Endoplasmic reticulum-localized CCX2 is required forosmotolerance by regulating ER and cytosolic Ca2+

dynamics in ArabidopsisMassimiliano Corsoa, Fabrizio G. Docculab, J. Romário F. de Meloa, Alex Costab,c,1,2, and Nathalie Verbruggena,1,2

aLaboratory of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles, 1050 Brussels, Belgium; bDepartment of Biosciences, University ofMilan, 20133 Milan, Italy; and cInstitute of Biophysics, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy

Edited by Julian I. Schroeder, University of California, San Diego, La Jolla, CA, and approved March 9, 2018 (received for review December 1, 2017)

Ca2+ signals in plant cells are important for adaptive responses toenvironmental stresses. Here, we report that the Arabidopsis CAT-ION/Ca2+ EXCHANGER2 (CCX2), encoding a putative cation/Ca2+

exchanger that localizes to the endoplasmic reticulum (ER), isstrongly induced by salt and osmotic stresses. Compared withthe WT, AtCCX2 loss-of-function mutant was less tolerant to os-motic stress and displayed the most noteworthy phenotypes (lessroot/shoot growth) during salt stress. Conversely, AtCCX2 gain-of-function mutants were more tolerant to osmotic stress. In addi-tion, AtCCX2 partially suppresses the Ca2+ sensitivity of K667 yeasttriple mutant, characterized by Ca2+ uptake deficiency. Remark-ably, Cameleon Ca2+ sensors revealed that the absence ofAtCCX2 activity results in decreased cytosolic and increased ERCa2+ concentrations in comparison with both WT and the gain-of-function mutants. This was observed in both salt and nonsaltosmotic stress conditions. It appears that AtCCX2 is directly in-volved in the control of Ca2+ fluxes between the ER and the cyto-sol, which plays a key role in the ability of plants to cope withosmotic stresses. To our knowledge, Atccx2 is unique as a plantmutant to show a measured alteration in ER Ca2+ concentrations.In this study, we identified the ER-localized AtCCX2 as a pivotalplayer in the regulation of ER Ca2+ dynamics that heavily influenceplant growth upon salt and osmotic stress.

Ca2+ transport | cation/Ca2+ exchanger family | NaCl | Cameleon sensors |abiotic stress

Plant tolerance to abiotic stress relies on a wide range ofphysiological and molecular mechanisms involving a fine

regulation of cytosolic ion homeostasis, including calcium (Ca2+)(1). In eukaryotes Ca2+ can act as a ubiquitous second messengerin triggered signal transduction cascades (2). In plants, severalstresses trigger Ca2+ influx in the cytosol that can involve theactivation of membrane-localized Ca2+-permeable channels (3).The homeostasis of [Ca2+]cyt depends on a fine regulation ofCa2+ influxes and effluxes that occur at the plasma membrane(PM) and membranes of various subcellular compartments (4–7). Downstream of [Ca2+]cyt elevation is the activity of variousCa2+ sensors (2) and reactive oxygen species (ROS)-producingenzymes that are synergistically activated by Ca2+ binding andphosphorylation by Ca2+-dependent protein kinases (3, 8, 9).Cameleon Ca2+ sensors have recently been developed to study

Ca2+ fluxes and dynamics in various organelles (4, 6, 10–13). Inplants, despite the development of this technology, there is still alack of molecular information regarding the regulation of [Ca2+]cytand the interplay between cytosol and cellular compartments,particularly the endoplasmic reticulum (ER), in the regulation ofCa2+ fluxes. Indeed, despite the important role of the ER in theregulation of Ca2+ dynamics in mammals (14) and in stress ad-aptation (15), little is known about its function in the controlof Ca2+ fluxes in plants (4, 7, 16). Only two ER-localized Ca2+

pumps have been partially characterized, namely ECA1 andACA2. Although their predicted function is to load cytosolic

Ca2+ into the ER lumen, their role in establishing ER-cytosolCa2+ dynamics is so far not clear (17, 18).In plants, three classes of membrane transporters are pre-

dicted to mediate Ca2+ fluxes: Ca2+-permeable channels, Ca2+-ATPases, and Ca2+/cation antiporters (CaCAs). The CaCAsuperfamily comprises five families, including cation/Ca2+ ex-changers (CCX) and Ca2+/proton exchangers (CAX) (5, 18, 19).Although Arabidopsis CAX members are well characterized (8,20–23), CCX’s function in plants remains largely unknown. Arecent characterization of Arabidopsis thaliana CCX1 supportsits role in leaf senescence, as well as a higher sensitivity of theccx1-1 mutant to Ca2+ deprivation (24). Functional character-ization based on heterologous expression in yeast suggests thatAtCCX3 and AtCCX4 have affinity for Mn2+, K+, and Na+ (25),while AtCCX5 appears to mediate K+ uptake and Na+ transport(26). Phylogenetic analysis showed that CCX5 and, to a lesserextent CCX3 and CCX4, genes are limitedly close to a mam-malian K+-dependent Na+/Ca2+ exchanger (NCKX6) (27, 28).CCXs are indeed annotated as cation/Ca2+ exchangers, even ifCCX3/4/5 are characterized as H+/K+ antiporters and do notexhibit affinity for Ca2+ (25, 26). The rice vacuolar cation/Ca2+

transporter OsCCX2 was recently characterized. This gene en-hanced tolerance of Saccharomyces cerevisiae cells in the pres-ence of excess Na+, Li+, Fe2+, Zn2+, and Co2+ (29).

