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A heat-activated calcium-permeable channel – Arabidopsiscyclic nucleotide-gated ion channel 6 – is involved in heatshock responses
Fei Gao1,2,3,†, Xiaowei Han1,†, Jianhai Wu1,†, Shuzhi Zheng1, Zhonglin Shang1, Daye Sun1, Rengang Zhou2,* and Bing Li1,*
1Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Science, Hebei Normal University, Shijiazhuang
050024, China,2Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, China, and3State Key Laboratory of Plant Cell and Chromosomal Engineering, Institute of Genetics and Developmental Biology, Chinese
Academy of Sciences, Beijing 100101, China
Received 25 June 2011; revised 13 February 2012; accepted 21 February 2012.*For correspondence (e-mail lbwxc@yahoo.com.cn or rengangzhou@yahoo.com.cn).†These authors contributed equally to this work.
SUMMARY
An increased concentration of cytosolic calcium ions (Ca2+) is an early response by plant cells to heat shock.
However, the molecular mechanism underlying the heat-induced initial Ca2+ response in plants is unclear. In
this study, we identified and characterized a heat-activated Ca2+-permeable channel in the plasma membrane
of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole-cell patch-clamp
technique. The results indicated that A. thaliana cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-
induced Ca2+ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of
thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana,
and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of
cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that
exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of
heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat-shocked plant
with activation of the Ca2+ channels and downstream expression of HSP genes sheds some light on how plants
transduce a heat stimulus into a signal cascade that leads to a heat shock response.
Keywords: Ca2+-permeable channel, CNGC6, heat shock response, thermotolerance, cAMP, Arabidopsis.
INTRODUCTION
High-temperature stress (a sudden increase in temperature)
disturbs cellular homeostasis and may result in a severe
retardation in growth and development, and possibly death
(Kotak et al., 2007; Timperio et al., 2008). Even small tem-
perature increments can induce a heat shock response
(HSR). The HSR is characterized by rapid reprogramming of
gene expression, leading to a transient accumulation of heat
shock proteins (HSPs) that is correlated with enhanced
thermotolerance (Vierling, 1991; Queitsch et al., 2000; Hong
and Vierling, 2001; Sanmiya et al., 2004; Miroshnichenko
et al., 2005; Charng et al., 2006; Li et al., 2007). HSP101,
HSP90, HSP70, HSP60 and HSP40 and some small molecular
HSPs are among the most abundantly expressed HSPs
(Vierling, 1991). It is thought that their biological role is to
allow cellular proteins to avoid and/or recover from stress-
induced protein aggregation (Miernyk, 1999; Nollen and
Morimoto, 2002; Mayer and Bukau, 2005).
The calcium ion (Ca2+) is probably the most versatile ion
found in eukaryotic organisms, and participates in nearly all
aspects of plant growth and development (Kudla et al.,
2010). It is known that heat shock (HS) induces a transient
increase in [Ca2+]cyt, the concentration of free Ca2+ in the
cytosol of plants (Biyaseheva et al., 1993; Gong et al., 1998;
Liu et al., 2003, 2006), and that the Ca2+–CaM (calmodulin)
complex regulates the activity of HS transcription factors,
expression of HSP genes and thermotolerance in plants (Liu
et al., 2003, 2005, 2008; Li et al., 2004; Zhang et al., 2009).
This evidence suggests that Ca2+ is involved in the HS signal
transduction pathway. However, the molecular mechanism
behind the heat-induced increase in [Ca2+]cyt in plants is
ª 2012 The Authors 1The Plant Journal ª 2012 Blackwell Publishing Ltd
The Plant Journal (2012) doi: 10.1111/j.1365-313X.2012.04969.x
unclear. It is well known that the major sources of increased
cytosolic free Ca2+ are intracellular Ca2+ pools and extra-
cellular Ca2+ stores. Gong et al. (1998) used inhibitors to
show that, in Nicotiana tabacum, HS mobilizes cytosolic
Ca2+ from both intracellular and extracellular sources. Liu
et al. (2006) used a pharmacological approach to show that,
in Arabidopsis thaliana, phospholipase C (PLC)/inositol
1,4,5-trisphosphate (IP3) mediates the heat-induced increase
in [Ca2+]cyt, and is involved in HSR. Recently, Zheng et al.
(2012) used reverse genetic analysis to show that PLC9 is
involved in the heat-induced increase in [Ca2+]cyt. In addi-
tion, Saidi et al. (2009) showed that a putative specific Ca2+-
permeable channel in the plasma membrane (PM) is
involved in a heat-induced increase in [Ca2+]cyt and regulates
the HSR in the moss Physcomitrella patens. The question
has been raised as to whether the PM Ca2+-permeable
channel contributes to the heat-induced increase in [Ca2+]cyt
in higher plants. A hyperpolarization-activated, voltage-
dependent Ca2+-permeable channel has been detected in
higher plants by patch-clamp analysis (Pei et al., 2000; Very
and Davies, 2000; Demidchik et al., 2002; Shang et al., 2005;
Qu et al., 2007; Wu et al., 2007). However, no product of a
heat-sensitive PM Ca2+-permeable channel gene has been
identified in plants. The aim of the present study was to
identify plant Ca2+-permeable channels that are sensitive to
heat.
Twenty of the 57 cation channel genes in the A. thaliana
genome encode cyclic nucleotide-gated cation channels
(CNGCs) (Ma et al., 2006). The CNGC proteins contain six
putative transmembrane domains and a binding site for
CaM that overlaps with a C-terminal binding site for cyclic
nucleotide monophosphate (cNMP) (Kohler et al., 1999).
