JXB Advance Access originally published online on November 7, 2005
Journal of Experimental Botany 2005 56(422):3051-3060; doi:10.1093/jxb/eri302
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RESEARCH PAPER |
Functional analysis of a putative Ca2+ channel gene TaTPC1 from wheat
National Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
To whom correspondence should be addressed: Fax: +86 10 64873428. E-mails: jszhang{at}genetics.ac.cn; sychen{at}genetics.ac.cn
Received 16 February 2005; Accepted 2 September 2005
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Abstract |
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The cytosolic free-calcium concentration [Ca2+](cyt) transiently increases under abiotic stresses and the proteins that control this process are gradually disclosed. The Ca2+-permeable channel is one type of these proteins in plants. In the present study, a novel Ca2+-permeable channel gene TaTPC1 encoding a putative membrane protein was cloned from wheat. It was induced under high salinity, polyethylene glycol, low temperature (4 °C), and abscisic acid. Expression of TaTPC1 in the yeast mutant lacking CCH1 can recover its growth under lithium stress through functional complementation. TaTPC1 transgenic plants exhibited more stomatal closing in the presence of Ca2+ than the control, supporting a role for the calcium channel in regulating plant responses to environmental change.
Key words: Calcium channel, stomatal closing, stress induction, transgenic plants, wheat
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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High salinity, drought and low temperature adversely affect plant growth and crop production. Plants have common mechanisms in physiological responses to reduce the damage by abiotic stresses. One early response to these stresses in plant cells is an increase in cytosolic free-calcium concentration [Ca2+](cyt) by means of infusion from the apoplastic space and release from internal stores (Sanders et al., 1999
; Xiong et al., 2002). The transient increases of cytosolic Ca2+ can be perceived by various Ca2+-binding proteins, such as CDPKs (Ca2+-dependent protein kinases) (Harmon et al., 2001), CBL1 (Cheong et al., 2003), and the SOS3 family of Ca2+ sensors, which are involved in coupling and transducing stress signals by specific protein phosphorylation cascades, and follow the plant stress response (Sheen, 1996). It is revealed that Ca2+, as a second messenger, plays an important role in stress signal transduction in plants (Sanders et al., 2002). The transport of Ca2+ is regulated by various Ca2+ channels, Ca2+-ATPases, and/or H+/Ca2+-antiporters on cell membranes. The entry of the Ca2+ into plant cells is through the function of Ca2+ channels in the plasma membrane, whereas the maintenance of an optimal Ca2+ concentration in cells is performed by Ca2+-ATPases and/or H+/Ca2+-antiporters. Therefore, calcium channels, like the Ca2+ transporters, were also involved in abiotic signal transduction by regulating the change in [Ca2+](cyt) (White, 2000; White and Broadley, 2003).
Calcium channels have been reported to localize in the plasma membrane, tonoplast, endoplasmic reticulum, chloroplast, and nuclear membranes according to electrophysiological studies (Sanders et al., 2002). There exist different types of gating mechanisms of Ca2+ channels in the plant, such as ligand-, volage-, and stretch-activated (Demidchik et al., 2002). The electrophysiological and biochemical characteristics of Ca2+-permeable channels of plant cells are well known, but only a limited number of genes encoding Ca2+-permeable channels have been isolated and functionally studied (White et al., 2002). A wheat gene LCT1, encoding a low-affinity cation transporter, can complement a yeast mutant with a disruption in the MIDI gene, which encodes a stretch-activated Ca2+-permeable non-selective cation channel (Amtmann et al., 2001). The homologues of nucleotide-gated cation channels (CNGCS) have been cloned from barley, tobacco, and Arabidopsis. In Arabidopsis, CNGCSs comprise a gene family, one of which was expressed in human embryonic kindney cells, and a cyclic nucleotide-dependent increase in Ca2+ permeability was demonstrated. AtTPC1 from Arabidopsis encodes a two-pore voltage-gated channel with high affinity for Ca2+ permeation, and rescued the Ca2+ uptake activity of a yeast mutant cch1 (encoding an homologous L-type Ca2+ channel). [Ca2+](cyt) was enhanced by overexpression of AtTPC1 or suppressed by its antisense expression under sucrose stress (Furuichi et al., 2001). Most recently, the AtTPC1 protein has been found to be ubiquitous in plant vacuoles and this channel may regulate seed germination and stomatal movement (Peiter et al., 2005). Its homologue OsTPC1 from rice has also been identified and characterized (Hashimoto et al., 2004; Kurusu et al., 2004). OsTPC1-overexpressing plants showed reduced growth and abnormal greening of roots (Kurusu et al., 2004). From tobacco BY-2 cells, two homologous genes, NtTPC1A and NtTPC1B, were also identified. These two genes complemented the growth of the yeast mutant defective in CCH1, and co-suppression of them resulted in the inhibition of a rise in cytosolic free-Ca2+ concentration in response to sucrose and a fungal elicitor (Kadota et al., 2004). These results suggest that the Ca2+-permeable channels play an important role in mediating the spatial and temporal variation of Ca2+, and are involved in regulating the growth and development of plants.