Significance

Given the lack of a nervous system in plants, signaling molecules,such as Ca2+, are of vital importance for perceiving environmentalstimuli. However, little information on the genes that control thisnetwork is available. Our demonstration that Arabidopsis CCX2,localized to the endoplasmic reticulum, modulates osmotic stressresponses through the regulation of cytosolic and endoplasmicreticulum Ca2+ concentrations is onemore step toward identifyingall the players involved in plant stress responses. The elucidationof these pathways will help to meet the necessary requirementsto face global challenges, such as high salinity on the world’ssurface, including arable land. This work sheds light on an as yetuncharacterized and key player that tunes intracellular Ca2+ ho-meostasis, with particular significance for plants under salt stress.

Author contributions: M.C., A.C., and N.V. designed research; M.C., F.G.D., J.R.F.d.M., andA.C. performed research; M.C., F.G.D., and A.C. contributed new reagents/analytic tools;M.C., A.C., and N.V. analyzed data; and M.C., A.C., and N.V. wrote the paper withcomments from all authors.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1A.C. and N.V. contributed equally to this work.2To whom correspondence may be addressed. Email: alex.costa@unimi.it or nverbru@ulb.ac.be.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720422115/-/DCSupplemental.

Published online March 26, 2018.

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In this study, we describe the role of AtCCX2 (At5g17850),which encodes a putative cation/Ca2+ exchanger. We show thatAtCCX2 is an ER-localized protein whose activity impactsboth cytosolic and ER [Ca2+] regulation under both standardand NaCl stress conditions and plant growth during salt andosmotic stresses.

ResultsAtCCX2 Is Induced by a Wide Range of Osmotic Stresses in A. thaliana.The expression of the five CCXs genes was retrieved from theelectronic Fuorescent Pictograph (eFP) (30) database (Fig. S1 Aand B). These data indicate that salt and mannitol treatmentscaused the highest induction of CCX1, -2, and -3 gene expressionin roots and shoots. Interestingly, CCX2 showed the earliestchanges (after 30 min of treatment) and the strongest inductionupon salt and mannitol treatments in roots and shoots. Thesedata suggest the possible involvement of CCX2 in salt andosmotic-stress responses, which were further investigated.

AtCCX2 Mutants Showed Contrasting Responses upon Salt andOsmotic Stress. To test whether CCX2 is involved in stress tol-erance, both loss- and gain-of-function approaches were fol-lowed. CCX2 knockout (ccx2), two ccx2 + p35S::AtCCX2-GFPcomplemented lines (ccx2-C), and four independent lines ofp35S::AtCCX2 [CCX2-overexpressed (OE)] Col-0 Arabidopsisplants (Fig. S1 C–G) were exposed to different stress conditions.While no significant differences in growth parameters were no-ticed in the control condition compared with the WT (Fig. S1 E–G), clearly sensitive and resistant phenotypes were observed forccx2 and CCX2-OE plants, respectively, when exposed to bothsalt and osmotic stresses (Fig. 1 and Fig. S2). ccx2 root growthwas strongly impaired at all tested NaCl concentrations, showinga reduction in the length of lateral roots compared with WT,while ccx2-C lines showed root growth similar to the WT uponNaCl stress (Fig. 1 A and B). Conversely, the root length of theCCX2-OE lines was longer upon NaCl treatment, compared withthe WT. In particular, lateral roots were on average two- tothreefold longer in CCX2-OE lines with respect to the WT,depending on NaCl concentration (Fig. 1 B and D). The sametrend was observed for shoot area in the ccx2, ccx2-C, and CCX2-OE plants (Fig. 1 C and D). ccx2 and CCX2-OE plants were also

tested for their ability to grow in the presence of KCl, mannitol(Fig. S2 A–D, G, and H), and of EGTA Ca2+ chelator (Fig. S2 E,F, and I). The results pinpointed a lateral root growth reductionin ccx2 with respect to WT, while CCX2-OE showed longer rootsand a larger shoot area compared with the WT. Together, theseresults support a role for CCX2 in salt and osmotic stress re-sponses, with a possible function in regulating Ca2+ homeostasis.The most noteworthy phenotype for CCX2 mutants was ob-

served in response to NaCl. We focused on this stress andmonitored the expression of CCX1, -2, and -3 genes (Fig. S2J), inaddition to others reported to play a role in salt stress and Ca2+

homeostasis (Fig. S2K). WT, ccx2, and CCX2-OE plants weretransferred to the control and 300 mM NaCl solutions andsampled at 2 and 6 h after stress initiation (HSI). CCX2 showed a10-fold increase in expression in NaCl-treated WT plants at 6HSI compared with control plants. Interestingly, CCX2 tran-script level was still increased, even in NaCl-treated CCX2-OEseedlings, with respect to the control condition as early as 2 HSI.CCX1 and CCX3 genes were also induced upon NaCl stress inWT plants, respectively, at 2 and 6 HSI, but to a lesser extentcompared with CCX2 (Fig. S2J). RD29B.1 and SOS1 showed nosignificant differences in expression between WT and the CCX2mutants (Fig. S2K). Conversely, at 6 HSI, the SOS3 calciumsensor (31) gene was induced by NaCl treatment in CCX2-OEplants but not ccx2 plants. Finally, ACA2, which encodes an ER-localized Ca2+ pump, was more highly expressed at 6 HSI inCCX2-OE mutants, with respect to WT and ccx2.