CNGCs are non-selective inward cation channels that medi-
ate transport of cations, including Ca2+, during essential
plant physiological processes (Leng et al., 1999, 2002; White
et al., 2002; Balague et al., 2003; Hua et al., 2003; Lemtiri-
Chlieh and Berkowitz, 2004; Gobert et al., 2006; Ali et al.,
2007; Urquhart et al., 2007; Kugler et al., 2009; Ma et al.,
2009; Guo et al., 2010). A. thaliana CNGC proteins have been
divided into five sub-groups (White et al., 2002), and appear
to be differentially expressed between tissues (Talke et al.,
2003). Involvement in the response to pathogens is the most
obvious physiological role of A. thaliana CNGCs. Reverse
genetic studies showed that CNGC2, CNGC 4 and CNGC11/
12 chimeras produce a ‘defense, no death’ phenotype
(Clough et al., 2000; Balague et al., 2003; Jurkowski et al.,
2004; Yoshioka et al., 2006; Ali et al., 2007; Urquhart et al.,
2007). However, the function of CNGCs appears not to be
restricted to biotic stress. It is known that CNGC1, 2, 7, 8,
10–12, 16 and 18 play roles in plant growth and development
processes (Chan et al., 2003; Bock et al., 2006; Ma et al.,
2006; Borsics et al., 2007; Frietsch et al., 2007; Chaiwongsar
et al., 2009; Urquhart et al., 2011). CNGC3, 10, 19 and 20
have been implicated in regulation of salt-stress adaptation
(Gobert et al., 2006; Guo et al., 2008; Kugler et al., 2009), and
CNGC1 and 2 are involved in plant adaptation to heavy metal
toxicity or high calcium stress (Sunkar et al., 2000; Chan
et al., 2003). It is still not known whether CNGCs, as Ca2+
channels, participate in the adaptation of plants to heat
stress.
CNGCs are activated by the binding of cNMP (cAMP and/
or cGMP) (Kaplan et al., 2007; Kudla et al., 2010). In animals
and microbes, cAMP is an important second messenger in a
wide range of physiological processes (Kaplan et al., 2007),
but the existence of cAMP in plants has been intensely
debated for decades. However, over the past 10 years, cAMP
has been unequivocally shown to participate in various plant
developmental processes (Jin and Wu, 1999; Moutinho
et al., 2001; Ohmori and Okamoto, 2004; Witters et al.,
2004), and there is ample evidence that cNMPs are involved
in controlling ion homeostasis in plants. Kurosaki and Nishi
(1993) and Volotovski et al. (1998) showed that stimulation
of Ca2+ influx and the Ca2+ cascade are regulated by cAMP in
cultured carrot (Daucus carota) cells and tobacco (Nicotiana
plumbaginofolia) protoplasts . Using single-channel patch-
clamp analysis, Lemtiri-Chlieh and Berkowitz (2004) showed
that the opening of Ca2+-permeable channels is triggered by
cNMPs in A. thaliana leaf guard cells and mesophyll cells. In
addition, cAMP was found to mediate responses to patho-
gens (Delledonne et al., 1998; Ali et al., 2007; Ma et al., 2009)
and protect plants against salt stress (Maathuis and Sanders,
2001). However, the role of cAMP in the plant HS signal
transduction pathway is still unclear.
In this study, using reverse genetic analysis and the
whole-cell patch-clamp technique, a member of A. thaliana
CNGC family, CNGC6, was identified and characterized.
Furthermore, we show that CNGC6 is a heat- and cAMP-
activated PM Ca2+-permeable channel that is involved in
HSRs. In addition, the role of cAMP in the HS signal
transduction pathway in A. thaliana was determined. Taken
together, the data presented here shed light of how plants
transduce a heat stimulus into a signal cascade, leading to
an HSR.
RESULTS
Characterization of Ca2+-permeable channels in the PM of
A. thaliana root protoplasts by patch-clamp analysis
The whole-cell patch-clamp technique was used to examine
the activity of Ca2+-permeable channels in the PM of
A. thaliana root protoplasts. Whole-cell voltage clamp
recordings from root protoplasts (12–14 lm) exposed to
basal bath solution (see Experimental Procedures) showed a
large, voltage-dependent, inward-rectified current ()172 pA
at )200 mV) at hyperpolarized potentials (negative poten-
tials >)100 mV) (Figure 1a,e). However, when AlCl3, GdCl3or LaCl3, which block plant Ca2+-conducting channels
(Lemtiri-Chlieh and Berkowitz, 2004; Wu et al., 2007), were
2 Fei Gao et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
added to the bath solution (final concentration 50 lM), the
current was severely inhibited (Figure 1b–d), and the mean
current intensity (Imean) at )200 mV was just )37 pA for
AlCl3, )28 pA for GdCl3 and )45 pA for LaCl3 (Figure 1e).
Based on the ion gradient between the pipette solution (see
Experimental Procedures) and the basal bath solution, the
Nernst equilibrium potential was +151 mV for Ca2+ (ECa) and
)78 mV for Cl) (ECl). Tail current analysis showed that the
reversal potential (Erev) for currents recorded in the patch
configuration was approximately +32 mV (Figure 1f,g),
close to ECa but not ECl, and in good agreement with the Erev
(ranging from +10 to +40 mV) for hyperpolarization-acti-
vated Ca2+ channels in various plant cells (Very and Davies,
2000; Demidchik et al., 2002; Shang et al., 2005; Qu et al.,
2007; Wu et al., 2007). Furthermore, the effects of the K+
channel blockers barium (Ba2+), cesium (Cs+) and tetra-
ethylammonium chloride (White and Lemtiri-Chlieh, 1995)
on conductance were measured. When 5 mM BaCl2, CsCl or
tetraethylammonium chloride was added to the bath solu-
tion, the current intensity was not inhibited (Figure S1). This
indicates that there is an inward-rectified Ca2+-permeable
channel in the PM of A. thaliana root protoplasts.
