A Na+/Ca2+ Exchanger-like Protein (AtNCL) Involved in Salt Stress in Arabidopsis*

Background: Na+/Ca2+ exchangers were found in animals, but no Na+/Ca2+ exchanger had been reported in plants. Results: AtNCL showed Na+/Ca2+ exchanger-like activity and regulated stress response. Conclusion: Functional Na+/Ca2+ exchanger-like protein exists in plants. Significance: Na+/Ca2+ exchange also play a role in Ca2+ homeostasis under abiotic stress in plants. Calcium ions (Ca2+) play a crucial role in many key physiological processes; thus, the maintenance of Ca2+ homeostasis is of primary importance. Na+/Ca2+ exchangers (NCXs) play an important role in Ca2+ homeostasis in animal excitable cells. Bioinformatic analysis of the Arabidopsis genome suggested the existence of a putative NCX gene, Arabidopsis NCX-like (AtNCL), encoding a protein with an NCX-like structure and different from Ca2+/H+ exchangers and Na+/H+ exchangers previously identified in plant. AtNCL was identified to localize in the Arabidopsis cell membrane fraction, have the ability of binding Ca2+, and possess NCX-like activity in a heterologous expression system of cultured mammalian CHO-K1 cells. AtNCL is broadly expressed in Arabidopsis, and abiotic stresses stimulated its transcript expression. Loss-of-function atncl mutants were less sensitive to salt stress than wild-type or AtNCL transgenic overexpression lines. In addition, the total calcium content in whole atncl mutant seedlings was higher than that in wild type by atomic absorption spectroscopy. The level of free Ca2+ in the cytosol and Ca2+ flux at the root tips of atncl mutant plants, as detected using transgenic aequorin and a scanning ion-selective electrode, required a longer recovery time following NaCl stress compared with that in wild type. All of these data suggest that AtNCL encodes a Na+/Ca2+ exchanger-like protein that participates in the maintenance of Ca2+ homeostasis in Arabidopsis. AtNCL may represent a new type of Ca2+ transporter in higher plants.

Calcium ions (Ca 2ϩ ) are required for many physiological functions. Numerous stimulations produce change in the cyto-solic concentration of Ca 2ϩ [Ca 2ϩ ] cyt 3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The mechanisms of production and elimination of [Ca 2ϩ ] cyt have become a key topic in Ca 2ϩ signaling and related functions. Various environmental stresses can stimulate the opening of Ca 2ϩ -permeable channels in the plasma or inner membrane, leading to an influx of Ca 2ϩ along its electrochemical gradient (12,13). After this influx, the cytosolic level of Ca 2ϩ could be returned to its resting state through the activity of several types of Ca 2ϩ transport proteins that extrude Ca 2ϩ out of the cytosol.
In animal cells, Ca 2ϩ transport proteins include Ca 2ϩ -ATPase pumps that use ATP directly and transporters that are driven indirectly by ATP using ion gradients (14 -19). Na ϩ /Ca 2ϩ exchangers (NCXs) are due to the later Ca 2ϩ transport proteins. Na ϩ /Ca 2ϩ exchange was first reported in guinea pig cardiac muscle (20). Since then, NCX proteins have been identified in the plasma membrane of many types of cells (21)(22)(23)(24)(25)(26)(27). NCX proteins, which consist of 9 -11 transmembrane domains with a large intracellular hydrophilic loop, play important roles in adjusting the [Ca 2ϩ ] cyt (28). For example, NCXs maintain the [Ca 2ϩ ] cyt during the intrinsic excitation-contraction cycle in cardiac muscle cells (29). NCXs are bidirectional Ca 2ϩ transporters, and the direction of Ca 2ϩ flux is dependent on the electrochemical Na ϩ gradient (30 -33). Thus, NCXs play important roles in Ca 2ϩ signaling and homeostasis in animal cells.
Until recently, only Ca 2ϩ /H ϩ exchangers (CAXs) and Ca 2ϩ -ATPases were reported to transport Ca 2ϩ through the membrane in plant cells (1,34 -39), whereas Na ϩ /H ϩ exchangers (NHXs) were reported to drive Na ϩ through the plasma membrane based on a H ϩ gradient (40,41). Thus, we sought to address whether a protein similar to animal NCXs exists in planta. Wang et al. suggested the existence of Na ϩ -dependent Ca 2ϩ uptake activity in vacuolar membrane vesicles from wheat (42). Here one NCX-like gene in Arabidopsis (AtNCL) encoded a protein similar to animal NCXs in structure, had Na ϩ /Ca 2ϩ exchange activity, and was involved in Ca 2ϩ homeostasis under salt stress in Arabidopsis.

