Journal of Plant Research

, Volume 125, Issue 4, pp 555–568

Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells

  • Takamitsu Kurusu
  • Takuya Yamanaka
  • Masataka Nakano
  • Akiko Takiguchi
  • Yoko Ogasawara
  • Teruyuki Hayashi
  • Kazuko Iida
  • Shigeru Hanamata
  • Kazuo Shinozaki
  • Hidetoshi Iida
  • Kazuyuki Kuchitsu
Regular Paper

DOI: 10.1007/s10265-011-0462-6

Cite this article as:
Kurusu, T., Yamanaka, T., Nakano, M. et al. J Plant Res (2012) 125: 555. doi:10.1007/s10265-011-0462-6

Abstract

To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca2+-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca2+ uptake defects of yeast mutants lacking mechanosensitive Ca2+ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca2+ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca2+ uptake, and significantly alleviated growth inhibition under Ca2+ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca2+ influx through the plasma membrane.

Keywords

Calcium ion Cell proliferation Hechtian strand Mechanosensitive ion channel Plasma membrane Tobacco (Nicotiana tabacum) BY-2 cells 

Introduction

Plants respond to mechanical stimuli, such as touch, wind, gravity, pathogen attack, and cellular deformation during development (Reddy 2001; Sanders et al. 2002). Mechanical stimuli often increase the cytosolic concentration of free Ca2+ ([Ca2+]cyt) (Knight et al. 1991), which is largely mediated by Ca2+-permeable mechanosensitive channels (Fasano et al. 2002; Braam 2005; Toyota et al. 2008; Dodd et al. 2010). However, the structure and physiological functions of mechanosensitive channels have not been fully elucidated.

Some mechanosensitive channel candidates have been reported in Arabidopsis thaliana. MSL9 and MSL10, the Arabidopsis homologs of the bacterial mechanosensitive channel MscS, are responsible for the mechanosensitive channel activity in the plasma membrane of root cells, although both proteins show higher permeability to Cl than to Ca2+ (Haswell et al. 2008).

Recently, mid1-complementing activity (MCA) proteins have also been reported as mechanosensitive channel candidates in A. thaliana (Nakagawa et al. 2007; Yamanaka et al. 2010). MCA1 (At4g35920) and MCA2 (At2g17780) proteins have 72.7% identity and 89.4% similarity to each other. These proteins are able to partially complement the yeast mid1 mutant, lacking a putative Ca2+-permeable mechanosensitive channel component. However, each protein seems to play distinct roles in planta; MCA1 has been suggested to play a role in touch sensing in the primary root, whereas the mca2-null mutant is defective in Ca2+ uptake from roots (Nakagawa et al. 2007; Yamanaka et al. 2010). Despite these studies, the subcellular and physiological functions of plant MCA family proteins remain largely unknown.

Ca2+ plays many roles in a variety of cellular events. Ca2+ uptake by plasma membrane Ca2+ channels is a prerequisite for environmental adaptation, growth and developmental processes, and cell proliferation in plants as well as animals (Conn and Gilliham 2010; Dayod et al. 2010; Dodd et al. 2010). Cell culture is suitable for studying the relationship between Ca2+ uptake and cell proliferation (Smith 1978; Kurusu et al. 2004; Sano et al. 2006). The Nicotiana tabacum BY-2 cell system is superior when studying the intracellular localization and dynamics of proteins and organelles as well as cell cycle-related phenomena (Nagata et al. 1992; Higaki et al. 2011). Overexpression of various types of putative Ca2+-permeable channels has been shown to affect Ca2+ accumulation and the Ca2+-dependency of cell proliferation (Kim et al. 2001; Chan et al. 2003; Kurusu et al. 2004). BY-2 cells have been successfully used to study the cell biological functions of Ca2+-permeable channels in relation to cell proliferation.

In the present study, we have cloned and characterized two MCA orthologs from BY-2 cells. Subcellular localization was characterized using MCA-GFP constructs, mechanical stress-induced gene expression was evaluated using NtERF4 as a probe gene, and Ca2+ uptake activity and sensitivity to hypo-osmotic shock was characterized both in yeast and BY-2 cells overexpressing either NtMCA1 or NtMCA2. We discuss the physiological functions of the MCA family proteins in Ca2+ uptake, Ca2+-dependent cell proliferation as well as in mechanical signal transduction.

Materials and methods

Isolation of NtMCA1 and NtMCA2 cDNAs

By comparing the conserved regions of A. thalianaMCA1 (locus name, At4g35920), MCA2 (locus name, At2g17780) and rice (Oryza sativa) OsMCA1 (locus name, Os03g0157300), two primers corresponding to homologous regions among these sequences were synthesized, and fragments of MCA1 homologs from tobacco BY-2 (N. tabacum L. cv. Bright Yellow-2) cells were amplified by PCR. Total RNA was extracted from BY-2 cells using Trizol Reagent (Life Technologies, Carlsbad, CA, USA). First-strand cDNA that was used as the PCR template was synthesized from 1 μg of total RNA using Superscript II (Life Technologies). We obtained two independent cDNA fragments encoding MCA1 and MCA2 homologs, termed NtMCA1 and NtMCA2. Full-length cDNA sequences for NtMCA1 and NtMCA2 were obtained using an RNA PCR Kit (AMV) ver. 2.1 (Takara, Otsu, Japan) and a 5′-Full RACE core Kit (Takara). An additional TAIL-PCR (Li et al. 2006) was performed to determine the 5′-end of NtMCA2 cDNA.