Heat shock activates the Ca2+-permeable channel in the PM
To investigate the contribution of the PM Ca2+-permeable
channel to the change in [Ca2+]cyt during HS, we generated
a stable transgenic line that constitutively expressed apo-
aequorin, a bioluminescent protein from the coelenterate
Aequorea victoria. When 10-day-old reconstituted trans-
genic seedlings were kept at 22�C, [Ca2+]cyt remained
constant within the duration of the experiment. A signifi-
cant increase in [Ca2+]cyt was observed during a single
temperature increase from 22 to 37�C. The start of this
increase in [Ca2+]cyt occurred within 3 min of the HS. At the
18th min of HS, [Ca2+]cyt reached a maximum increase (1.6-
fold). GdCl3 and LaCl3 markedly reduced the heat-induced
increase in [Ca2+]cyt (Figure 2a,b). This result strongly
suggests that the heat-induced increase in [Ca2+]cyt is
mediated by a putative Ca2+-permeable channel in the PM
of A. thaliana cells.
Figure 1. Patch-clamp analysis was used to char-
acterize a putative Ca2+-permeable channel in the
PM of A. thaliana root cell protoplasts.
The Ca2+ current was recorded by step voltage
clamping. The trace is a representative current
from six protoplasts. The protocols used are
indicated in the inset.
(a) Whole-cell recording from a protoplast show-
ing a large voltage-dependent inward-rectified
current at hyperpolarized potentials (Control).
(b–d) Application of AlCl3 (b), GdCl3 (c) or LaCl3(d) (final concentration 50 lM) severely inhibited
the current.
(e) Current–voltage relationship (I–V) for (a)–(d)
(mean � SD, n = 6).
(f) Whole-cell tail current test.
(g) I–V curve for (f) (mean � SD, n = 6).
CNGC6 is a heat-activated calcium channel 3
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
The whole-cell patch-clamp technique was used to deter-
mine whether temperature increases triggered the activation
of Ca2+-permeable channels in the PM of A. thaliana root
protoplasts. A significant increase in inward current was
observed when the temperature was increased from 22 to
37�C (Figure 2c,d), and the Imean at )200 mV increased from
)190 to )393 pA (Figure 2g). The start of this increase in
current occurred within 1 min of HS. Slow-ramp voltage
clamping confirmed the role of HS in activation of a Ca2+
channel (Figure S2). When 50 lM GdCl3 or LaCl3 was applied,
a slight increase in inward current was observed when the
temperature was increased from 22 to 37�C (Figures 1c,d and
2e,f), and the Imean at )200 mV increased from )28 to )74 pA
for GdCl3 and from )45 to )75 pA for LaCl3 (Figures 1e and
2g), indicating that most of the heat-induced increase in Ca2+
current was inhibited by GdCl3 or LaCl3. The results further
demonstrate the presence of a putative, heat-activated PM
Ca2+-permeable channel in A. thaliana.
CNGC6 facilitates the acquisition of thermotolerance in
Arabidopsis
To screen out a heat-sensitive PM Ca2+-permeable channel, a
group of homozygous T-DNA insertion mutants of A. thali-
ana CNGC genes were isolated and identified. These
mutants were tested for differences in acquired thermotol-
erance by comparison with wild-type (WT) plants. The re-
sults showed that plants lacking CNGC6 (At2g23980), i.e.
cngc6 (SALK_042207) (Figure 3a), were more sensitive to
heat stress than WT (Figure S3). To test whether the ther-
mosensitivity of cngc6 plants may be attributed to loss of the
CNGC6 gene, complementation and over-expression analy-
ses were performed. Complementation experiments
showed that the survival rates for WT seedlings and for
seedlings of lines COM12 and COM25 (two cngc6 lines
complemented with CNGC6) after HS (45�C for 130 min)
were 70, 78 and 77%, respectively, whereas that for cngc6
Figure 2. Characterization of a putative heat-
activated Ca2+-permeable channel.
(a,b) Ca2+ channel blockers inhibited the heat-
induced increase in [Ca2+]cyt. Seedlings express-
ing apoaequorin were incubated in Ca2+ buffer
with or without 100 lM GdCl3 or LaCl3 at 22�C for
6 h, and then placed in a luminometer at 37�C(HS) or 22�C (control). The Ca2+ concentration
before HS (approximately 0.3 lM) was set to 1
and used for normalization. (a) Time course of
the increase in [Ca2+]cyt induced by HS. (b)
Comparison of [Ca2+]cyt among treatments at
the 18th min of HS. This experiment was re-
peated three times, with the same trend being
shown each time. Values are means � SD of
three independent experiments. The different
letters indicate significant differences (ANOVA,
post-hoc least significant difference (LSD),
P < 0.05).
(c–g) Ca2+ channel blockers inhibited the heat-
induced Ca2+ influx. The Ca2+ current was
recorded by step voltage clamp. Each trace is a
representative current from six protoplasts. (c)
Ca2+ current before HS (control); (d) Ca2+ current
after HS (HS); (e) 50 lM GdCl3 inhibited most of
the Ca2+ current under HS (HS + GdCl3); (f) 50 lM
LaCl3 inhibited most of the Ca2+ current under HS
(HS + LaCl3). (g) Current–voltage relationship (I–
V) for total current (mean � SD, n = 6).
4 Fei Gao et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
seedlings was just 50% (Figure 3b,c), indicating that genetic
complementation of cngc6 plants with CNGC6 restored the
WT phenotype to the mutant plants. The over-expression
experiments showed that the survival rates for seedlings of
lines OE4 and OE8 (two CNGC6 over-expression lines) after
HS (45�C for 140 min) were 72 and 74%, respectively,
whereas the survival rates for WT seedlings and EV seed-
lings (control plants transformed with an empty vector) were
only 52 and 55%, respectively (Figure 4a,b). These results
suggest that CNGC6 facilitates acquisition of thermotoler-
ance in A. thaliana seedlings. To ensure that the phenotypic
changes observed were caused by the CNGC6 gene,
real-time quantitative RT-PCR was used to analyze the level
of expression of the CNGC6 gene in seedlings with various
genotypes. The results showed that expression of the
CNGC6 gene was eliminated in cngc6, but increased in
COM12, COM25, OE4 and OE8 lines by 1.8-, 1.7-, 2.1- and
2.2-fold, respectively, compared to WT (Figures 3d and 4c).
Taken together, the data indicate that the acquired thermo-
tolerance in the seedlings correlates positively with the
expression level of the CNGC6 gene.