In the present study, a putative Ca2+-permeable channel gene TaTPC1 was cloned from wheat. Its expressions were induced in roots under abscisic acid (ABA) and various stress treatments. Expression of TaTPC1 in the yeast mutant lacking CCH1 (homologous to the 1-subunit of a voltage-gated Ca2+ channel) can recover its growth through functional complementation, and TaTPC1-overexpressing plants exhibited more stomatal closing under Ca2+ when compared with the control plants.
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Materials and methods |
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Plant materials and treatments
Wheat (China spring, Triticum aestivum) seeds were germinated and maintained on wet cheesecloth, supplemented with water, under continuous illumination at 25 °C. For cold treatment, the 10-d-old seedlings were kept at 4 °C for various periods and both shoots and roots were harvested for RNA isolation. For ABA, NaCl, and polyethylene glycol (PEG) treatments, the seedlings were transferred into solutions containing 100 µM ABA, 250 mM NaCl, and 25% PEG (w/v), respectively, treated for various periods, and then harvested.
Construction of a wheat cDNA library and yeast one-hybrid screening
A cDNA library was constructed with the mRNA from the salt- and PEG-treated wheat seedlings. According to the manufacturer's (Stratagene) protocol, the first-strand cDNAs were synthesized and followed by second-strand synthesis. The uneven termini of the double-stranded cDNA were filled in with Pfu DNA polymerase, and EcoRI adapters were ligated to the blunt ends. After XhoI digestion, the double-stranded cDNAs were ligated to the Uni-ZAP XR vector, and then packaged by Gigapack III Gold packaging extract. The library was amplified and converted to the pAD-GAL4 library by mass excision. For one-hybrid screening, the pAD-GAL4 plasmids containing wheat cDNA inserts were transformed into the Saccharomyces cerevisiae strain YRG2 harbouring the dual DRE-controlled reporter genes His3 and LacZ (Liu et al., 1998). Transformants were plated on medium lacking histidine, ura, and leucine but containing 3-AT (a competitive inhibitor of the HIS3 gene product). Approximately 1x107 clones were screened. Colonies larger than 1 mm were obtained and streaked on the same medium. After growing for 36–72 h, the colonies were further tested for the expression of ß-galactosidase by filter lift assays (Stratagene). Plasmids isolated from the putative yeast colonies were transformed into Escherichia coli. The cDNA inserts in these plasmids were sequenced according to standard procedures.
5'-RACE for the 5' sequence of TaTPC1
5'-RACE was performed because the isolated cDNA (TaTPC1) was partial in its 5'-end. Two specific primers were designed according to the partial cDNA sequence of TaTPC1 (GSP: 5'-CCAGCCAATTCTGCTACTTTCTGCAG-3'; NGSP: 5'-CTCGCGGTAGCAGCCAAGGATGGT-3'). 5'-RACE was carried out according to the SMART12 RACE cDNA Amplification Kit User Manual (Clontech). PCR reaction was performed for five cycles of 94 °C for 5 s, 72 °C for 2 min; five cycles of 94 °C for 5 s, 68 °C for 10 s, 72 °C for 2 min; 30 cycles of 94 °C for 5 s, 64 °C for 10 s, 72 °C for 2 min; and finally 72 °C for 10 min. Subsequently, the dilution of the PCR product was used in a nested PCR reaction. The nested PCR conditions were identical to the initial reaction. The products were analysed on a 1.2% agarose/EtBr gel and the corresponding DNA band was recovered and directly sequenced.