Subcloning of NtMCA1 and NtMCA2 cDNAs in a yeast expression vector

NtMCA1 and NtMCA2 cDNAs with a BamHI site at the 5′-end and a NotI site at the 3′-end were synthesized by PCR using the cDNAs as templates and the following primers: 5′-GGATCCATGGCAACGTGGGAACAC-3′ and 5′-GCGGCCGCTTAGGATTCCATAAATTG-3′ for NtMCA1, and 5′-GGATCCATGGCTTCATGGGAAC-3′ and 5′-GCGGCCGCTCAGGATTCCATTAAC-3′ for NtMCA2, where the BamHI and NotI sites are underlined and the initiation and termination codons are in bold. The resulting products were cut with BamHI and NotI and inserted into the BamHI–NotI site of the YEplac181-based, multicopy expression vector, YEpTDH (XBS) (Gietz and Sugino 1988; Hashimoto et al. 2004), in which a cDNA of interest is transcribed under the control of the yeast TDH3 promoter. The resulting plasmids were named YEpTDH(XBS)-NtMCA1 and YEpTDH(XBS)-NtMCA2.

Yeast strains and medium

The yeast strains used were H311 (MATamid15::HIS3; Tada et al. 2003) and H319 (MATamid15::HIS3cch1Δ::HIS3; Nakagawa et al. 2007). The low Ca2+ medium SD.Ca100 (Iida et al. 1990, 1994), which contains 100 μM CaCl2 as the Ca2+ source, was used in most of the experiments. The yeast strains were transformed with YEpTDHXho-MCA1 (Nakagawa et al. 2007), YEpTDHXho-MCA2 (Yamanaka et al. 2010) and YEpTDH(XBS)-NtMCA1/2, as previously described (Mount et al. 1996).

Cell viability assay in yeast

Yeast cells were grown to the exponentially growing phase (2 × 106 cells/mL) in SD.Ca100 medium at 30°C, and incubated with 6 μM α-factor for 0, 4 and 8 h, after which viability was determined by the methylene blue method (Iida et al. 1990).

Ca2+ accumulation in yeast

Yeast cells were grown to 2 × 106 cells/mL in SD.Ca100 medium at 30°C, and incubated for 2 h with 185 kBq/mL 45CaCl2 (1.8 kBq/nmol; Cat. No. NEZ013, PerkinElmer, Waltham, Massachusetts, USA). Samples (100 μL) were filtered with Millipore filters (Type HA, 0.45 μm, Millipore, Billerica, MA, USA) presoaked with 5 mM CaCl2 and washed five times with the same solution. The radioactivity retained on each filter was counted as previously described (Iida et al.1990).

Measurement of cytosolic [Ca2+] changes in yeast

Yeast cells carrying the apoaequorin-expressing plasmid, pGAPAQ1 (Nakajima-Shimada et al. 1991), were grown to the exponential phase (~6 × 106 cells/mL) in 2 mL of SD medium supplemented with 1 M sorbitol at 30°C, harvested by centrifugation for 5 min at 3,000 rpm, and resuspended in 300 μL of SD/1 M sorbitol medium containing 5 μM coelenterazine. The suspension was incubated in the dark for 20 min to reconstitute aequorin, and then centrifuged as above. The pelleted cells were washed once with 300 μL of SD/1 M sorbitol medium and suspended in 300 μL of the same medium. An aliquot (100 μL) of the suspension was transferred to a cuvette and set in a luminometer (Lumi-counter 2500, Microtech Nition, Funabashi, Japan). One minute after the start of the luminescence intensity monitoring, 600 μL of SD medium with or without sorbitol was added to the suspension to induce a hypo-osmotic shock, and the monitoring was continued for another 2 min. At the end of the experiment, 700 μL of 4% Triton X-100/4 M CaCl2 solution were added to the suspension to measure the maximum luminescence intensity (Lmax).

Plant cell material and transformation

Suspension-cultured cells of BY-2 were grown in modified Linsmaier and Skoog (LS) medium as previously reported (Nagata et al. 1992). The cell suspension was agitated in the dark on a rotary shaker at 110 rpm and 27°C, and maintained by weekly dilution (1/60–1/15).

To generate transgenic BY-2 cells expressing NtMCA1/2-GFP and NtMCA1/2, the coding regions were subcloned into the pENTR/D-TOPO cloning vector (Life Technologies), and then cloned into the pH7WGF2 vector encoding an N-terminal EGFP fusion or the pH7WG2 vector (Karimi et al. 2002), using the LR clonase reaction.

The transformation of BY-2 cells was carried out in accordance with An (1985) with the following minor modifications: 4 mL of a 3-day-old exponentially growing culture was transferred to a 90 mm Petri dish and incubated at 28°C with 100 μL of a fresh overnight-culture of Agrobacterium tumefaciens pGV2260, containing the binary vectors. After a 48-h co-cultivation, the BY-2 cells were washed and plated on LS agar medium containing hygromycin (50 μg/mL) and carbenicillin (250 μg/mL). Every 3–4 weeks, the transformants were selected and transferred onto fresh medium for continued selection.