CNGC6 promotes expression of HSP genes during heat
shock
To understand whether CNGC6 is involved in the HSR,
real-time quantitative RT-PCR was used to analyze the
expression level of three A. thaliana HSP genes (HSP18.2,
HSP25.3 and HSP70) after HS in seedlings with various
genotypes. Ten-day-old seedlings grown at 22�C were
treated with 5 mM CaCl2 for 1 h, and then incubated at
37�C for 1 h. After HS, the expression levels of HSP18.2
(At5g59720), HSP25.3 (At4g27670) and HSP70 (At3g12580)
genes in the cngc6 mutant were lower by 31, 36 and 33%,
respectively, compared to WT; the expression levels of
these three HSP genes in COM12 were similar to that in
WT, and the expression levels of the three HSP genes in
OE8 were 3.4-, 2.8- and 2.1-fold higher, respectively, than
in WT (Figure 5). In conclusion, expression of HSP18.2,
HSP25.3 and HSP70 correlates positively with expression
of the CNGC6 gene, indicating that CNGC6 promotes
expression of HSP genes.
CNGC6 is a heat-activated Ca2+-permeable channel and
mediates Ca2+ influx
To determine the role of CNGC6 in mediating Ca2+ influx
during HS, the Ca2+ current in the PM before HS (unheated)
and after HS (heated) was compared among WT, cngc6,
COM and OE lines using the whole-cell patch-clamp tech-
nique. Under non-HS conditions, the Ca2+ current in cngc6
was lower than that in WT, COM12 and OE8. HS at 37�Cactivated an inward Ca2+ current in WT, COM12 and OE8
plants, and the Imean at )200 mV after HS in OE8 ()501 pA)
was much higher than that in WT ()393 pA) and COM12
()420 pA). However, this heat-activated current is absent
from cngc6 root cell protoplasts (Figure 6). The results
suggest that CNGC6 is a heat-activated Ca2+-permeable
Figure 3. Transformation of the cngc6 mutant
with CNGC6 rescued the thermotolerance phe-
notype.
WT, wild-type plants; cngc6, CNGC6 mutant;
COM12 and COM25, two cngc6 lines comple-
mented with CNGC6.
(a) Intron/exon organization of the CNGC6 gene
(coding region) and location of the T-DNA
inserted into the CNGC6 gene. Filled boxes,
exons; thin lines, introns; triangle, T-DNA inser-
tion position.
(b, c) Seedling phenotype (b) and survival rate (c)
of WT, cngc6, COM12 and COM25 plants after
HS. This experiment was repeated using five
batches with similar results. Values are
means � SD from eight independent experi-
ments (30 seedlings per experiment).
(d) Real-time quantitative RT-PCR analysis of
CNGC6 transcripts in WT, cngc6, COM12 and
COM25 seedlings. This experiment was repeated
three times, with the same trend being shown
each time. Values are means � SD from three
independent experiments. The experiment was
performed using CNGC6-specific primers (Table
S1). The expression level of CNGC6 in WT
seedlings was set to 1 and used for normaliza-
tion.
Asterisks indicate significant differences com-
pared to WT (Student’s t test: *P < 0.05,
**P < 0.01).
CNGC6 is a heat-activated calcium channel 5
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
channel that mediates the influx of Ca2+ from an extracellular
source during HS.
Tissue-specific expression of the CNGC6 gene
and subcellular localization of the CNGC6 protein
Tissue-specific expression of the CNGC6 gene was deter-
mined by real-time quantitative RT-PCR using RNA from
various A. thaliana tissues. The expression level of the
CNGC6 gene was in the order flowers > leaves > young
siliques > roots > seedlings > stems (Figure 7a). A GUS
reporter assay was used to monitor the detailed expression
pattern of the CNGC6 gene. GUS expression driven by the
CNGC6 promoter (2000 bp) is shown in Figure 7(b). The data
indicate that the CNGC6 gene is ubiquitously expressed in
A. thaliana.
Yellow fluorescent protein (YFP) reporter analysis was
used to evaluate the transient expression of CNGC6. Onion
epidermal cells were transformed with the pAVA321-CNGC6-
YFP fusion construct (Data S1). Onion epidermal cells trans-
formed with empty vector pAVA321-YFP were used as a
control. After plasmolysis, YFP fluorescence was distributed
throughout the cell in control plants, but coincided with the
cell periphery in CNGC6–YFP plants (Figure S4). Further-
more, green fluorescent protein (GFP) reporter analysis was
used to assess the stable expression of CNGC6. To determine
the PM, root tissues were incubated in 5 lM FM4-64 (a PM
marker) solution for 15 min. Co-localization of GFP fluores-
cence (green) and FM4-64 fluorescence (red) in transgenic
plants stably expressing pCAMBIA1300-CNGC6-sGFP before
and after plasmolysis indicated that the CNGC6–sGFP
(synthetic GFP, S65T) fusion protein is restricted to the PM
of root cells. Plants expressing the empty vector pCAM-
BIA1300-sGFP served as controls, and GFP fluorescence was
distributed in the nuclei, PM and cytoplasm. Using the same
confocal imaging conditions, no green fluorescence was
observed in root cells of WT (Figure 7c).
CNGC6 is a cAMP-activated Ca2+-permeable channel
To evaluate the role of cAMP in regulating the activity of
CNGC6, the effect of cAMP on Ca2+ conductance through
the PM was examined by the whole-cell patch-clamp
technique. Application of the exogenous lipophilic cAMP
analog dibutyryl-cAMP (db-cAMP), activated an inward
Ca2+ current in WT A. thaliana root cell protoplasts, and the
Imean at )200 mV after addition of db-cAMP to the bath
solution (final concentration 50 lM) was 3.6-fold higher
than in the control. This cAMP-activated current was
absent from cngc6 root cell protoplasts (Figure 8). An
inhibitor of cNMP phosphodiesterase, 1-methyl-3-(2-meth-
ylpropyl)-7H-purine-2,6-dione (IBMX), was used to increase
the level of endogenous cAMP. The results showed that
application of IBMX activated an inward Ca2+ current in
(b) (c)
(a)
Figure 4. Improved thermotolerance in A. thaliana due to CNGC6 over-expression.