Northern analysis
RNA extraction was performed as described previously (Zhang et al., 1996). Total RNA (30 µg) was fractionated in a 1.2% agarose gel containing formaldehyde and blotted onto Hybond-N+ nylon membrane for northern analysis. The full-length of TaTPC1 cDNA was labelled as a probe and used in the hybridization. The membranes were washed in 2x SSC plus 0.1% SDS at 45 °C for 15 min and in 1x SSC plus 0.1% SDS at 45 °C for 5 min. The membranes were then autoradiographed using a phosphoimaging system (Amersham Pharmacia).
Generation of yeast mutant for the CCH1 gene
The S. cerevisiae strain used in this study was W303.1A (MATa leu2-3112 ura3-1 trp1-92 his3-11,15 ade2-1 can1-100 GAL SUC mal), which contains the yeast CCH1 gene. The CCH1 gene encodes a protein homologous to the 
1-subunit of a voltage-gated L-type Ca2+ channel from mammalian cells. To knock out the CCH1 gene, a 706 bp fragment encoding a Trp marker gene was amplified from the pBD vector of the yeast two-hybrid system through PCR with 5' primer: 5'-AGATCATCGTGGAATAGAATAGATCTGGTATCTTCTGFCAGTATTAAGCACACAAAGGCAGC-3' and 3' primer: 5'-CTGATTCGCCTTAAGCTTAAAATATTTCCAGTAGCCTGTTACAGTAATAACCTATTTCTTAGC-3' (1513–2223 bp). The procedure was performed as described previously (Wang et al., 2005). The transformed yeast cells were screened on SD medium lacking Trp (SD–Trp medium). The wild-type yeast cells could not grow on SD–Trp medium, while the mutant cch1 with the mutation in the CCH1 gene could grow on it.
TaTPC1 functional complementation assay
The yeast plasmid pYES2 with the GAL1 promoter was used as an expression vector. The TaTPC1 coding region was amplified from the original plasmids with two primers 5'-GCAGGATCCAGAGAGATGAGCGAAGCGAG-3' (sense primer) and 5'-AGTGAATTCTCAAGAGTTTTGAGATCCATC-3' (antisense primer). The amplified product was digested with KpnI plus XhoI and ligated to KpnI/XhoI-digested pYES2 to construct the expression vector pYES2-TaTPC1. The plasmid pYES2-TaTPC1 or pYES2 vector were transformed into wild-type or mutant yeast cells, respectively, generated as in the section above, using the lithium acetate method. The transformant with pYES2 vector was used as a control. The transformant was cultured in SD liquid medium lacking Ura (SD–Ura medium) until OD600=1.2, and then diluted to an OD600 value of 0.01 or 0.001. Five microlitres of each dilution was dripped on basic YPGAL (1% yeast/2% peptone/2% galactose; Difco) medium or YPGAL medium containing 5 mM or 10 mM lithium chloride (LiCl), and cultured at 30 °C for 3 d. The growth status of the yeast cells was observed.
Arabidopsis transformation and stomatal aperture analysis
A BamH1/Kpn1 fragment encoding the full-length TaTPC1 was inserted downstream of the 35S promotor in the plant expression vector pBin438, and introduced into Arabidopsis plants by the vacuum infiltration technique. Independent homozygous transgenic lines were obtained after selection of T3 progeny on MS medium containing 50 mg l–1 kanamycin. Ten-day-old seedlings of the transgenic progenies and wild-type plants are cultured on MS medium and used for molecular analysis. Twenty-day-old seedlings of these plants grown in pots containing vermiculite were used to observe stomatal aperture. The lower epidermis was ripped off different leaves and floated in a solution consisting of 50 µM KCl, 10 mM MES-TRIS, and 100 µM, 1 mM, or 10 mM Ca2+, respectively. The peels were incubated under light at 25 °C for 1.5 h. Then the stomatal apertures were measured under an optical microscope.