Northern blot and RT-PCR analyses

Total RNA was extracted from each frozen sample of BY-2 cells using Trizol reagent according to the manufacturer’s protocol (Life Technologies). For northern blot analysis, denatured total RNA (20 μg) was subjected to electrophoresis on a 1% agarose gel containing 5.5% formaldehyde, and then transferred onto a Hybond-N + membrane (GE Healthcare, Horten, Norway). DIG-labeled NtMCA1 and NtMCA2 anti-sense RNA probes were prepared from template DNA fragments containing a T7 promoter and the 5′-half of NtMCA1 (1–679 nt) or NtMCA2 (1–690 nt), using a DIG RNA-labeling kit according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany). Pre-hybridization was performed for 1 h at 42°C in high-SDS hybridization buffer [50 mM sodium polyphosphate, 5 × SSC, 7% SDS, 0.1% N-lauroylsarcosine, 2% Blocking Reagent (DIG Wash and Block Buffer Set; Roche) and 50% formamide]. Hybridization was performed overnight at 68°C in high-SDS hybridization buffer with denatured DIG probes. The filter was washed twice with 1% SDS/2 × SSC for 5 min and twice with 0.1% SDS/0.1 × SSC for 15 min at room temperature. The hybridization signal was visualized using LAS-3000mini (Fuji Film Co., Tokyo, Japan).

For reverse transcriptase-PCR (RT-PCR), 3 μg of total RNA were subjected to cDNA synthesis using an oligo-dT primer. Then, PCR amplifications were performed using a specific primer set (5′-CACCATGGCAACGTGGGAACAC-3′ and 5′-TTAGGATTCCATAAATTGAGAAGGC-3′ for NtMCA1; 5′-ATTCAAGTATGCCTGGGTGCTTGAC-3′ and 5′-TTCGTTGTCGAGGACCATGC-3′ for EF1α), the synthesized cDNA as a template and Ex-Taq DNA polymerase (Takara).

Quantification of mRNA by real-time RT-PCR

Each mRNA was quantified by real-time RT-PCR as described by Kurusu et al. (2010). First-strand cDNA was synthesized from 3 μg of total RNA using an oligo-dT primer and reverse transcriptase. Real-time PCR was performed using an ABI PRISM 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) with the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) and the gene specific primers: NtMCA1-RealF, 5′-CGATTGGCATTTTAGGGCTAAT-3′; NtMCA1-RealR, 5′-TGCATGAATCCAGTAGTGACAAGTT-3′; NtMCA2-RealF, 5′-TTAAAAAGCATTATACGGAGGTAACG-3′; NtMCA2-RealR, 5′-AAGGAAGATATGAACACGCAAAGA-3′; NtERF4-RealF, 5′-GAAGAAAGTCTGAGGTGAAATCAAGA-3′; NtERF4-RealR, 5′-AAGACACATCCCTGGCCTATTC-3′; NtERF2-RealF, 5′-TGTATTATGTCTATGGCGTGTAAAGTGT-3′; NtERF2-RealR, 5′-TCAATCCAAAGTGAGGACAAGAAA-3′; NtActin-RealF, 5′-GGGTTTGCTGGAGATGATGCT-3′; NtActin-RealR, 5′-GCTTCATCACCAACATATGCAT-3′. Relative mRNA abundances were calculated using the standard curve method and normalized to the corresponding NtActin gene levels. Standard samples of known template amounts were used to quantify the PCR products.

Ca2+ uptake in BY-2 cells

BY-2 cell suspensions at three days after subculturing in LS medium, containing 0.1 mM Ca2+, were used for measurement of Ca2+ uptake. Ten milliliters of cell culture aliquots were transferred to a 50 mL tube and incubated for 1 day. Cells were weighed and diluted to 0.1–0.2 mg/mL, and 45CaCl2 [30 MBq/mmol; PerkinElmer, Cat. No. NEZ013] was added to a final concentration of 33 kBq/g. Then, cells were agitated at 25°C and 1 mL of cells was collected at 0, 15 and 30 min after 45CaCl2 addition. Cells were filtered using Whatman GF/C filters presoaked with 5 mM CaCl2 and 1 mM LaCl3, and washed five times with an ice-cold solution of the same composition to remove 45Ca2+ from the cell wall. The radioactivity retained on each filter was counted as previously described (Iida et al. 1990).

Growth assay and hypo-osmotic treatment of BY-2 cells

Cell growth assay was performed basically as described by Kurusu et al. (2004). Ten milliliters of stationary culture of BY-2 cells were transferred to a 50 mL tube containing Ca2+-free LS medium and pre-incubated for 3 h. Cells (fresh weight 0.5 g) were transferred to LS medium (final concentration 0.01 mg/mL) with a standard (3 mM) or low Ca2+ concentration (0.1 or 0.06 mM). After culturing for 0, 3, 5 or 7 days, the fresh weight of cells was measured.

To test the effect of hypo-osmotic treatment, the growth medium of BY-2 cells was replaced by diluted medium 3 days after subculturing, and gene expression was analyzed as described in the legend for Fig. 7.

Subcellular localization of NtMCA1-GFP and NtMCA2-GFP

GFP fluorescence images were observed using an LSM 5 EXCITER confocal fluorescence microscope with a C-Apochromat 40×/1.2W corr (water; Carl Zeiss, Oberkochen, Germany) objective lens. Images were processed with the Zeiss LSM Image Browser (Carl Zeiss) and Adobe Photoshop Elements 7.0 (Adobe Systems, San Jose, CA, USA). The fluorescent styryl membrane probe FM4-64 (Molecular Probes, Carlsbad, CA, USA) was kept as a 17 mM stock solution in sterile water, and used at a final concentration of 4.25 μM to label the vacuolar membrane (tonoplast). Three-day-old BY-2 cells were treated with FM4-64 for 3 h and washed twice with the culture medium.