WT, wild-type plants; EV, empty vector control; OE4 and OE8, two CNGC6 over-expression lines.
(a, b) Seedling phenotype (a) and survival rate (b) of WT, EV, OE4 and OE8 plants after HS. This experiment was repeated using five batches with similar results.
Values are means � SD from nine independent experiments (30 seedlings per experiment).
(c) Real-time quantitative RT-PCR analysis of CNGC6 transcripts in WT, EV, OE4 and OE8 seedlings. This experiment was repeated three times, with the same trend
being shown each time. Values are means � SD from three independent experiments. The experiment was performed using CNGC6-specific primers (Table S1).
The expression level of CNGC6 in WT seedlings was set to 1 and used for normalization.
Asterisks indicate significant differences compared to WT (Student’s t test: **P < 0.01).
6 Fei Gao et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
WT, and the Imean at )200 mV after addition of IBMX to the
bath solution (final concentration 750 lM) was 2.5-fold
higher than in the control. This IBMX-activated current was
absent from the cngc6 mutant (Figure 8). These data indi-
cate that CNGC6 is a cAMP-activated Ca2+-permeable
channel.
Levels of cytosolic cAMP are increased by a mild heat shock
and is involved in heat shock responses
The level of cytosolic cAMP during HS was analyzed in
10-day-old seedlings grown at 22�C and then exposed to
37�C for 0, 1, 2, 3 or 4 min. The results showed that HS at
37�C induced an increase in the level of cAMP. The start of
this increase occurred within 1 min of the start of the HS.
The cAMP level reached a maximum 2.9-fold increase at
2 min after the start of the HS, then began to decrease after
3 min, returning to the initial level after 4 min (Figure 9a).
The role of adenylyl cyclase (AC) in the heat-induced
increase in cAMP was evaluated using 2¢,5¢-dideoxy-
adenosine, an inhibitor of AC. The result indicated that the
heat-induced increase in cAMP was inhibited by pre-
treatment with 20 lM 2¢,5¢-dideoxyadenosine solution,
suggesting that heat-induced cAMP increases may be
dependent on AC (Figure 9b).
The effects of exogenous cAMP on expression of HSP
genes were analyzed using real-time quantitative RT-PCR.
Ten-day-old WT seedlings, grown at 22�C, were incubated
for 2 h in 0, 25, 50, 100 or 150 lM db-cAMP solution
containing 5 mM CaCl2. Expression of HSP18.2 and
HSP25.3 in treated seedlings was detected using real-time
quantitative RT-PCR. The results showed that expression of
HSP18.2 and HSP25.3 was induced by 25 or 50 lM db-cAMP,
and reached a maximum 1.5- or 1.7-fold increase (Fig-
ure 9c,d). These results indicate that cAMP is likely to play an
important role in the HS signal transduction pathway.
DISCUSSION
CNGC6 is a heat-activated PM Ca2+-permeable channel
involved in HSRs
A. thaliana CNGC proteins have been reported to be asso-
ciated with a diverse range of physiological phenomena.
However, the functions of A. thaliana CNGC6 have not been
documented. In this investigation, the tissue expression
pattern, subcellular localization and functional properties of
CNGC6 were studied. A tissue expression assay showed that
CNGC6 was expressed throughout the plant (Figure 7a,b).
The CNGC6 protein has a predicted structure of six trans-
membrane domains (Kaplan et al., 2007). This study con-
firmed PM localization of CNGC6 by assay of the transient
and stable expression of CNGC6 (Figures 7c and S4). Using
patch-clamp analysis, a large, voltage-dependent, inward-
rectified current at hyperpolarized potentials in the PM of
A. thaliana was detected in root cell protoplasts exposed to a
basal bath solution containing 10 mM CaCl2 (Figure 1a).
A pharmacological approach showed that this current was
intensively inhibited by the Ca2+ channel blockers AlCl3,
GdCl3 and LaCl3 (Figure 1b–d), but not by the K+ channel
blockers BaCl2, CsCl or tetraethylammonium chloride (Fig-
ure S1). This suggests that the channel detected in the PM of
A. thaliana root cell protoplasts is likely to be a Ca2+-per-
meable channel. In contrast, addition of 5 mM BaCl2 slightly
increased the conductance (Figure S1e), supporting previ-
ous observations that Ba2+ can permeate Ca2+-permeable
channels (White, 2000; Lemtiri-Chlieh and Berkowitz, 2004).
Furthermore, this study found that CNGC6 was a heat-acti-
vated PM Ca2+-permeable channel (Figure 6), and that
CNGC6 was involved in the expression of HSP genes (Fig-
ure 5) and acquisition of thermotolerance (Figures 3 and 4),
Figure 5. CNGC6 promotes the expression of HSP genes during HS.
WT, wild-type plants; cngc6, CNGC6 mutant; COM12, a cngc6 line comple-
mented with CNGC6; OE8, a CNGC6 over-expression line.
(a) Expression of HSP18.2 in WT, cngc6, COM12 and OE8.
(b) Expression of HSP25.3 in WT, cngc6, COM12 and OE8.
(c) Expression of HSP70 in WT, cngc6, COM12 and OE8.
Real-time quantitative RT-PCR was performed using HSP18.2-, HSP25.3- or
HSP70-specific primers (Table S1). The expression level of HSP genes in WT
seedlings was set to 1 and used for normalization. Values are means � SD
from three independent experiments. Asterisks indicate significant differ-
ences compared to WT (Student’s t test: *P < 0.05, **P < 0.01).
CNGC6 is a heat-activated calcium channel 7
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
indicating that extracellular Ca2+ stores are the source of the
increase in [Ca2+]cyt in response to HS. The data presented
here show that the survival rate of seedlings (Figure 3) and
the expression of HSP genes (Figure 5) in the cngc6 mutant
decreased by approximately 30% compared to WT.