EXPERIMENTAL PROCEDURES
Plant Materials and Growth Conditions-The Arabidopsis thaliana stocks described in this work were of the Columbia (Col) ecotype. atncl-1 and atncl-2 correspond to Syngenta Arabidopsis Insertion Library (SAIL)_791_D12 and SAIL_ 770_A10, respectively. For salt stress, 7-day-old seedlings were transferred to plates containing 1/2 MS medium or 1/2 MS medium supplemented with 150 mM NaCl. After 7 days, the seedlings were photographed, and the survival rate was determined based on the number of plants with two to four true leaves that had become completely white in color; the chlorophyll content was measured as reported previously (43). Freezing tolerance was assayed as described (44), 10-day-old seedlings were frozen at Ϫ6°C for 3 h and thawed at 4°C for 12 h. After a 5-day recovery, survival rate and chlorophyll contents were measured. Heat stress was performed as described previously (45). 7-day-old seedlings were exposed to 45°C for 75 min and recovered at 22°C for 7days.
Yeast Functional Complementation Test-A CAX function test was carried out in Saccharomyces cerevisiae strain K667 (MATa cnb1::LEU1 pmc1::TRP1 vcx1⌬) as described (36). The coding sequences of AtNCL, sAtNCL, and OsCAX3 were isolated and cloned into p416GPD then transformed into K667 cells using the LiAc/PEG method. Saturated liquid cultures of K667 containing the various plasmids were diluted to A 600 ϭ 1.0 then spotted onto selection medium (-Ura), yeast extract peptone dextrose (YPD) medium, or YPD containing 200 mM CaCl 2 . The dishes were incubated at 30°C for 3 days before taking pictures.
Quantitative RT-PCR-qRT-PCR was employed to measure the transcript levels. The RNA samples were pretreated extensively with an RNase-free DNase to remove any contaminating genomic DNA prior to use. The PCR primers are listed in supplemental Table 1. qRT-PCRs were performed in 96-well blocks with an Applied Biosystems 7500 real-time PCR system using the SYBR Green I mix in a volume of 20 l. The specificity  of the PCRs was determined by melt curve analysis of the amplified products using the standard methods installed in the system. The comparative ⌬⌬CT method was employed to evaluate relative quantities of each product amplified from the samples. All qRT-PCRs were performed in biological triplicates using RNA samples extracted from three independent plant materials grown under identical growth conditions. ␤-Glucuronidase (GUS) Assay-Histochemical staining and fluorometric assays were performed according to the method reported by Jefferson (66).
Bacterial Recombinant Protein Expression and 45 Ca 2ϩ Overlay Assays-The putative EF-hand Ca 2ϩ binding domain (CaBD) sequence from AtNCL was cloned into pET-30(a) (Novagen) then transformed into Escherichia coli BL21 (DE3) cells (Novagen). His-tagged recombinant AtNCL was separated using Ni-affinity column (Novagen).The binding of 45 Ca 2ϩ to CaBD of AtNCL was assayed as described (47). The purified protein was resolved on a 12 or 18% SDS-polyacrylamide gel then transferred to PVDF membrane and incubated for 30 min with 100 mCi/ml 45 CaC1 2 . The membrane was then washed and exposed to a storage PhosphorImage screen for 12 h. Images were collected using a Typhoon 9210 imager (Amersham Biosciences).