Cell cycle synchronization of BY-2 cells

Cell cycle synchronization was performed as described by Kadota et al. (2005). A stationary culture of BY-2 cells was diluted 1/10 in fresh modified LS medium supplemented with 5 μg/mL aphidicolin (Wako Pure Chemical, Osaka, Japan). After 24 h the aphidicolin was removed by extensive washing and the cells were resuspended in fresh medium. Cell division percentages were obtained by determining the mitotic index by staining DNA with SYTOX Green (Life Technologies), which was detected using fluorescence microscopy (AxioImager.A1, Carl Zeiss).

Results

Isolation of NtMCA1 and NtMCA2 genes and phylogenetic analysis of the MCA family

Based on conserved amino acid sequences between MCA1, MCA2 and their sole rice (O. sativa) ortholog, OsMCA1 (Os03g0157300), we designed primers for RT-PCR to amplify MCA cDNA from BY-2 cells. Two putative clones, named NtMCA1 and NtMCA2 (GenBank Accession Nos. AB622811 and AB622812, respectively), were obtained. RACE PCR and TAIL-PCR were used to determine the full-length sequences of these cDNAs. Both NtMCA1 and NtMCA2 encode polypeptides of 419 amino acid residues with predicted molecular masses of 47,785 and 47,982, respectively. The deduced amino acid sequences of NtMCA1 and NtMCA2 are quite similar to each other (83.1% identity), and are 69.0%/68.0% identical to MCA1, 63.0%/61.5% identical to MCA2, and 67.4%/66.2% identical to OsMCA1, respectively. Each N-terminal region contains a domain of unknown function, which is specifically conserved in plant genes. Each C-terminal half possesses a cysteine-rich domain of unknown function, called the Plac8 or DUF614 motif (Fig. 1a, b, Galaviz-Hernandez et al. 2003).
Fig. 1

Alignment of MCA family proteins. a MCA family proteins from A. thaliana, tobacco (N. tabacum), and rice (O. sativa). The alignment was performed by ClustalW and shaded by Boxshade. The N-terminal domain is marked with a dotted line. The double lines, dot lines, and black lines under the MCA sequences indicate a coiled-coil region, a serine-rich region and a carboxy-terminal respectively. This cysteine-rich region of carboxy-terminal is similar to the Plac8 motif found in plant and animal proteins. b The Plac8 family proteins. The alignment was performed by ClustalW and shaded by Boxshade. The lines under the Plac8 sequences represent the protein-binding motif of ONZIN (Rogulski et al. 2005)

We searched for the MCA family proteins in various organisms, and found orthologs in all land plants including ferns and mosses, but not in algae or animals, suggesting that the function of the MCA proteins may be fundamental to land plants. Using the phytozome database (http://www.phytozome.net/), we collected the full-length amino acid sequences of the MCA family proteins from various land plants and conducted a phylogenic analysis. The phylogenetic tree of the MCA family (Fig. 2) clearly indicates that genetic diversity of MCA genes occurred relatively recently. Interestingly, only one MCA gene is present in various Poaceae (Fig. 2).
Fig. 2

A phylogenetic tree of the MCA family proteins based on amino acid sequences constructed using the Tree View software by the neighbor-joining method. The scale bar (0.1) represents the relative branch length. Full binominal names are as follows: Aquilegia coerulea (AcoGoldSmith_v1.005671m.g), Arabidopsis lyrata (Al491044, Al480672), Arabidopsis thaliana (At4G35920, At2G17780), Brachypodium distachyon (Bradi1g74650), Brassica rapa (KBrH009I12), Carica papaya (evm.TU.supercontig_6.294), Glycine max (Glyma01g36860, Glyma11g08430, Glyma02g05130, Glyma16g23240), Hordeum vulgare (FLbaf94d24), Manihot esculenta (cassava4.1_008447m.g, cassava4.1_008443m.g), Medicago truncatula (Medtr5g022570, Medtr8g089340), Nicotiana tabacum (AB622811, AB622812), Oryza sativa (AB601973), Physcomitrella patens (XP_001768983, XP_001785848), Populus trichocarpa (POPTR_0005s11200), Prunus persica (ppa006351m.g), Selaginella moellendorffii (Smo93428), Setaria italica (SiPROV010555m.g), Solanum lycopersicum (C02SLm0020N19.1_0000003), Sorghum bicolor (Sb01g046630), Vitis vinifera (GSVIVG01006329001), and Zea mays (GRMZM2G027821)

Complementation of yeast Ca2+-requiring mutants

Saccharomyces cerevisiae mid1 mutants are defective in a putative Ca2+-permeable mechanosensitive channel component, and show lethality upon exposure to the mating pheromone (Iida et al. 1994; Kanzaki et al. 1999). Thus, we tested the functional complementation of mid1 mutants by NtMCA1 and NtMCA2 overexpression. The mid1 mutants were transformed with the yeast expression plasmids YEpTDHXho-NtMCA1 or YEpTDHXho-NtMCA2, containing NtMCA1 or NtMCA2 cDNAs, respectively. Quantitative viability assays showed that NtMCA1 and NtMCA2 were able to partially rescue the mating pheromone-induced death of mid1 cells (Fig. 3a).
Fig. 3