Although these differences were statistically significant
(P < 0.05), the effect of CNGC6 on thermotolerance was
limited, implying complexity and diversity in HS signal
transduction pathways. Two lines of evidence suggest that
intracellular Ca2+ pools are likely to play an important role in
the HS signal transduction pathway. First, when extracellu-
lar free Ca2+ was eliminated by EGTA, the expression level of
HSP18.2 after HS in the cngc6 mutant was higher than that in
WT (Figure S5), suggesting that Ca2+ release from the
intracellular Ca2+ pool was induced to compensate for the
loss of CNGC6. Second, application of 1-[6-[((17b)-3-Meth-
oxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-
dione (U-73122), an antagonist of PLC, decreased the
thermotolerance of WT, cngc6, COM12 and COM25 seedlings
(Figure S6). Taken together, the evidence suggests that HS
mobilizes cytosolic Ca2+ from both extracellular and intra-
cellular sources.
It is known that all animal CNGCs are heterotetramers,
comprising more than one CNGC gene product (Kaupp and
Seifert, 2002; Zhong et al., 2002; Zheng and Zagotta, 2004),
and plant CNGCs may also be heterotetrameric in native
membranes (Talke et al., 2003; Ma et al., 2009). The presence
of 20 members of the CNGC family in A. thaliana suggests
the probability of a large diversity of heteromeric channels,
leading to functional variety and specialization (Talke et al.,
2003). Thus, we speculate that the heat- and cyclic nucleo-
tide-activated currents recorded from the root cell mem-
brane may be mediated by a CNGC channel complex that
includes the CNGC6 polypeptide. However, increasing evi-
dence shows that translational arrest of one plant CNGC can
alter conductance properties across native plant cell mem-
branes and affect the functions of plants (Jurkowski et al.,
2004; Ali et al., 2007; Borsics et al., 2007; Chaiwongsar et al.,
Figure 6. Patch-clamp analysis identified a heat-activated Ca2+-permeable channel in the PM of root cell protoplasts of WT, COM12 and OE8; this current was absent
from cngc6 root cell protoplasts.
WT, wild-type plants; cngc6, CNGC6 mutant; COM12, a cngc6 line complemented with CNGC6; OE8, a CNGC6 over-expression line. The Ca2+ current before HS
(unheated) and after HS (heated) was compared in WT, cngc6, COM12 and OE8 plants. The Ca2+ current was recorded by step voltage clamp. Each trace is a
representative current from six protoplasts. Currents in unheated (at 22�C) and heated (at 37�C) protoplasts are shown in the left and middle columns. The I–V curve
is shown in the right column (mean � SD, n = 6).
8 Fei Gao et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
Figure 7. Expression pattern and subcellular localization of CNGC6.
(a) Expression of the CNGC6 gene in various tissues by real-time quantitative RT-PCR. The experiment was performed using CNGC6-specific primers (Table S1). The
expression level of CNGC6 in roots was set to 1 and used for normalization. Values are means � SD from six independent experiments.
(b) GUS staining of transgenic plants harboring PCNGC6:GUS: (1) 7-day-old seedling; (2) 14-day-old seedling; (3) rosette leaf; (4) cauline leaf; (5) stem; (6) anthotaxy;
(7) flower; (8) young silique.
(c) Subcellular localization of CNGC6 protein in root tissue from wild-type plants (A–D), transgenic plants expressing sGFP (E–H), and transgenic plants expressing
CNGC6–sGFP before plasmolysis (I–L) and after plasmolysis (M–P). The channels of GFP fluorescence, FM4-64 fluorescence and bright field were imaged
simultaneously. GFP fluorescence (A, E, I, M); FM4-64 fluorescence (B, F, J, N); bright-field images (C, G, K, O); combined GFP, FM4-64 and bright-field images (D, H,
L, P).
CNGC6 is a heat-activated calcium channel 9
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
2009). Perhaps the absence of one CNGC protein affects
assembly of the natural channel complex and results in
formation of deviant channels in mutants (Ma et al., 2009).
cAMP is involved in the heat shock signal transduction
pathway
Plant CNGCs contain a putative binding site for cNMP at the
C-terminus (Kudla et al., 2010), and it is known that A. tha-
liana CNGC2 and CNGC4 are activated directly by cAMP
(Balague et al., 2003; Ali et al., 2007). This study determined
whether the activity of CNGC6 is also gated by cAMP. To
evaluate this, the role of endogenous cAMP in PM Ca2+
conductance in WT and the cngc6 mutant was investigated.
Although genes encoding enzymes that synthesize
cAMP (adenylyl cyclase) and breakdown cAMP (cNMP
phosphodiesterase) in plants have not been identified
(Martinez-Atienza et al., 2007), AC activity and cNMP phos-
phodiesterase activity are present in plants (Brown et al.,
1980; Cooke et al., 1994; Witters et al., 2004). An inhibitor of
cNMP phosphodiesterase, IBMX, was used to increase the
level of endogenous cAMP. Application of IBMX activated an
inward Ca2+ current in WT but not in cngc6 (Figure 8), sug-
gesting that CNGC6 is activated by endogenous cAMP. Fur-
thermore, it was found that the level of endogenous cAMP
was increased by a mild HS (Figure 9a), and that exogenous
cAMP may activate CNGC6 (Figure 8) and promote expres-
sion of the HSP18.2 and HSP25.3 genes (Figure 9c,d), like HS,
indicating that HS is likely to activate CNGC6 by increasing
the level of endogenous cAMP.
Based on the results presented here, we propose a model
for how plants transduce a heat stimulus into a signal cascade
resulting in a HSR. The effects of heat stimulus on the CNGC6-
dependent current may occur via activation of an AC in the
cytoplasm, thus leading to an increase in cAMP that activates
CNGC6 (or CNGC complexes containing CNGC6). Activation
of CNGC6 results in influx of Ca2+ into the cell, facilitating
expression of HSP genes and acquisition of thermotolerance.