Subcellular Localization of AtCCX2. To determine the subcellularlocalization of CCX2, we analyzed the localization of the C-terminalGFP-tagged CCX2 fusion protein in the ccx2 complemented line(Fig. 1). Hence, Fig. 2 A–F provides representative confocal mi-croscope images of ccx2-C Arabidopsis seedlings, demonstrating thatthe chimeric protein was expressed in leaf (Fig. 2A and Movie S1),hypocotyl (Fig. 2B), and root cells (Fig. 2C). The GFP signal dec-orated structures resembling the typical ER morphology and theassociated nuclear envelope (32). To confirm CCX2 localization,the same construct was transiently expressed in agro-infiltrated to-bacco leaf (Fig. 2G–J). In this case, the AtCCX2-GFP signal largelycolocalized with the mCherry-HDEL ER-marker (Fig. 2 I and J andMovie S2). Moreover, coexpression of AtCCX2-GFP with untaggedRFP (cytosolic and nuclear localization) demonstrated that theGFP signal only surrounded the nucleus and was absent from nu-cleoplasm (Fig. 2 K–M), further confirming its membrane associa-tion. However, in this latter case the pixel scatter-plot analysis stillrevealed a partial colocalization of GFP and free RFP, but this wasassumed to be the presence of the large vacuole that pushes boththe ER and cytoplasm against the PM (Fig. 2N). Overall, our resultsclearly indicate that CCX2 is an ER-localized protein.

AtCCX2 Shows Ca2+ Transport Properties. Despite AtCCX2 beingpredicted to be a cation/Ca2+ exchanger, no data have beenprovided about its transport properties. Hence, to assess CCX2Ca2+ transport capability in vivo, we expressed AtCCX2 in thelow-affinity Ca2+ uptake-deficient triple S. cerevisiae yeast mu-tant K667, which lacks the vacuolar ATPase (PMC1), the vacu-olar exchanger (VCX1), and the cytosolic regulatory subunit(CNB1). The K667 triple mutant has been reported to show aCa2+ uptake deficiency and reduced growth at high external[Ca2+] (29). This evidence was indeed confirmed by us and,importantly, the K667 Ca2+-sensitivity was partially suppressedby AtCCX2 expression (Fig. S3), hence supporting its role inCa2+ transport.

AtCCX2 Regulates Cytosolic and ER Ca2+ Fluxes. The overall resultssuggest a role for CCX2 in Ca2+ transport and convinced us toanalyze in vivo Ca2+ dynamics in the cytosol of WT, ccx2, andCCX2-OE plants transformed with the NES-YC3.6 Cameleon

Fig. 1. In vitro growth test conducted on Col-0 WT, ccx2, ccx2-Com-plemented (ccx2-C) and CCX2-OE (OE) lines subjected to NaCl treatment.Relative primary (PR, A) and lateral (LR, B) root length (L) and shoot area (SA,C) were recorded after 7 d of treatment. Histograms represent relativegrowth values with respect to WT. Error bars represent the SD (n = 25–45 forroot; n = 15–35 for shoot). Asterisks indicate statistically significant differ-ences (P = 0.05) between WT and mutants by ANOVA Duncan’s new multi-ple-range test. (D) WT, ccx2, ccx2-C and CCX2-OE plants 7 d after 100 mMNaCl treatment.

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sensor (12, 33) subjected to NaCl (Fig. S4 A–L and V). Cellsfrom the root maturation zone were considered for the analysis(Movies S3–S5). Interestingly, the Cameleon ratios, expressed ascpVenus/CFP values that report Ca2+ level (higher ratio =higher Ca2+ concentration), showed significant differences in thecells of the region of interest (ROI) among the three genotypesalready under resting conditions (Fig. S4W). Particularly, thecytosolic resting Ca2+ level was significantly lower and higher inccx2 and CCX2-OE roots, respectively, in comparison with WT.Moreover, to study NaCl-induced Ca2+ increase in the threegenotypes, a custom perfusion set-up for in vivo fluorescencemicroscopy of Arabidopsis roots was employed (as in refs. 4 and34). Seedlings were challenged with 100 and 200 mM NaCl stress(8 min of treatment) and a first cytosolic [Ca2+] peak was ob-served (only at 200 mM NaCl), followed by a second and higherpeak when the NaCl solution was replaced with a control solu-tion (Fig. S4V). Because the resting Ca2+ concentrations weredifferent among the genotypes (Fig. 3 B, E, H, and K), thenormalized ratio changes were compared (Fig. 3 C and I and Fig.S5 A–C), and this revealed that the first peak (NaCl adminis-tration) showed a milder slope in the FRET curve and a lowermaximum normalized peak in ccx2 at 200 mM (Fig. 3 D–F).Despite CCX2-OE plants showing a higher basal Ca2+ levelcompared with the WT (Fig. 3B and Fig. S4W), the CCX2-OEnormalized FRET ratios were similar to the WT in response toNaCl (Fig. 3C). NaCl removal caused lower and higher maximalfree [Ca2+]cyt (Fig. 3G) and maximum normalized peaks (Fig. 3 Iand L) in ccx2 and CCX2-OE mutants, respectively, comparedwith the WT. The slope of normalized FRET curves suggeststhat Ca2+ accumulation kinetics were slower in ccx2 and faster inCCX2-OE, with respect to WT (Fig. 3J).We then tested a milder stress by treating the seedlings with