Function of NtMCA1 and NtMCA2 in yeast cells. a NtMCA1 and NtMCA2 rescue cell viability after exposure of the mid1 mutant to α-factor. NtMCA1 or NtMCA2 cDNA in a multicopy plasmid was expressed under the control of the TDH3 promoter in the yeast mid1 mutant. The parental strain of the mutant was used as a positive control. Data are the mean ± SD of three independent experiments. *P < 0.05 versus the vector (control) in each mutant. b NtMCA1/2 rescues Ca2+ uptake activity in the mid1 cch1 double mutant. Ca2+ uptake was measured by the method described by Iida et al. (1990). MCA1, MCA2, NtMCA1, or NtMCA2 cDNA in a multicopy plasmid was expressed under the control of the TDH3 promoter in the yeast mid1 cch1 double mutant. Data are the mean ± SD of three independent experiments. *P < 0.05 versus the vector control in the mutant

In yeast, Mid1 and Cch1, a homologue of the α1 subunit of the mammalian voltage-gated Ca2+ channels, constitute the sole high-affinity Ca2+ influx system and therefore the phenotypes of mid1 mutants are quite similar to mid1 cch1 double mutants with respect to Ca2+ uptake and mating pheromone-induced death (Fischer et al. 1997; Paidhungat and Garrett 1997; Muller et al. 2001). To eliminate the possibility that NtMCA1 and NtMCA2 activate Cch1 to induce Ca2+ influx, instead of forming a Ca2+-permeable channel, we examined whether NtMCA1 and NtMCA2 could complement the low Ca2+ uptake phenotype of mid cch1 mutants. As previously demonstrate for MCA1 and MCA2 from A. thaliana (Yamanaka et al. 2010), both NtMCA1 and NtMCA2 complemented the low Ca2+ uptake activity of the double mutant (Fig. 3b), suggesting that each one of them mediates Ca2+ influx in yeast cells, independently of Mid1 and Cch1.

NtMCA1 and NtMCA2 mediate hypo-osmotic shock-induced changes in cytosolic [Ca2+] in yeast cells

To test the involvement of NtMCA1 and NtMCA2 in the regulation of mechanical stress-induced Ca2+ influx, we generated NtMCA1/2-overexpressing yeast cells harboring the Ca2+-sensitive photoprotein, aequorin. As shown in Fig. 4a and b, a transient increase in [Ca2+]cyt was induced by hypo-osmotic shock in yeast cells, and NtMCA1 and NtMCA2 overexpression enhanced the response. In contrast, isotonic treatment did not induce a significant Ca2+ influx both in control cells and in NtMCA1- and NtMCA2-overexpressing cells (Fig. 4b). These results suggest that NtMCA1 and NtMCA2 participate in hypo-osmotic shock-triggered Ca2+ influx in yeast cells.
Fig. 4

NtMCA1 and NtMCA2 enhance hypo-osmotic shock-induced [Ca2+]cyt increase in yeast cells. Cells of the H319 strain (mid1 cch1) bearing YEpNtMCA1, YEpNtMCA2, or an empty vector (YEpTXBS) were transformed with an apoaequorin-expressing plasmid, pGAPAQ1. Each transformant was challenged by hypo-osmotic shock and monitored for the aquorin luminescence intensity, as described in “Materials and methods”. a Changes in [Ca2+]cyt, as revealed by the aequorin luminescence intensity. A typical result is shown for each transformant. Essentially, the same results were obtained with each transformant in three independent experiments. The arrow represents the start of hypo-osmotic shock. b Summary of the three independent experiments. The peak value of the [Ca2+]cyt increase obtained from the three independent experiments was averaged and represented with a bar graph. Standard deviation is shown on the bars. ***P < 0.0001 versus the vector control

Effects of NtMCA1 and NtMCA2 overexpression on Ca2+ sensitivity, Ca2+ uptake and cell growth in BY-2 cells

To reveal the function of NtMCA1 and NtMCA2 in plant cells, we constructed BY-2 cell lines overexpressing NtMCA1 or NtMCA2 under the control of the cauliflower mosaic virus 35S promoter (Fig. S1). We then tested whether NtMCA1- and NtMCA2-overexpression affect Ca2+ sensitivity of growth in BY-2 cells. BY-2 cells overexpressing NtMCA1 and NtMCA2 grew less than control cells in a regular medium containing 3 mM Ca2+ (Fig. 5). When the Ca2+ concentration of the medium was decreased to 0.1 and 0.06 mM, the growth of control cells was significantly restricted, whereas the growth of NtMCA1- and NtMCA2-overexpressors was not (Fig. 5). Eventually, NtMCA1-overexpressors showed a higher growth rate than the control line, and NtMCA2-overexpressors grew at a growth rate comparable to the control line in the Ca2+-deficient medium. Overexpression of NtMCA1 and NtMCA2 did not show a significant effect on cell size (data not shown).
Fig. 5

Ca2+ sensitivity of BY-2 cells overexpressing NtMCA1 and NtMCA2. Seven-day-old cultured cells were assayed. Cells (fresh weight 0.5 g) were transferred to LS medium with standard or low Ca2+ concentration. After culturing for 0, 3, 5 or 7 days, the fresh weight of the cells was measured. Data are the mean ± SD of three independent experiments. *P < 0.05 versus the control