This study has linked plant heat perception to cytosolic cAMP
elevation, a cAMP-activated Ca2+ channel and downstream
HSR. We have provided evidence for the involvement of
cAMP in the plant HS signal transduction pathway, and have
given an example of CNGC linking two important signaling
systems in plant cells: cAMP and Ca2+ signaling. However, it
is still not known how heat perception is linked to the
increased level of cytosolic cAMP in plant cells, although this
possibly occurs through a receptor in the PM. In addition, this
study did not assess the role of CaM in regulation of CNGC6,
and this will be examined in future studies.
EXPERIMENTAL PROCEDURES
Plant materials and growth conditions
Arabidopsis thaliana (ecotype Columbia-0) seeds were surface-sterilized using 75% v/v ethanol and deep-sterilized using 10% w/vsodium hypochlorous acid. They were then plated on MS medium
Figure 8. Patch-clamp analysis identified a
cAMP-activated Ca2+-permeable channel in the
PM of WT root cell protoplasts; this current was
absent from cngc6 root cell protoplasts.
Ca2+ current before (control) and after addition of
db-cAMP or IBMX to the bath solution was
compared in wild-type plants (WT) and the
CNGC6 mutant (cngc6). The Ca2+ current was
recorded by a step voltage clamping protocol.
Each trace is a representative current from six
protoplasts. The I–V curve is shown at the bottom
(mean � SD, n = 6).
10 Fei Gao et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
containing 3.0% w/v sucrose and 0.8% w/v agar, and incubated at4�C in darkness for 3 days. Following incubation, the plates wereplaced vertically or horizontally in a growth chamber at 22�C underlong-day conditions (16 h light/8 h dark) with a light intensity ofapproximately 100 lmol photons m)2 sec)1. After 2 weeks, thehorizontally cultivated seedlings were transplanted into soil andcultured at 22�C under long-day conditions.
Preparation of protoplasts and electrophysiology analysis
Protoplasts were isolated as described by Demidchik and Tester(2002) from 1 cm long of root tips of A. thaliana cultivated verticallyat 22�C for 6–7 days. Whole-cell voltage patch-clamping was per-formed as described by Wu et al. (2007). Patch-clamp pipettes (1.5/1.1 mm diameter, from WPI, http://www.wpiinc.com/) were pulledon a vertical electrode puller (PB-7; Narishige, http://www.narishige.co.jp/english/). The electrode was filled with pipette solution(0.5 mM CaCl2, 4 mM Ca(OH)2, 2 mM Mg-ATP, 0.5 mM Tris-ATP,10 mM EGTA and 15 mM HEPES/Tris, pH 7.2, adjusted to anosmolality of 300 mOsm/kg with sorbitol; free Ca2+ concentration100 nM). The basal external (bath) solution comprised 10 mM CaCl2and 5 mM MES/Tris, pH 5.8, adjusted to an osmolality of 300 mOsm/Kg with sorbitol. The resistance of the electrode in the bath solutionwas approximately 20 MX. Seal resistances were up to 2 GX. Afterholding the whole-cell high seal resistances for at least 15 min,currents were recorded and data were sampled at 1 kHz and filteredat 200 Hz. Membrane potentials were corrected for liquid junctionpotentials and series resistance. An Axon 200B amplifier (AxonInstruments, Sunnyvale, CA, USA) controlled by PCLAMP 9.0 soft-ware (Axon Instruments) was used to record the current signal.
Basal currents were recorded at room temperature (20–22�C). HStreatment (37 � 2�C) was performed by continuous bath perfusion.
In vivo reconstitution of aequorin and Ca2+ measurement
In vivo reconstitution of the aequorin was performed by incubating10-day-old A. thaliana seedlings expressing apoaequorin in Ca2+
buffer (0.1 mM KCl, 1 mM CaCl2, 10 mM MES, pH 5.0, and 2.5 lM
coelenterazine h) at 22�C for 6 h, and then washing three times (1min/time) with buffer. The luminescence of the seedlings wasrecorded using a microplate luminometer (Centro LB 960, Berthold,http://www.berthold.com). At the end of each monitoring period,discharging solution (1 M CaCl2 and 10% ethanol) was added. Theconcentration of cytosolic Ca2+ was obtained by calculating the pCausing the equation described by Plieth (2006).
Identification and isolation of the cngc6 mutant
Seeds of a T-DNA insertional mutant for A. thaliana cyclic nucle-otide-gated channel 6 (CNGC6), SALK_042207, were obtainedfrom the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). The homozygous CNGC6 mutant (cngc6) wasscreened by PCR as described by the Salk Institute GenomicAnalysis Laboratory (http://signal.salk.edu/T-DNA_Genotyping_Procedure.ppt). PCR was performed using genomic DNA from T3
generation seedlings using CNGC6-specific genomic primers(left, 5¢-CCACAAAGCCCCAACAATCTGC-3¢; right, 5¢-ATGCATGGCGTGGGACAACC-3¢) and a left border primer for the T-DNAinsertion (LBb1: 5¢-GCGTGGACCGCTTGCTGCAACT-3¢). The loca-tion of the T-DNA insertion was determined by sequencing
Figure 9. Cytosolic cAMP is involved in the HSR.
(a) Time course of heat-induced cAMP generation in A. thaliana seedlings. Ten-day-old seedlings grown at 22�C were heated at 37�C for the length of time indicated.
The level of cAMP in the cells of unheated control seedlings was normalized to 1.
(b) 2’,5’-dideoxyadenosine (DDA) blocked the heat-induced increase in cAMP levels. Control, 22�C; HS, heat shock at 37�C for 2 min; DDA + HS, treatment with 20 lM
DDA solution for 1 h followed by HS at 37�C for 2 min. The level of cAMP in the cells from unheated control seedlings was normalized to 1.
(c, d) Effect of db-cAMP on expression of HSP18.2 (c) or HSP25.3 (d) using real-time quantitative RT-PCR. Ten-day-old WT seedlings grown at 22�C were incubated for
2 h in various concentrations of db-cAMP solution that included 5 mM CaCl2. The experiment was performed using HSP18.2- or HSP25.3-specific primers (Table S1).