100 mM NaCl. In this latter case we observed smaller differencesamong the genotypes, which were not statistically different.However, CCX2-OE plants still showed the tendency of a highermaximum Ca2+ peak compared with the WT, hence essentiallyconfirming the behavior observed at 200 mM NaCl. Therefore,this phenotype was dependent upon the magnitude of the stim-ulus (Fig. S5 A–C). We argue that the absence of CCX2 or itsoverexpression leads, respectively, to loss- and gain-of-functionphenotypes with respect to the maximum [Ca2+]cyt accumulation

in response to both NaCl administration and removal. Given theabove data on [Ca2+]cyt homeostasis (Fig. 3) and the fact thatCCX2 is localized to the ER (Fig. 2), an additional step was toassess ER luminal [Ca2+] ([Ca2+]ER) response to NaCl stressusing the CRT-D4-ER Cameleon sensor (4) (Fig. S4 M–U andX). The CRT-D4-ER expression was lower compared with NES-YC3.6, which forced us to use a higher-magnification objective(20× instead of 4×) for ER luminal Ca2+ analyses. However, thesame ROI was considered for ER and cytosol Ca2+ measure-ments (Fig. S4 V and X). Under resting conditions, WT and ccx2genotypes did not show significant differences in the [Ca2+]ER(Fig. S4Y) and NaCl addition did not induce any clear change in[Ca2+]ER in WT or ccx2 plants (Fig. 4A). This result may dependon the sensitivity of the sensor used or the Ca2+ threshold re-quired for its accumulation in the ER, similarly to that reportedfor mitochondria. Nevertheless, noteworthy results were ob-served for FRET ratios in CRT-D4-ER ccx2 mutants when NaClwas removed (Fig. 4 A and B). NaCl removal caused a gradualrise in [Ca2+]ER (Movies S6 and S7), and notably, the FRETratios significantly differed between WT and ccx2 seedlings,showing a higher maximal free [Ca2+]ER in ccx2 (P = 0.009).Differences between WT and ccx2 in ER [Ca2+] were also in thiscase more pronounced at 200 mM than at 100 mM NaCl treat-ment (Fig. 4 B–E and Fig. S5 D–F).Finally, we tested cytosolic and ER Ca2+ dynamics in response to

external ATP (eATP), that has no direct effect on the solutionosmotic potential and induces a strong Ca2+ transient in the cytosolof root tip cells. No differences were observed for [Ca2+]cyt betweenthe WT and CCX2 loss- and gain-of-function mutants (Fig. S5 G–Jand Movies S8–S10). Conversely, striking differences in the con-centration, maximum peak, and accumulation kinetics of [Ca2+]ERwere observed between WT and ccx2, (Fig. S5 K–P and Movies S11and S12), as already observed for NaCl treatment.In conclusion, the ccx2 mutant showed lower cytosolic Ca2+

increase and higher ER Ca2+ accumulation, a phenotype thatwas particularly remarkable upon NaCl removal.

Growth and Ca2+ Accumulation Dynamics in ccx1 and ccx3 MutantsGive Insights into the Role of the AtCCX Family in Regulating NaClResponses. A step forward in the analysis was to shed light on therole of CCX1 and CCX3 genes, which are other members of the

Fig. 2. AtCCX2-GFP subcellular localization in the ccx2 Arabidopsis knockout background and in agro-infiltrated leaves of Nicotiana benthamiana. (A–C)Representative fluorescence images of AtCCX2-GFP in a selected focal plane of a ccx2/AtCCX2-GFP Arabidopsis plantlet in different tissues/cell types: coty-ledon epidermal cells (A); root mature zone cells (B); and root tip cells (C). (D–F) Bright-field images corresponding to the fluorescence images shown in A–C.(G) Fluorescence of AtCCX2-GFP in a representative tobacco epidermal cell coexpressed with mCherry-HDEL used as an ER+ marker. (H) Fluorescence ofmCherry-HDEL of the cell shown in G. (I) Merged fluorescent signals of GFP and mCherry. (J) Two-dimensional scatter plot visualizing the correlation of theGFP and mCherry pixel intensities over all pixels in the images in G and H. (K) Fluorescence of AtCCX2-GFP in a representative tobacco epidermal cellcoexpressed with free RFP used as cytosolic and nuclear-positive marker. (L) Fluorescence of RFP of the cell shown in K. (M) Merged fluorescent signals of GFPand RFP. (N) Two-dimensional scatter plot visualizing the correlation of the GFP and RFP pixel intensities over all pixels in the images in K and L. NE, nuclearenvelope. (Scale bars, 25 μm.)