We also examined the role of NtMCA1 and NtMCA2 in the Ca2+ uptake activity of BY-2 cells. In this experiment, we employed GFP-tagged derivatives of NtMCA1 and NtMCA2 (Fig. 6a). As shown in Fig. 6b, the Ca2+ uptake activity was higher in the NtMCA1-GFP and NtMCA2-GFP overexpressors than the control cells expressing GFP, suggesting that NtMCA1-GFP and NtMCA2-GFP are functional, at least in respect to the Ca2+ uptake activity across the plasma membrane in BY-2 cells.
Fig. 6

Effect of NtMCA1 and NtMCA2 overexpression on Ca2+ uptake in BY-2 cells. a RT-PCR of the NtMCA1/2 transcripts. EF1α mRNA was used as an internal control. b45Ca2+ uptake into tobacco (N. tabacum) BY-2 cells. The cells were agitated at 25°C and 1 mL of cells was collected at 0, 15 and 30 min after 45CaCl2 addition. Next, the cells were filtered using Whatman GF/C filters presoaked with 5 mM CaCl2 and 1 mM LaCl3, and washed five times with an ice-cold solution of the same composition. The radioactivity retained on each filter was counted. Data are the mean ± SD of three independent experiments. *P < 0.05 versus the GFP control line

Effect of NtMCA1 overexpression on hypo-osmotic shock-induced gene expression in BY-2 cells

To test whether NtMCA proteins are involved in mechanical stress signaling, we analyzed the mechanical stress-induced gene responses in BY-2 cells overexpressing NtMCA proteins. In tobacco (N. tabacum), some ERF (ethylene-responsive transcription factor) genes including NtERF4, but not NtERF2, are responsive to mechanical stress such as touch, osmotic shock and salinity (Ohme-Takagi et al. 2000).

NtERF4 was transiently induced by hypo-osmotic shock in BY-2 cells. Its transcript levels were significantly enhanced in BY-2 cells overexpressing NtMCA1, depending upon strength of hypo-osmotic shock (Fig. 7a, b). In contrast, the expression of NtERF2, which was not induced by hypo-osmotic shock, was scarcely affected by the overexpression of NtMCA1 (Fig. 7c). Taken together, NtMCA1-GFP cells are sensitized with respect to the expression of a hypo-osmotic shock-induced gene, suggesting the possible involvement of NtMCA1 in the regulation of mechanical stress-induced gene expression in BY-2 cells.
Fig. 7

Involvement of NtMCA1 in the regulation of hypo-osmotic shock-induced expression of NtERF4 in BY-2 cells. a, b Quantification of the expression level of NtERF4 in the control line and the NtMCA1-overexpressor by real-time quantitative PCR. Total RNA was isolated from the cells harvested at the indicated time points of hypo-osmotic shock. For hypo-osmotic shock, the growth medium was replaced by fourfold diluted medium at 0 min (a). 2 mL of water, medium, or diluted medium were added to the NtMCA1-overexpressor at 0 min to generate wide-ranging changes in extracellular osmotic pressure. Total RNA was isolated from the cells 30 min after the hypo-osmotic shock. c Quantification of the expression level of NtERF2 in the control line and NtMCA1-overexpressor. Total RNA was isolated from the cells harvested at the indicated time points of hypo-osmotic shock. The amount of each mRNA was calculated from the threshold point located in the log-linear range of the RT-PCR. The relative level of each gene in the control cells at time 0 (a, c) or medium treatment (b) was standardized as 1. Data are the mean ± SE of three independent experiments. **P < 0.005, significant difference compared with the control line

We also tried to examine the effect of overexpression of NtMCA1 and NtMCA2 on Ca2+ influx induced by mechanical stress. However, NtMCA1/2-overexpressing cells harboring aequorin showed abnormal morphology (data not shown) and were not suitable for proper monitoring of [Ca2+]cyt.

Subcellular localization of NtMCA1/2

NtMCA1-GFP fluorescence was detected predominantly in the plasma membrane of BY-2 cells (Figs. 8a–c, S2a), whereas GFP was localized at the nucleus and the cytoplasm (Fig. S2e, g). In addition, the fluorescence signals of NtMCA1-GFP and FM4-64 (a vacuolar membrane marker) were not co-localized (Fig. S2c). NtMCA2-GFP was also targeted to the plasma membrane (Fig. S3), suggesting that NtMCA1 and NtMCA2 are localized at the plasma membrane.
Fig. 8

Subcellular localization of NtMCA1-GFP in BY-2 cells. a, b Fluorescence and differential interference contrast (DIC) images of NtMCA1-GFP. c Fluorescence image of NtMCA1-GFP. e Fluorescence image of NtMCA1-GFP focused on the cell surface of (c). Fluorescence images of NtMCA1-GFP during cell division (d) and at telophase (f). g Fluorescence image of NtMCA1-GFP in plasmolysed cells. Scale bars 10 μm

The fluorescence signals of NtMCA1-GFP were also observed as punctuated structures on the cell surface (Fig. 8e), near the poles of dividing cells (Fig. 8d, arrowheads), and the immature cell plate at telophase (Fig. 8f, arrowhead). In plasmolysed cells treated with a high osmotic solution (4% NaCl), the fluorescence signals of NtMCA1-GFP were localized not only to the plasma membrane but also to the Hechtian strands, which connect the plasma membrane to the cell wall (Fig. 8g, arrows). We observed two types of Hechtian strands: one connects the plasma membrane and the cell wall in a straight line (Fig. 8g, left cell; arrow) and the other forms branched polygonal lines along the inner side of the cell wall (Fig. 8g, right cell; arrow). These fluorescence signals were also observed in BY-2 cells overexpressing the NtMCA2-GFP fusion protein (data not shown).