Measurement of Reverse-mode NCX Activity-Full-length AtNCL CDS with an extra Kozak sequence (GCCACC) before the initiation codon was cloned into pDsRed1-N1 using NdeI and AgeI. CHO-K1 cells were transfected with the resulting construct by electroporation as described (48). Reverse-mode NCX activity was measured as described (49). The fluorescence intensity was calculated at each time point before and after the addition of Ca 2ϩ containing loading buffer (F basal and F, respectively). The data for each cell were plotted as follows: Fluorescence Imaging-Fluorescence microscopy was performed using a Zeiss LSM 510 Meta microscope (Carl Zeiss Micro Imaging, Inc.). For yellow fluorescent protein (YFP) and propidium iodide (PI) imaging, an argon ion laser at 514 nm was used for excitation; emission was detected using a BP500 -550 nm filter for YFP and LP615 nm for PI. Fluo-3 quantitative analysis was done as described (50,51). Excitation of Fluo-3 was ] cyt level in the AtNCL-expressing cells increased. In comparison, the vector control and unloaded cells showed little change, whereas outside 5 mM Ni 2ϩ or 150 mM Na ϩ inhibited AtNCL activity. C, ion selectivity test of AtNCL. D, changing of outside pH from 7.4 to 6.4 having no effect on AtNCL-mediated Ca 2ϩ uptake. E, outside K ϩ not necessary for Ca 2ϩ uptake. F, outside 10 mM Mg 2ϩ inhibition Ca 2ϩ uptake. performed using an argon ion laser at 488 nm. Emitted light was detected through a BP505-530-nm filter from a 545-nm dichroic mirror. DsRed fluorescence was excited using a green helium-neon laser at 543 nm and was detected through a 545-nm dichroic mirror and LP560-nm filter. The images were processed using LSM 4.2 software (Carl Zeiss Micro Imaging, Inc.).
Plant Membrane Fractionation and Western Blotting-Protein extracts were prepared from the wild-type or transgenic seedlings and separated into soluble and membrane fractions by ultracentrifugation as described previously (52). The fractions were then analyzed by Western blotting using anti-GFP serum as the primary antibody.
Element Analysis by AAS-The pretreated or untreated seedlings were washed with deionized water, dried using a blast dryer, and weighed. The seedlings were then subjected to digestion by the aqueous method using the chloric acid/perchloric acid (4:1, v/v) method. Next, the digested samples were diluted with deionized water and analyzed by AAS (AA240FSϩ240Z, Varian).
Noninvasive Ion Flux Measurement Using an Ion-selective Microelectrode-The net fluxes of Ca 2ϩ and H ϩ in the root tips of 4-day-old seedlings grown on MS plates were measured noninvasively by SIET (54) using the BIO-001A SIET system (Xuyue (Beijing) Science and Technology Co., Ltd., Beijing, China). The SIET system measures static ionic/molecular concentrations and concentration gradients using ion-selective microelectrodes (55). The concentration gradient was measured by moving the electrode repeatedly between two positions along a predefined excursion (5-30 m) at a fixed frequency in the range of 0.3-0.5 Hz.

RESULTS
AtNCL Is a Putative NCX-AtNCL (At1g53210) was predicted as a sodium/calcium exchanger family protein by the TAIR database. AtNCL comprises seven exons and six introns, encoding a putative protein with 585 amino acids. The N-terminal-most 22 amino acids in AtNCL comprise a putative signaling peptide (analyzed by SignalP). The protein had 10 putative transmembrane domains, with a hydrophilic loop between the fifth and sixth transmembrane domains containing two predicted EF-hand domains (analyzed by SMART). The deduced structure of AtNCL is similar to HsNCX1 from Homo sapiens (supplemental Fig. S1). AtNCL was considered a relative of CAXs (56), but it could not function as CAX in yeast, even if they have similar localization in yeast cells (Fig. 1). CAXs always have a conserved N-terminal autoinhibitory sequence (57)(58)(59); however, N-terminal deleted sAtNCL also cannot recover the yeast K667 phenotype (Fig. 1). On the other hand, AtNCL cannot work as a NHX in yeast (Fig. 1C), so we wanted to know whether it was a NCX-like protein.
Ca 2ϩ Binding Ability of AtNCL-Because animal NCXs have intracellular loop with Ca 2ϩ binding activity, we have tested the Ca 2ϩ binding ability of AtNCL. Ca 2ϩ -dependent electrophoretic mobility shift and 45 Ca 2ϩ binding assays were used to examine the predicted the Ca 2ϩ binding ability of the putative EF-hand domain in AtNCL (amino acids 262-430; CaBD). The vector pET-30(a)-CaBD was constructed as shown in supplemental Table S1, and recombinant CaBD tagged with six His residues at its N terminus was expressed in E. coli. Before loading the sample, EGTA or divalent ions were added to the sample. By SDS-PAGE, CaBD ran faster in the presence of 5 mM Ca 2ϩ than it did in the presence of EGTA or the bivalent ions Mg 2ϩ and Mn 2ϩ (Fig. 2A). According to our 45 Ca 2ϩ binding assay results, recombinant CaBD bound Ca 2ϩ as did our positive control, recombinant Arabidopsis CaM isoform 2 (AtCaM2), a known Ca 2ϩ -binding protein, whereas our negative control, bovine serum albumin (BSA), did not (Fig. 2B).