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CCX family induced by osmotic and salt stresses (Fig. S1A). Theccx1 mutant displayed a reduced lateral root length comparedwith WT during 100 mM NaCl stress, while ccx3 showed nosignificant differences (Fig. S6 A and C). Similar results wereobserved after exposure to KCl and mannitol treatments (Fig. S6D, F, G, and I).Analysis of Ca2+ dynamics highlighted a significantly lower

and higher steady state [Ca2+]cyt in ccx1 and ccx3 mutants, re-spectively, with respect to the WT (Fig. S7B). Notably, ccx3 andccx1 showed contrasting responses in the regulation of Ca2+

homeostasis upon NaCl, with a maximum peak after NaCl stressin the former and considerably lower levels in the latter (Fig. S7C–M). Finally, ER Ca2+ measurements conducted on ccx1 plants(but not ccx3 because of silencing-related effects) showed similarresponses to those observed in ccx2 (Fig. S7 N–S).

DiscussionOur results shed light on the role of AtCCX2, a putative cation/Ca2+ exchanger that is localized to the ER (Fig. 2) and give in-sights into the role of the AtCCX family in the regulation of[Ca2+]cyt and [Ca2+]ER dynamics upon salt stress. Salt stress isusually associated with a rise of [Ca2+] in the cytosol and thesubcellular compartments of plant cells (35–37) and other re-sponses involving PM depolarization and ROS homeostasis (38).

In plants, the impact of ER on the regulation of Ca2+ fluxes andrelated responses have remained largely undecoded (7), andthere is little evidence for a role of the ER as a regulator of [Ca2+]cythomeostasis upon stress in vivo (4). CCX2 characterization addsanother piece to this puzzle and highlights the presence of a genethat impinges on the regulation of ER-cytosol intracellular Ca2+

dynamics upon abiotic stress.

AtCCX2 Loss-of-Function and Gain-of-Function Mutants DisplayContrasting Responses to Salt Stress. CCX2 loss- and gain-of-function mutants displayed impaired and enhanced growth com-pared with the WT upon NaCl stress and also upon osmotic stresstreatment (Fig. 1 and Fig. S2). As already observed for otherArabidopsis mutants (39), the impaired root growth upon EGTAtreatment may suggest that CCX2 is involved in NaCl-stress re-sponses in a Ca2+-dependent way. However, the EGTA effect onintracellular Ca2+ is not fully understood, and long treatments canbe misleading.The link between CCX2 and salt stress was further strength-

ened by results obtained from CCX gene-expression analysis onNaCl-stressed seedlings (Fig. S2J). As already reported for othergain-of-function mutants (40), the CCX2 transcript steady-statelevel was significantly induced upon NaCl in CCX2-OE seed-lings at 2 HSI, most likely indicating possible stress-dependent

Fig. 3. [Ca2+]cyt measured in root elongation zone in Col-0 WT, ccx2, CCX2-OE2 and CCX2-OE3 plants subjected to NaCl treatment. (A and G) Representativetrace of cytosolic Ca2+ kinetics recorded in plants subjected to NaCl administration (first peak, A–F) and removal (second peak, G–L). Small black arrow in-dicates analyzed peak. (B and H) Cytosolic Ca2+ FRET raw ratios. (C and I) Normalized cytosolic Ca2+ FRET ratios. (D and J) Slope of Ca2+ accumulation curves. (Eand K) Representative root-ratio images under perfusion before (140 and 630 s) and after (220 and 710 s) NaCl administration (E) and removal (K). (F and L)Maximum normalized Ca2+ peak. A t test was conducted to assess differences between WT and mutants (*P < 0.05, **P < 0.01). Error bars represent the SD(n = 10–12).

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posttranscriptional regulation of expression. The unchangedexpression of CCX1 and the slight induction of the CCX3 gene inthe ccx2 background upon NaCl treatment suggest a lack ofcompensation for CCX2 expression during salt stress. These datasupport nonoverlapping functions for the CCX transporters.Expression analysis did not suggest a role for CCX2 in the reg-ulation of RD29B.1 and SOS1 genes (Fig. S2K). As in our ex-periment, SOS1 was not induced, contrary to RD29B that is awell-known marker for osmotic stress, and the potential role ofCCX2 on SOS1 regulation is difficult to deduce. In contrast,SOS3 (31) was less expressed in ccx2 plants, and ACA2 (17) wasmore expressed in CCX2-OE under NaCl treatment with respectto WT. Given that SOS3 and ACA2 are well-known players inCa2+ regulation under NaCl stress, these results further suggestan important role for CCX2 in Ca2+ homeostasis. Interestingly,because ACA2-predicted function is to transport Ca2+ into theER (17), its higher expression in CCX2-OE mutants, upon NaClstress, might be related to the higher [Ca2+]cyt response observedin these plants.

