science Foundation grant
no. 15-14-30008 to V.D.
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Supporting Information
Additional supporting information may be found online in the
Supporting Information section at the end of the article
Fig. S1 Phylogenetic relationships of CNGCs.
Fig. S2 Evolutional phylogeny of CNGCs.
Fig. S3 Phylogenetic relationships of GLRs.
Fig. S4 Evolutional phylogeny of GLRs.
Fig. S5 Phylogenetic relationships of plant MscS-like (MSL)
channels.
Fig. S6 Phylogenetic relationships of plant Ca2+-ATPases.
Fig. S7 Phylogenetic relationships of plant Ca2+/H+ exchangers
(CAXs).
Table S1 Selected examples of Ca2+-ATPase involvement in abiotic
stress responses in plants
Table S2 Selected examples of involvement of CAX Ca2+/H+
exchangers in abiotic stress responses in plants
Please note: Wiley Blackwell are not responsible for the content or
functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
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