AtNCL Possesses NCX-like Activity in CHO-K1 cells-To assess whether AtNCL has NCX-like activity, a CHO-K1 cellbased heterologous expression system was used. CHO-K1 cells transfected with pAtNCL-DsRed were tested against CHO-K1 cells transfected with pDsRed1-N1 as a control. The [Ca 2ϩ ] cyt was indicated by Fluo-3 fluorescence. In Na ϩ -free buffer con- taining 2 mM Ca 2ϩ , the signal from the Na ϩ -loaded AtNCLexpressing cells rose to saturation within 5 min whereas the vector control showed little signal (Fig. 3B). In contrast, the [Ca 2ϩ ] cyt did not increase significantly if the cells were not loaded with Na ϩ (Fig. 3B). This suggests that the reverse NCX activity in the AtNCL-transfected cells (Fig. 3) was much higher than in the controls (Fig. 3). This activity was inhibited by 150 mM extracellular Na ϩ (the concentration was set approximately equal to the inner Na ϩ concentration to offset the Na ϩ gradient) or by 5 mM Ni 2ϩ , a NCX inhibitor used in animal cell (60) (Fig. 3B).
To confirm the ion selectivity for the exchange function of AtNCL, we performed another set of Ca 2ϩ flux assays using cells loaded with a buffer solution containing Na ϩ , K ϩ , or Li ϩ . The result demonstrated that Na ϩ and K ϩ both can drive the Ca 2ϩ flux and Na ϩ is more efficient than K ϩ . However, Li ϩ had no effect (Fig. 3C). Proton concentration was increased outside the cell by changing the bath buffer pH from 7.4 to 6.4, and the Na ϩ /Ca 2ϩ exchange activity was not significantly affected (Fig.  3D).
To reveal whether the activity of AtNCL is dependent on K ϩ -like Na ϩ /Ca 2ϩ -K ϩ exchanger, we tested the bath buffer with K ϩ or without K ϩ , respectively. The data indicated that outside K ϩ was not necessary for AtNCL activity (Fig. 3E).
Mg 2ϩ was reported not affect NCX activity (61). Here, we tested the Mg 2ϩ effect on AtNCL, by add 10 mM Mg 2ϩ to bath buffer. The result suggested that Mg 2ϩ can suppress the AtNCL activity (Fig. 3F). Overall, AtNCL has NCX-like Ca 2ϩ uptake activity in CHO-K1 cells.
Membrane Localization of AtNCL in Planta-AtNCL was reported to be localized to the vacuole by prediction and proteomics strategy (62)(63)(64)(65). In this study, p35S::AtNCL-YFP was constructed and transformed into Arabidopsis, the signal in the AtNCL-YFP transgenic seedlings was stronger near the plasma membrane after plasmolysis (Fig. 4A). Overlap detected between the PI and YFP signals indicated that AtNCL may localized to the plasma membrane in the transgenic root cells. However, AtNCL-YFP was also detected at the inner membrane.
Next, microsomal proteins were extracted from the p35S::AtNCL-YFP transgenic lines and probed with anti-GFP antibodies (Fig. 4B). A specific band was detected in microsome fraction but not in the soluble proteins from the transgenic lines or the microsomal proteins from wild-type seedlings.
AtNCL Expression in Arabidopsis-The expression pattern of AtNCL was examined using a GUS reporter gene fusion system (66). pAtNCL::AtNCL-GUS was constructed as described in supplemental Table S1 and transformed into Arabidopsis. GUS staining of 10 independent transgenic lines revealed that AtNCL was expressed broadly in the seedlings and flowers under normal growth conditions (Fig. 5A).
AtNCL expression was increased during abiotic stress, such as salt, ABA, heat shock, and cold stress, as indicated by qRT-PCR and GUS assay (Fig. 5, B-E). AtNCL expression increased within 1 h of exposure to 150 mM NaCl and reached its peak after approximately 5 h (Fig. 5B). Expression of the gene was unchanged following exposure to the same concentration of LiCl or in a mock sample under salt stress (data not shown). Exogenous ABA treatment caused a similar change in AtNCL expression (Fig. 5C). Heat shock and cold stress also increased AtNCL expression; however, heat shock had a greater effect than cold stress (Fig. 5, D and E).