AtCCX2 Regulates Cytosolic and ER Ca2+ Dynamics upon NaCl. Be-cause CCX2 showed its involvement in Ca2+ transport in yeast(Fig. S3) and is localized to the ER (Fig. 2), in vivo Ca2+ analysiswas carried out on WT and CCX2 mutant roots expressing cy-tosolic and ER Cameleon sensors (4, 12). Differences betweenWT and CCX2 mutants were already recorded for cytosolicresting prestimulus Ca2+ levels, which were lower and higher inccx2 and CCX2-OE, respectively, compared with the WT (Fig.S4W). Furthermore, the lower cytosolic Ca2+ peak (Fig. 3 F andL) and slower Ca2+ accumulation (Fig. 3 D and J) recorded upon200 mM NaCl treatment in the ccx2 mutant compared with WTcorrelated well with the observed plant growth (Fig. 1). Stimu-lation of cytosolic and ER Ca2+ accumulation by NaCl werepreviously reported (4, 36), and a positive role for Ca2+ in theregulation of salt stress-related gene expression has already beenestablished (38). Thus, differences in growth observed in NaCl-treated WT, ccx2, and CCX2-OE plants were most likely due todifferential regulation of Ca2+ homeostasis that led to lower andhigher [Ca2+]cyt in the CCX2 loss- and gain-of-function mutants,respectively (Fig. 3). The significantly higher steady state of[Ca2+]ER compared with [Ca2+]cyt (considering different in vitroCa2+ affinity for Ca2+ of the YC3.6 Kd = 250 nM and the D4Kd = 195 μM), and higher Ca2+ peak observed in NaCl-treatedccx2 roots with respect to the WT (Fig. 4 C–E), allowed theidentification of CCX2 as a pivotal player in the regulation of[Ca2+]ER and [Ca2+]cyt dynamics. Furthermore, ccx2 seedlingstreated with eATP were characterized by a higher [Ca2+]ER peakwith respect to WT, suggesting an alteration of ER Ca2+ ho-meostasis in the mutant even though no differences were ob-served in the cytosol (Fig. S5). Although other Arabidopsismutants showed different Ca2+ accumulation in subcellular

compartments (e.g., MICU knockout mutants showed alteredmitochondria Ca2+ levels compared with the WT), this did notlead to different cytosol Ca2+ dynamics (34). Indeed, in the caseof CCX2, coupled cytosolic and ER Ca2+ changes upon NaClremoval were measured. We believe that CCX2 is involved insalt stress tolerance through its role in Ca2+ transport regulationacross the ER and cytosol. The ER does not seem to be a maincontributor for stimulus-induced increases in cytosolic Ca2+

concentration (4). However, luminal ER Ca2+ elevations typi-cally follow cytosolic ones and when CCX2 is missing, this ER/Ca2+ dynamic is altered. The absence of AtCCX2 activity re-sults in decreased cytosolic and increased ER Ca2+ concentra-tions in comparison with WT and the gain-of-function mutantsin both salt and nonsalt osmotic stress conditions (Figs. 3 and 4and Fig. S8), resulting in higher plant stress sensitivity. Finally,as observed for the rice vacuolar transporter OsCCX2 (29),AtCCX2 partially suppresses the Ca2+ sensitivity of a yeast mu-tant deficient in low-affinity Ca2+ uptake (Fig. S3), supporting itsinvolvement in Ca2+ transport. The overall data suggest a directinvolvement of CCX2 in the control of Ca2+ fluxes between theER and the cytosol, which plays a key role in the ability of theplant to cope with osmotic stresses.

Toward Defining a Role of AtCCX Family. In addition to the extendedstudy of CCX2, CCX1 and CCX3 knockout mutants were analyzed.While CCX1 was proposed to be involved in modulating Ca2+

signaling (23), CCX3 was not previously associated with Ca2+ (24).Growth tests (Fig. 1 and Fig. S6) and in vivo Ca2+ measure-

ments (Fig. 3 and Fig. S7) of knockout mutants suggested thatCCX2 and CCX1 genes are likely to play similar roles in plantstress tolerance, while CCX3 seems to play a different role fromCCX1 and CCX2. In support of those results, a phylogeneticanalysis of the CCX family (20, 28) placed Arabidopsis CCX1 andCCX2 closer in the tree than CCX3 and CCX4.As already hypothesized for Arabidopsis, regulation of Ca2+

homeostasis is associated with different responses during saltstress (4, 36, 38), in which different members of the CCX familyseem to play a role. Taken together, our results give insights intothe unexplored role of the CCX gene family in planta andhighlight a major role for CCX2 in the regulation of [Ca2+]cyt and[Ca2+]ER, with downstream effects on salt stress tolerance.

MethodsWT, ccx1, ccx2, ccx3, and CCX2-OE plants were in the Col-0 background.Growth conditions and tolerance tests, generation of transgenic lines, yeastcomplementation test, transcriptomic experiment, measurement of Ca2+

dynamics, statistical methods, protocols used for subcellular localization, andother imaging measurements are reported in SI Methods.