The expression level in the seedlings treated with 5 mM CaCl2 was set to 1 and used for normalization. Values are means � SD from three independent experiments.
CNGC6 is a heat-activated calcium channel 11
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), doi: 10.1111/j.1365-313X.2012.04969.x
the PCR product. The transcript abundance of CNGC6 in seedlingsof various genotypes was determined by real-time quantitativeRT-PCR.
Plant transformation
Plasmid pCAMBIA1300-35S:CNGC6 was constructed for functionalanalysis of CNGC6. The coding region of CNGC6 was amplifiedusing cDNA from 10-day-old A. thaliana seedlings with forwardprimer 5¢-GCTCTAGAATGTTCGATACTTGTGGCCCA-3¢ and reverseprimer 5¢-CGAGCTCGTCAGTGATCTTCAGCAGAGAAATCTG-3¢.The PCR product was digested with XbaI/SacI and cloned into thebinary vector pCAMBIA1300-35S (http://www.cambia.org/daisy/cambia/585). Plasmid pCAMBIA1300-PCNGC6:GUS was con-structed in order to assess the expression pattern of CNGC6. TheCNGC6 promoter region was PCR-amplified using genomic DNAfrom A. thaliana seedlings, and was ligated into pCAMBIA1300-GUS (http://www.cambia.org/daisy/cambia/585) digested with PstI/XbaI. The plasmid pCAMBIA1300-CNGC6-sGFP was constructed inorder to assay the subcellular localization of CNGC6 protein. CNGC6was PCR-amplified from a plasmid containing the CNGC6 codingregion using forward primer 5¢-TCTAGAATGTTCGA-TACTTGTGGCCCA-3¢ and reverse primer 5¢-GGATCCGGTGATCTT-CAGCAGAGAAATCTG-3¢. The PCR product was fused topCAMBIA1300-sGFP digested with XbaI/BamHI. The resultingplasmids were transformed into Agrobacterium tumefaciens strainGV3101, and then introduced into cngc6 or WT by the floral-dipmethod (Clough and Bent, 1998). Transgenic seedlings werescreened on MS medium containing 25 lg ml)1 hygromycin.
Assay of acquired thermotolerance
A. thaliana seeds of various CNGC6 genotypes (30 seeds pergenotype) were plated on separate regions of the same plate con-taining half-strength MS, 1% w/v sucrose and 0.3% w/v Phytagel�(Sigma, http://www.sigmaaldrich.com/), pH 5.8. Seven-day-oldseedlings, grown at 22�C, were incubated for 1 h in sterilized 5 mM
CaCl2, acclimated at 37�C for 30 min, returned to 22�C for 2 h, thenchallenged at 45�C for 130 or 140 min, and finally allowed to recoverat 22�C for 4–6 days. The seedling survival rate was calculated.Plants that were still green and producing new leaves were con-sidered to have survived.
Histochemical staining for GUS expression
Histochemical staining for GUS expression was performed asdescribed by Jefferson et al. (1987).
Assay of cAMP level
For extraction of cAMP, 10-day-old A. thaliana seedlings grown at22�C were exposed to 37�C for 0, 1, 2, 3 or 4 min, and then ground inliquid nitrogen. Then 0.4 ml PBS was added to 0.2 g ground frozentissue. After centrifugation at 12 000 g for 15 min, the supernatantwas used to assay the cAMP level. Changes to the cytosolic cAMPlevel during HS were analyzed using a cAMP-Glo� assay kit (Pro-mega, http://www.promega.com) and a microplate luminometer(Centro LB 960, Berthold), according to Promega’s instructions.
Real-time quantitative RT-PCR
Total RNA was isolated from seedlings with various genotypesusing TRIzol� reagent (Invitrogen, http://www.invitrogen.com/) inorder to compare the transcript abundance of HSP and CNGC6genes. Total RNA was isolated from A. thaliana tissues using TRI-zol� reagent (Invitrogen) for an analysis of the CNGC6 expressionpattern. Total RNA (400 ng) was used for first-strand cDNA syn-
thesis using the ExScript� RT reagent kit (Takara, http://www.takara.com) according to the manufacturer’s instructions. Real-timequantitative RT-PCR was performed as described by Zhang et al.(2009).
Microscopy
Root tissues of 5-day-old seedlings were incubated in 5 lM FM4-64for 15 min in the dark. Fluorescence was observed using a laserscanning confocal microscope (Zeiss LSM 510, http://www.ze-iss.com) with 543 nm excitation from a HeNe laser, a 458/477/488/514 nm dichroic mirror, 505-530 BP emission filtration and 560 nmLP emission filtration). In order to induce plasmolysis, the rootswere treated with 6% w/v NaCl.
Statistical analysis
Statistical analyses were performed using STATISTICA 6.0(StatSoft,http://www.statsoft.com). The significance of differences was testedat P < 0.05 using ANOVA with post-hoc least significant difference(LSD) or Student’s t test, as indicated.
ACKNOWLEDGEMENTS
This research was financed by grants from the Key Research SpecialFunds of the Ministry of Agriculture, China (2009ZX08009-017B), theNatural Science Foundation of China (30870201 and 31070257) andthe Natural Science Foundation of Hebei Province, China(C2009000280).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Effects of K+ channel blockers on the inward-rectifiedconductance.Figure S2. Effect of HS on the PM Ca2+ conductance.Figure S3. Knocking out the CNGC6 gene decreased the thermotol-erance of seedlings.Figure S4. Fluorescence images of YFP and CNGC6–YFP fusionproteins transiently expressed in onion epidermal cells.Figure S5. Effects of EGTA on expression of HSP18.2 in WT, mutantand COM plants under HS.Figure S6. Effects of PLC inhibitor on the thermotolerance of plants.Table S1. Primers used for real-time PCR amplifications.Data S1. Construction and transformation of pAVA321-CNGC6-YFP.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.
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