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Results |
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Cloning of the TaTPC1 gene from wheat
The wheat cDNA library was screened for stress-inducible homologues of the CBF/DREB gene by the yeast one-hybrid method. Whereas one sequence turned out to be the homologous gene of CBF/DREB (Shen et al., 2003
), another sequence from a weak positive colony exhibited similarity to voltage-gated Ca2+-permeable two-pore channel genes. Because Ca2+ channels were also involved in stress responses (White and Broadly, 2003), the current putative Ca2+ channel gene, named TaTPC1 (Triticum asetivum two-pore channel 1), was investigated further. Since the inserted sequence still represents the 3' part of the TaTPC1 gene, 5'-RACE was carried out and a fragment of 1351 bp was obtained, which overlapped with the sequence of TaTPC1. The combined sequence represented a full-length cDNA of TaTPC1.
The TaTPC1 was 2841 bp in length with an open reading frame of 2229 bp flanked by the 117 bp 5'-untranslated region (UTR) and 495 bp 3'-UTR plus a poly (A) tail (Fig. 1A). The open reading frame encoded a putative protein of 743 amino acids with a predicated molecular mass of 85.6 kDa. By analysis via the SMART program (Letunic et al., 2002) and comparison with the amino acid sequence from other Ca2+-permeable channel proteins, several conserved domains were identified. As shown in Fig. 1B, the TaTPC1 has two homologous domains (nos 1 and 2) similar to other TPCs (Furuichi et al., 2001; Hashimoto et al., 2004; Kurusu et al., 2004). Both domains have six transmembrane segments, S1 to S6 (Fig. 1A, B). There is a pore loop (P) between S5 and S6 in each domain. S4 contains charged residues and may function as a voltage sensor. S1, S2, S3, S5, and S6 segments of each domain have distinct hydrophobic indices while S4 and pore loop regions have smaller indices. Both N- and C-termini are hydrophilic. The connecting segment between domains II and I is two Ca2+-binding EF-hand motifs, which are also hydrophilic and may regulate its activity. The overall amino acid sequence shared 52%, 77%, and 85% identity with AtTPC1 (AB053952
[GenBank]
, Arabidopsis; Furuichi et al., 2001), OsTPC1 (AB100696
[GenBank]
, Oryza sativa; Hashimoto et al., 2004), and HvTPC1 (AY465119
[GenBank]
, Hordeum vulgare), respectively (Fig. 2). These results indicated that TaTPC1 was more closely related to the two-pore voltage-gated channel for Ca2+ from monocots.
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Expression of the TaTPC1 gene under different abiotic stresses
The wheat seedlings were subjected to various treatments and expression of the TaTPC1 gene was examined. As shown in Fig. 3, an increase of TaTPC1 mRNA was detected in wheat seedling roots after treatment with high salinity, PEG, low temperature (4 °C), and ABA. However, the expression pattern was different. Under ABA treatment, the expression of TaTPC1 was induced early when compared with the patterns under NaCl and PEG treatments. When treated with low temperature, the expression of TaTPC1 was not detected until 24 h after the initiation of the treatment. TaTPC1 expression was not detected in shoots of the wheat seedlings under these treatments. These results indicate that TaTPC1 may play specific roles in roots in response to various treatments.