ACKNOWLEDGMENTS. We thank Dr. Andrea Rosanoff (Center for Magne-sium Education & Research) for the critical reading; Jérôme Gennen for helpwith the genetic constructs; and Prof. Kyle W. Cunningham (The Johns

Fig. 4. [Ca2+]ER measured in root elongation zone in Col-0 WT and ccx2 plants subjected to NaCl treatment. (A) Representative trace of cytosolic Ca2+ kineticsrecorded in plants subjected to NaCl administration (first peak, not considered) and removal (second peak, black arrow). (B) Representative root-ratio imagesunder perfusion before (680 s) and after (730 s) NaCl removal. (C) ER Ca2+ FRET raw ratios. (D) Normalized ER Ca2+ FRET ratios. (E) Maximum normalized Ca2+

peak. A t test was conducted to assess differences between WT and mutant (n.s. = not significant; P = P value). Error bars represent the SD (n = 12–13).

3970 | www.pnas.org/cgi/doi/10.1073/pnas.1720422115 Corso et al.

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Hopkins University) for providing yeast strains. This work was supported byFonds National de la Recherche Scientifique Grant PDR T.0206.13 (to N.V.);European Molecular Biology Organization short-term Fellowship ASTF 604-

2015 (to M.C.); the Ministero dell’Istruzione, dell’Università e della RicercaGrant FIRB 2010 RBFR10S1LJ_001 (to A.C.); and the Università degli Studi diMilano, Piano di Sviluppo di Ateneo 2015-2016 (to A.C.).

1. Golldack D, Li C, Mohan H, Probst N (2014) Tolerance to drought and salt stress inplants: Unraveling the signaling networks. Front Plant Sci 5:151.

2. Dodd AN, Kudla J, Sanders D (2010) The language of calcium signaling. Annu RevPlant Biol 61:593–620.

3. Kurusu T, Kuchitsu K, Tada Y (2015) Plant signaling networks involving Ca(2+) andRboh/Nox-mediated ROS production under salinity stress. Front Plant Sci 6:427.

4. Bonza MC, et al. (2013) Analyses of Ca2+ accumulation and dynamics in the endo-plasmic reticulum of Arabidopsis root cells using a genetically encoded Cameleonsensor. Plant Physiol 163:1230–1241.

5. Bonza MC, De Michelis MI (2011) The plant Ca2+ -ATPase repertoire: Biochemicalfeatures and physiological functions. Plant Biol (Stuttg) 13:421–430.

6. Loro G, et al. (2012) Targeting of Cameleons to various subcellular compartmentsreveals a strict cytoplasmic/mitochondrial Ca2+ handling relationship in plant cells.Plant J 71:1–13.

7. Navazio L, et al. (2000) Calcium release from the endoplasmic reticulum of higherplants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phos-phate. Proc Natl Acad Sci USA 97:8693–8698.

8. Baliardini C, Meyer C-L, Salis P, Saumitou-Laprade P, Verbruggen N (2015) CATIONEXCHANGER1 cosegregates with cadmium tolerance in the metal hyperaccumulatorArabidopsis halleri and plays a role in limiting oxidative stress in Arabidopsis Spp.Plant Physiol 169:549–559.

9. Baliardini C, Corso M, Verbruggen N (2016) Transcriptomic analysis supports the roleof CATION EXCHANGER 1 in cellular homeostasis and oxidative stress limitationduring cadmium stress. Plant Signal Behav 11:e1183861.

10. Costa A, Kudla J (2015) Colorful insights: Advances in imaging drive novel break-throughs in Ca2+ signaling. Mol Plant 8:352–355.

11. Costa A, Candeo A, Fieramonti L, Valentini G, Bassi A (2013) Calcium dynamics in rootcells of Arabidopsis thaliana visualized with selective plane illumination microscopy.PLoS One 8:e75646.

12. Krebs M, et al. (2012) FRET-based genetically encoded sensors allow high-resolutionlive cell imaging of Ca2+ dynamics. Plant J 69:181–192.

13. Loro G, et al. (2016) Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleonfluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in thestroma. Plant Physiol 171:2317–2330.

14. Wang M, Kaufman RJ (2014) The impact of the endoplasmic reticulum protein-fold-ing environment on cancer development. Nat Rev Cancer 14:581–597.

15. Howell SH (2013) Endoplasmic reticulum stress responses in plants. Annu Rev PlantBiol 64:477–499.

16. Stefano G, Brandizzi F (2017) Advances in plant ER architecture and dynamics. PlantPhysiol 176:178–186.

17. Anil VS, Rajkumar P, Kumar P, Mathew MK (2008) A plant Ca2+ pump, ACA2, relievessalt hypersensitivity in yeast. Modulation of cytosolic calcium signature and activationof adaptive Na+ homeostasis. J Biol Chem 283:3497–3506.

18. Liang F, Cunningham KW, Harper JF, Sze H (1997) ECA1 complements yeast mutantsdefective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase inArabidopsis thaliana. Proc Natl Acad Sci USA 94:8579–8584.