Phenotypic Analysis of atncl Mutant and Overexpression Lines-The phenotypes of several atncl loss-of-function mutants and overexpression lines under salt stress were investigated (Fig. 6). Two AtNCL T-DNA insertion lines, atncl-1 and atncl-2, were isolated from the SAIL collection. The T-DNA was inserted into the sixth and third introns of AtNCL in atncl-1 and atncl-2, respectively. Full-length AtNCL mRNA was not detected in either of the mutant lines (Fig. 6, A and B). Both the atncl-1 and atncl-2 alleles were backcrossed with wildtype Col-0. BASTA resistance was observed among the F2 progeny at a 3:1 ratio, indicating that both alleles may contain a single T-DNA insertion with a functional selection marker. Next, p35S::AtNCL-GUS was transformed into Arabidopsis ecotype Col-0 and selected using Hygromycin B. atnclox, a single-copy insertion line, was included in the experiment.
No differences were detected in atncl and atnclox in terms of seed germination, plant growth, and flowering time under normal growth conditions compared with wild-type Col-0. However, under salt stress conditions, the atncl mutant seedlings were less sensitive than wild-type or the overexpression lines. The survival rate and chlorophyll contents were measured after treatment with 150 mM NaCl (Fig. 6C). The survival rate for atncl-2 was 83.2% compared with 62.5 and 42.8% for wild-type and the overexpression lines, respectively (Fig. 6C). The average total chlorophyll content for atncl-2 after salt stress was 0.308 mg/g fresh weight, compared with 0.261 and 0.246 mg/g fresh weight for wild-type and the overexpression lines, respectively (Fig. 6C), whereas the length of roots of atncl-2 showed no difference with wild type and overexpression lines. The survival rate for atncl-1 was similar to that for atncl-2 (supplemental Fig. S4). The survival rate and chlorophyll contents of mutant seedlings were also slightly higher than wild type after heat shock and freezing stress (supplemental Fig. S5).

atncl Mutants Show an Altered Ca Content and Ca 2ϩ Flux
Activity during Salt Stress-AAS was used to verify the total elemental Na and Ca contents in plants with or without NaCl stress (Fig. 7A). Under normal growth conditions, the total Na content in the atncl mutant seedlings was slightly lower than that in the wild-type or atnclox seedlings, whereas the level of Ca was higher. The ion content change in the atnclox lines was opposite that in the mutant lines. Under conditions of NaCl stress, the Na and Ca content change was different (Fig. 7A). The Ca content in the atncl loss-of-function mutants was largely unchanged, whereas that in wild-type and the overexpression lines was decreased. In comparison, the Na content in wild-type and the overexpression lines was much higher than that in atncl. Transgenic p35S::aequorin seedlings were used to monitor the [Ca 2ϩ ] cyt in response to salt stress (Fig. 7B). Aequorin luminescence in the atncl and wild-type seedlings quickly increased approximately 2-fold following exposure to 50 mM NaCl. However, atncl showed stronger luminescence than wild type during their return to the resting level, which took about 600 s. This indicates that the [Ca 2ϩ ] cyt is higher in the atncl seedlings compared with the wild-type seedlings.
To determine whether AtNCL participates in Na ϩ /Ca 2ϩ transport, SIET was used to monitor the flux of Ca 2ϩ at the root tip during salt stress (Fig. 7C). After the addition of 50 mM NaCl to the test system, an outward Ca 2ϩ flux exceeding 800 pmol⅐cm Ϫ 2 s Ϫ1 was recorded in wild-type seedlings, atncl and atnclox. The Ca 2ϩ flux reached the resting level within 10 min in wild type and atnclox, whereas in the atncl mutant seedlings, the flux lasted for approximately 30 min before returning to the resting level. In comparison, the H ϩ flux patterns in atncl and wild type were similar following treatment with 50 mM NaCl (supplemental Fig. S6). The H ϩ flux increased rapidly, reaching its peak within 5 min; thereafter, the flux decreased gradually, returning to the resting level within 20 min.