The expression pattern of NtMCA1 and NtMCA2 along the cell cycle phases

To date, the transcriptional control of MCA genes is largely unknown. We monitored the transcript levels of NtMCA1 and NtMCA2 at the cell cycle phases by real-time RT-PCR. The transcript level of NtMCA2 was the highest at the G1 phase and the lowest at the S phase. In contrast, NtMCA1 transcript levels were rather constant throughout the cell cycle (Fig. 9a, b).
Fig. 9

Expression of NtMCA1 and NtMCA2 in each cell cycle phase in synchronous-cultured BY-2 cells. a Quantification of the expression levels of NtMCA1 and NtMCA2 in synchronized BY-2 cells at 0.5 h (S phase), 5.5 h (G2 phase), 8 h (M phase) and 11 h (G1 phase) after aphidicolin release were analyzed by real-time quantitative PCR. Total RNA was isolated from the cells harvested at the indicated time points. The amount of each mRNA was calculated from the threshold point located in the log-linear range of the RT-PCR. The relative level of each gene in BY-2 cells at the S phase was standardized as 1. The error bars indicate the SEM of three independent experiments. *P < 0.05, significant difference compared with the expression at the S phase. b Changes in the mitotic index of synchronous BY-2 cells. Data are the mean ± SE of three independent experiments

Discussion

Phylogenetic study and domain structure of the MCA family proteins

The amino acid sequence identity of the MCA proteins was relatively high throughout the entire sequence, except for the short serine-rich region in the middle, which showed substantial diversity among these proteins. The N-terminal region (NtMCA1/2, 1–179 aa) has a plant-specific sequence with unknown function (Fig. 1a). Proteins containing this region, other than the MCA family, have only been found in grass species, and all of them are protein kinases. This region also shows a weak similarity to the UND domain of plant U-box proteins (Mudgil et al. 2004) and the stem region of some motor proteins. This may reflect its potential coiled-coil function and possible interaction with the cytoskeleton.

The C-terminal half possesses a cysteine-rich domain of unknown function, called the Plac8 or DUF614 motif (Fig. 1a, b; Galaviz-Hernandez et al. 2003). Proteins containing a Plac8 motif (called the PLAC8 superfamily) are widely distributed among viruses, protozoa and higher eukaryotes including plants and animals. The functions of these proteins are largely unknown, but some Plac8 proteins have been reported in sensitivity to divalent cations (Song et al. 2004; Nakagawa et al. 2007; Tanaka et al. 2007; Yamanaka et al. 2010), interaction with cell proliferation-related proteins (Rogulski et al. 2005; Cong and Tanksley 2006; Li et al. 2006), and localization at plasma membrane microdomains (Iomini et al. 2006; Lefebvre et al. 2007). A region corresponding to the ONZIN protein-binding domain (Rogulski et al. 2005; Li et al. 2006) is highly conserved among members of the Plac8 family (Fig. 1b, underline), suggesting that the MCA family proteins may interact with other proteins via this Plac8 motif.

Intracellular localization of NtMCA1 and NtMCA2

NtMCA1 and NtMCA2 appear to be localized at the plasma membrane, Hechtian strands, cell plate and perinuclear vesicles (Figs. 8, S2, S3). Hechtian strands are fibrous structures connecting between the plasma membrane and the cell walls (Buer et al. 2000). Localization at the Hechtian strands has also been observed with other typical plasma membrane ion channels, including A. thaliana SLAC1 and MSL10 (Haswell et al. 2008; Vahisalu et al. 2008). NtMCAs may have a crucial role as mechanosensitive Ca2+-permeable channels at Hechtian strands.

We detected NtMCA1-GFP fluorescence near the poles and at the immature cell plate during cell division (Fig. 8d, f). Because cell plate formation requires membrane vesicles and the cytoskeleton (Müller et al. 2009), NtMCA1 localized in the perinuclear membrane vesicles may have a role in the regulation of cell division and cell plate formation together with the cytoskeleton. Tomato (Solanum lycopersicum) FW2.2, containing the Plac8 motif, has been reported to be involved in cell division (Cong and Tanksley 2006). Accordingly, Plac8 motif-containing proteins may be of interest in future cell proliferation studies.

The NtMCA1-GFP protein was also localized at punctuated structures on the cell surface (Fig. 8e), which might be related to plasma membrane microdomains. The inward rectifying K+ channel, KST1, also forms clusters in plasma membranes, and its GFP-tagged derivatives were observed as patches in both endomembranes and plasma membranes (Ehrhardt et al. 1997; Zimmermann et al. 1998). Recently, we showed that A. thaliana MCA1 and MCA2 form a homo-oligomer in yeast cells (Nakano et al. 2011). NtMCA1 and NtMCA2 may form clusters or complexes with other signaling molecules in planta, such as other ion channels.

Involvement of NtMCA1 and NtMCA2 in Ca2+ uptake and cell division regulation

Our results demonstrate that NtMCA1 and NtMCA2 mediate Ca2+ uptake in both yeast (Fig. 3b) and BY-2 cells (Fig. 6b), which is consistent with the A. thaliana orthologs. Ca2+ has been postulated to be involved in the regulation of cell division (White and Broadley 2003). Lowering the extracellular Ca2+ concentration resulted in prolonged metaphase in stamen hair cells of Tradescantia (Hepler 1985). Chromosome motion in the anaphase is accelerated when intracellular [Ca2+] is resting around 1 μM, whereas it is inhibited when intracellular [Ca2+] is above or below the resting level (Zhang et al. 1990).