19. Cai X, Lytton J (2004) The cation/Ca(2+) exchanger superfamily: Phylogenetic analysisand structural implications. Mol Biol Evol 21:1692–1703.

20. Pittman JK, Hirschi KD (2016) Phylogenetic analysis and protein structure modellingidentifies distinct Ca(2+)/Cation antiporters and conservation of gene family structurewithin Arabidopsis and rice species. Rice (N Y) 9:3.

21. Shigaki T, Hirschi KD (2006) Diverse functions and molecular properties emerging forCAX cation/H+ exchangers in plants. Plant Biol (Stuttg) 8:419–429.

22. Zhao J, et al. (2009) Interaction between Arabidopsis Ca2+/H+ exchangers CAX1 andCAX3. J Biol Chem 284:4605–4615.

23. Mei H, et al. (2009) Root development under metal stress in Arabidopsis thalianarequires the H+/cation antiporter CAX4. New Phytol 183:95–105.

24. Li Z, et al. (2016) CCX1, a putative Cation/Ca2+ exchanger, participates in regulation ofreactive oxygen species homeostasis and leaf senescence. Plant Cell Physiol 57:2611–2619.

25. Morris J, et al. (2008) AtCCX3 is an Arabidopsis endomembrane H+ -dependent K+

transporter. Plant Physiol 148:1474–1486.26. Zhang X, Zhang M, Takano T, Liu S (2011) Characterization of an AtCCX5 gene from

Arabidopsis thaliana that involves in high-affinity K+ uptake and Na+ transport inyeast. Biochem Biophys Res Commun 414:96–100.

27. Shigaki T, Rees I, Nakhleh L, Hirschi KD (2006) Identification of three distinct phylo-genetic groups of CAX cation/proton antiporters. J Mol Evol 63:815–825.

28. Emery L, Whelan S, Hirschi KD, Pittman JK (2012) Protein phylogenetic analysis ofCa(2+)/cation antiporters and insights into their evolution in plants. Front Plant Sci 3:1–19.

29. Yadav AK, et al. (2015) A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+

/cation transport in yeast. Sci Rep 5:17117.30. Winter D, et al. (2007) An “Electronic Fluorescent Pictograph” browser for exploring

and analyzing large-scale biological data sets. PLoS One 2:e718.31. Gong D, Guo Y, Schumaker KS, Zhu J-K (2004) The SOS3 family of calcium sensors and

SOS2 family of protein kinases in Arabidopsis. Plant Physiol 134:919–926.32. Brandizzi F, Fricker M, Hawes C (2002) A greener world: The revolution in plant

bioimaging. Nat Rev Mol Cell Biol 3:520–530.33. Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A (2004) Expanded dynamic

range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescentproteins. Proc Natl Acad Sci USA 101:10554–10559.

34. Wagner S, et al. (2015) The EF-Hand Ca2+ binding protein MICU choreographs mi-tochondrial Ca2+ dynamics in Arabidopsis. Plant Cell 27:3190–3212.

35. Stephan AB, Kunz H-H, Yang E, Schroeder JI (2016) Rapid hyperosmotic-induced Ca2+

responses in Arabidopsis thaliana exhibit sensory potentiation and involvement ofplastidial KEA transporters. Proc Natl Acad Sci USA 113:E5242–E5249.

36. Choi W-G, Toyota M, Kim S-H, Hilleary R, Gilroy S (2014) Salt stress-induced Ca2+

waves are associated with rapid, long-distance root-to-shoot signaling in plants. ProcNatl Acad Sci USA 111:6497–6502.

37. Evans MJ, Choi W-G, Gilroy S, Morris RJ (2016) A ROS-assisted calcium wave de-pendent on AtRBOHD and TPC1 propagates the systemic response to salt stress inArabidopsis roots. Plant Physiol 171:1771–1784.

38. Choi W-G, Hilleary R, Swanson SJ, Kim S-H, Gilroy S (2016) Rapid, long-distanceelectrical and calcium signaling in plants. Annu Rev Plant Biol 67:287–307.

39. Bidzinski P, et al. (2014) Physiological characterization and genetic modifiers of ab-errant root thigmomorphogenesis in mutants of Arabidopsis thaliana MILDEW LO-CUS O genes. Plant Cell Environ 37:2738–2753.

40. Chinnusamy V, et al. (2003) ICE1: A regulator of cold-induced transcriptome andfreezing tolerance in Arabidopsis. Genes Dev 17:1043–1054.

41. Meyer C-L, et al. (2011) Isolation and characterization of Arabidopsis halleri andThlaspi caerulescens phytochelatin synthases. Planta 234:83–95.

42. Ruberti C, Costa A, Pedrazzini E, Lo Schiavo F, Zottini M (2014) FISSION1A, an Ara-bidopsis tail-anchored protein, is localized to three subcellular compartments. MolPlant 7:1393–1396.

43. Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markersfor co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136.

44. Waadt R, Kudla J (2008) In planta visualization of protein interactions using bi-molecular fluorescence complementation (BiFC). Cold Spring Harb Protoc 2008:pdb.prot4995.

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