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Functional complementation of the yeast mutant cells by TaTPC1
To analyse the function of TaTPC1, a complementation strategy was adopted. The CCH1 gene of the yeast strain (W303.1A) (Fisher et al., 1997), which shares high homology with the
1-subunit of a voltage-gated Ca2+ channel from mammalian cells (Paidhungat and Garrett, 1997), was knocked out through the insertion of a Trp marker gene by homologous recombination (Wang et al., 2005). The yeast cch1 mutant strain was thus obtained by its survival on SD–Trp medium. The wild-type yeast cells cannot grow on the same medium. The CCH1 gene is involved in Ca2+ uptake and is instrumental in the response of a wild-type strain to ion stress (Fisher et al., 1997; Paidhungat and Garrett, 1997). Disruption of the CCH1 gene resulted in the blocking of the Ca2+ uptake in the cch1 mutant and then led to the sensitivity of the mutant to LiCl (Paidhungat and Garrett, 1997). Taking advantage of this feature of the cch1 mutant, the present TaTPC1 gene can be transformed into the mutant to test if the gene can fulfil a similar function as the CCH1 did. The open reading frame of TaTPC1 was thus ligated to the expression vector pYES2, and the recombinant plasmid pYES2-TaTPC1 was transformed into the mutant cells. The pYES2 vector itself was also transformed into the wild-type and the mutant cells as control experiments. To compare the growth status of these transformants, a series of overnight cultures grown in the SD–Ura liquid medium were diluted and dropped onto YPGal or YPGal plus 5 or 10 mM LiCl medium. On normal YPGal medium, all of the transformants showed normal growth (Fig. 4A). On 5 mM or 10 mM LiCl medium, the mutant cch1 cells harbouring the pYES2-TaTPC1 can grow normally, similar to wild-type yeast cells harbouring the pYES2 vector (Fig. 4B, C). On the contrary, the cch1 mutant with the pYES2 vector cannot grow on the same medium. These results indicate that the TaTPC1 protein can complement the function of Ca2+ channel CCH1 and may then regulate the level of [Ca2+](cyt) to maintain the normal growth of yeast mutants under lithium stress.
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Transgenic Arabidopsis plants overexpressing the TaTPC1 gene
To analyse the function of TaTPC1 in the plant, the TaTPC1 gene was cloned into binary vector pBin438 under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 5A), and then transformed into Arabidopsis plants using the vacuum infiltration method. Homozygous T3 lines were obtained for four lines and the integration of TaTPC1 into the Arabidopsis genome was confirmed by Southern blot (data not shown). The TaTPC1 mRNA level in transgenic plants was examined and it was found that all the four independent lines showed significantly increased mRNA levels compared with the wild-type plants (Fig. 5B). The phenotype of the transgenic plants was also examined and no significant change was found when it was compared with wild-type plants under normal or stress (salt, drought, or ABA) conditions (data not shown). Because TaTPC1 is a putative Ca2+ channel protein, it may thus facilitate the entry of Ca2+ into the plant cells in the transgenic plants overexpressing the TaTPC1 gene. However, no significant alterations in the Ca2+ contents were detected in the aerial part of the transgenic plants in comparison with the wild-type plants under both normal conditions and CaCl2 treatment (data not shown). Although no significant change in overall Ca2+ content was observed in the transgenic plants, transient variations may still exist in various tissues or cells.
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Stomatal aperture analysis of the TaTPC1 transgenic plants
Calcium channel plays an important role in regulating the Ca2+ influx, and the transient change of [Ca2+] was involved in stomatal movement (McAinsh et al., 1990
). To examine whether overexpression of TaTPC1 in the transgenic line affected stomatal movement under different concentrations of CaCl2, the stomatal aperture of wild-type and TaTPC1-overexpressing plants was analysed in vitro using isolated epidermal peels. Under a concentration of 100 µM Ca2+ that should promote stomatal opening, the distribution of open stomata in transgenic lines 3 and 4 peaked at 1.6 µM, while the control peaked at 1.9 µM (Fig. 6A), indicating that the apertures of the stomata from the transgenic lines are generally smaller than those from the wild-type plants. Under a concentration of 1 mM Ca2+, transgenic lines 3 and 4 and the control plants all showed a peak at 1.3 µM for stomatal aperture (Fig. 6B). When the plants were treated further with 10 mM CaCl2, the distribution peak of stomatal aperture for transgenic lines 3 and 4 moved further toward 1.0 µM, whereas the distribution peak for the control plants was still localized at 1.3 µM (Fig. 6C). These results indicate that overexpression of TaTPC1 most likely promoted the closing of stomata in the presence of Ca2+.