Ca2+ uptake mediated by MCA proteins appears to affect cell proliferation in BY-2 cells. Restricted cell proliferation of NtMCA1- and NtMCA2-overexpressors (Fig. 5) suggests that constitutively higher Ca2+ uptake activity may result in higher [Ca2+]cyt, and hence restrict cell proliferation. On the other hand, overexpression of NtMCA1 significantly maintained the cell growth even in Ca2+-deficient media (Fig. 5). This may be attributed to the higher Ca2+ uptake activity of the overexpressors (Fig. 6b), which is sufficient to maintain [Ca2+]cyt permissive for cell proliferation under Ca2+ deficiency. Taken together, the growth of BY-2 cells may depend, at least partially, on Ca2+ uptake mediated by NtMCA1 and NtMCA2.

The Ca2+ uptake activity of NtMCA2-overexpressors was higher than NtMCA1-overexpressors (Fig. 6b). NtMCA1 and NtMCA2 showed different effects on cell growth under Ca2+ limitation (Fig. 5). These results focus again on the importance of [Ca2+]cyt for cellular functions. Cell growth depends on Ca2+ homeostasis, which is determined by the balance between extracellular [Ca2+] and Ca2+ uptake activity. Excess and deficiency of Ca2+ could lead to inhibition of cell growth.

We showed that the level of NtMCA2 transcripts changed along the different phases of the cell cycle, displaying its maximum at the G1 phase, when cell elongation and expansion occurs, and its minimum at the S phase (Fig. 9a, b). In BY-2 cells, K+ channel activity is regulated by the cell cycle and is involved in the regulation of cell elongation (Sano et al. 2009). In plant cells, cell elongation/expansion is associated with ion uptake and mechanical stimulation at the plasma membrane. NtMCAs may be involved in mechanosensing and regulation of Ca2+ homeostasis at the plasma membrane and Hechtian strands, especially during the G1 phase, to regulate cell elongation.

Possible roles of NtMCAs in mechanosensing and mechanical signal transduction

The transient hypo-osmotic shock-induced [Ca2+]cyt increase in yeast cells was enhanced by overexpression of NtMCA1/2 (Fig. 4a, b). Furthermore, NtMCA1-overexpressing BY-2 cells are sensitized with respect to the expression of a hypo-osmotic shock-induced gene, NtERF4 (Fig. 7b). This is consistent with the previous observation that MCA1-overexpressing plants showed constitutively elevated expression of another touch-inducible gene, the A. thaliana TCH3/CML12 (Nakagawa et al. 2007). A possible explanation is that NtMCA1 and NtMCA2 are involved, at least partially, in Ca2+ influx across the plasma membrane, and may play a role in the regulation of mechanosensing and mechanical signal transduction pathways. However, our system was constructed using NtMCA-overexpressors, and the exact correlation between NtMCA protein levels and the regulation of Ca2+ uptake remains to be evaluated, possibly using NtMCA1/2-suppressed cell lines, an approach that may be important in future projects to further unveil the roles of the NtMCA proteins in tobacco (N. tabacum) cells.

Acknowledgments

We would like to thank Dr. Yasuhiro Kadota, Ms. Yui Miki and Mr. Yasuhiro Sakurai for technical assistance, and Dr. Dierk Wanke for discussion. This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (21200067) to T. K., for Scientific Research on Priority Area (21026009 and 23120509) to H. I., for Scientific Research B (19370023) to K. K. and (21370017) to H. I., for Exploratory Research (21658118) to K. K., and by grants from Japan Science and Technology Agency, for CREST to H. I. and K. K.

Supplementary material

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Supplementary Figures (TIF 4.88 mb)
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Supplementary Figures (TIF 25.6 mb)
10265_2011_462_MOESM3_ESM.tif (12 mb)
Supplementary Figures (TIF 11.9 mb)

Copyright information

© The Botanical Society of Japan and Springer 2011

Authors and Affiliations

  • Takamitsu Kurusu
    • 1
    • 2
  • Takuya Yamanaka
    • 1
  • Masataka Nakano
    • 3
    • 6
  • Akiko Takiguchi
    • 1
  • Yoko Ogasawara
    • 1
  • Teruyuki Hayashi
    • 1
    • 7
  • Kazuko Iida
    • 4
  • Shigeru Hanamata
    • 1
  • Kazuo Shinozaki
    • 5
  • Hidetoshi Iida
    • 3
  • Kazuyuki Kuchitsu
    • 1
    • 2
  1. 1.Department of Applied Biological ScienceTokyo University of ScienceNodaJapan
  2. 2.Research Institute for Science and Technology (RIST)Tokyo University of ScienceNodaJapan
  3. 3.Department of BiologyTokyo Gakugei UniversityKoganeiJapan
  4. 4.Laboratory of BiomembraneTokyo Metropolitan Institute of Medical ScienceTokyoJapan
  5. 5.RIKEN Plant Science CenterTsukubaJapan
  6. 6.United Graduate School of Agricultural ScienceTokyo University of Agriculture and TechnologyFuchuJapan
  7. 7.Division of Plant SciencesNational Institute of Agrobiological SciencesTsukubaJapan

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