DISCUSSION
AtNCL May be a Novel Exchanger in Plant-AtNCL was once predicted to be a CAX-like protein (56); however AtNCL cannot work like CAXs in yeast to suppress the Ca 2ϩ -hypersensitive phenotype in K667, even if they share similar localization in yeast (Fig. 1). The AtNCL-transformed yeast growth was not affected by moderate change in pH (supplemental Fig. S3). By increasing the outside H ϩ , the Ca 2ϩ uptake of AtNCL-expressing cells was not affected. AtNCL also cannot work as a NHX in yeast (Fig. 1C), and AtNCL showed NCX-like activity in CHO-K1 cells (Fig. 3). By SIET, H ϩ flux patterns in atncl and wild type were similar during salt stress. These data suggested that AtNCL was probably a NCX-like protein rather than a CAX.
Recently, the structure of a NCX family member NCX_Mj had been resolved (27), and some critical site for ion binding and transport was identified. Sequence alignment analysis of NCXs and AtNCL showed that some critical amino acids that were conserved in NCXs also could be found in AtNCL, such as in ␣1 and ␣2 repeat, conservative glutamic acid or aspartic acid for bidentate Ca 2ϩ coordination, conservative serine or threonine for extracellular Na ϩ binding site, and some Thr, Ser, or asparagine sites forming the Na ϩ site on the intracellular side were also found in AtNCL (supplemental Fig. S2). This suggests that AtNCL may share a similar ion exchange mechanism with NCXs.
However, there were also some differences between AtNCL and mammalian NCXs. For instance, unlike the NCXs (27,61), the Ca 2ϩ uptake activity of AtNCL can be driven by the K ϩ gradient and inhibited by Mg 2ϩ (Fig. 3), but outside K ϩ was not necessary for the Ca 2ϩ uptake (Fig. 3). This suggested that AtNCL might not be a Na ϩ /Ca 2ϩ -K ϩ exchanger-like protein.
AtNCL May Contribute to Ca 2ϩ Homeostasis under Abiotic Stress-The results of a previous microarray analysis and expression level analysis indicate that salt stress, heat shock, cold stress, and ABA treatment can induce AtNCL expression (Fig. 5). In addition, it is known that Ca 2ϩ signaling can be evoked by various stimuli. We found that salt and ABA stress induced greater AtNCL expression than temperature stimuli; however, we focused on the salt stress phenotype in our subsequent experiments to determine the function of AtNCL in plants. AtNCL was expressed broadly in Arabidopsis seedlings, flowers, and root tips (Fig. 5). Measurement of the elemental Ca content by AAS showed that under normal conditions, the atncl, wild-type, and overexpression seedlings all maintained a balance between Ca 2ϩ and Na ϩ (Fig. 7A), whereas during salt stress the Ca 2ϩ signal was ended by transfer of the Ca 2ϩ from the cytoplasm to the apoplast or vacuole. During this process, AtNCL may be activated to extrude Ca 2ϩ ; thus, atncl showed a higher [Ca 2ϩ ] cyt and root surface Ca 2ϩ flux by SIET than wild type (Fig. 7C). The enhanced Ca 2ϩ signal may evoke a stronger response to salt stress, which may explain the improved growth of the atncl mutant lines compared with wild-type or the overexpression lines under conditions of salt stress.
To better understand the role of AtNCL, aequorin was used to monitor the [Ca 2ϩ ] cyt level. We found that during salt stress, the atncl seedlings had higher [Ca 2ϩ ] cyt than wild type. This apparent defect in Ca 2ϩ extrusion was likely caused by the lack of AtNCL. Measurement of the root surface ion flux by SIET revealed a slower Ca 2ϩ flux recovery process in the atncl mutant seedlings, which might be a consequence of the slower recovery of the [Ca 2ϩ ] cyt .
It has been shown that the root surface ion flux might originate from ion exchange at the cell wall (67); however, the manner of the ion flux differed between the atncl and wild-type seedlings (Fig. 7C), possibly due to the difference in ion transport activity. In some cases, the detected ion flux might reflect a combination of ion transport across the cell wall and membrane. Even if AtNCL is absent, there are other transporters capable of terminating the Ca 2ϩ signal, such as CAXs or Ca 2ϩ pumps in the endomembrane and plasma membrane. Thus, the mutant seedlings showed no difference from wild type under normal conditions. AtNCL may behave like an animal NCX (68), regulating Ca 2ϩ homeostasis under abnormal conditions. However, further experiments are needed to clarify how many Na ϩ ions AtNCL exchanges for one Ca 2ϩ and the regulating mechanism of its exchanger activity in planta.