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Discussion |
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Under normal growth condition, the [Ca2+](cyt) in plant cells is maintained at about a level of 100 nM through the activity of Ca2+-ATPase, Ca2+-permeable channel, and Ca2+/H+ antiporters in cell membranes (White and Broadley, 2003
). Under abiotic stress conditions, [Ca2+](cyt) was enhanced. An explanation for this increase could be membrane depolarization under abiotic stress treatments, which leads to the opening of Ca2+-permeable channels (White and Broadley, 2003). A second reason could be that expression of genes encoding Ca2+-permeable channel were induced under abiotic stress. This latter case has been proved from the increase of [Ca2+](cyt) after overexpression of AtTPC1 (Furuichi et al., 2001). In the present study, a calcium channel gene TaTPC1 was cloned and characterized from wheat. The expression of TaTPC1 was induced under high salinity, PEG, low temperature (4 °C), and ABA, implying that TaTPC1 is involved in plant stress responses. The spatial and temporal distribution of Ca2+ may be changed compared with that under normal conditions. However, the level of [Ca2+](cyt) is strictly controlled because excessive Ca2+ may cause cell death. Excessive Ca2+ may be effluxed to extracellar space or stored in the vacuoles and endoplasmic reticulum, etc. through a Ca-ATPase (Chung et al., 2000) and a Ca2+/H+ antiporter (Ueoka-Nakanishi et al., 2000), whose expression was also induced by abiotic stress treatments. It should be noted that TaTPC1 was inducible in the root but not the shoot of wheat seedlings, implying that TaTPC1 may play a major role in the root by responding to stimuli from the soil. By contrast to the induction of TaTPC1 by various stresses, other TPC1 genes such as AtTPC1 from Arabidopsis and OsTPC1 from rice appeared to be ubiquitously expressed in the whole plant (Furuichi et al., 2001; Kurusu et al., 2004). The different expression patterns may result from different homologues of the TPC1 genes. In tobacco, two homologues, NtTPC1A and NtTPC1B, have been identified (Kadota et al., 2004). In wheat, two expressed sequence tags (BQ904560
[GenBank]
and CA617077
[GenBank]
) were also identified (data not shown), which showed homology to the present TaTPC1. The two expressed sequence tags exhibited similarity to different parts of the TaTPC1 gene and are not overlapping (data not shown). This analysis indicates that the wheat genome may contain other homologous TPC1 genes.
Although the TaTPC1 gene can be induced by various stresses and ABA, the TaTPC1-overexpressing plants did not show significant alterations in sensitivity to these treatments (data not shown). This observation is different from that obtained in the AtTPC1-overexpressing Arabidopsis. Peiter et al. (2005) reported that AtTPC1 overexpression or knockout in Arabidopsis regulated seed germination in response to ABA. The discrepancy may be due to the fact that the present wheat TaTPC1 gene is expressed in a heterologous Arabidopsis system. Alternatively, the wheat TaTPC1 gene may not be a real orthologue of the Arabidopsis AtTPC1 gene since their proteins shared only about 50% identity.
To date, most of the results on the localization of the Ca2+-permeable channel were based on electrophysiological and biochemical methods (Sanders et al., 2002). The TaTPC1-GFP fusion protein was used to study the localization of the TaTPC1 protein in onion epidermal cells and it was found that TaTPC1 appeared to be localized in the plasma membrane (data not shown). This localization is in contrast with the result obtained by Peiter et al. (2005), who reported that the Arabidopsis AtTPC1 protein was localized in the vacuolar membrane. The reason for this difference is not known. It is possible that the discrepancy may result from the different expression systems used. Alternatively, the present TaTPC1 may represent a homologue of TPC1 in wheat, as discussed above, and different homologues may have various functions and membrane locations. Other biochemical methods should be used to demonstrate the precise location of the TaTPC1.
Yeast has been proposed as a model system to study the function of plant genes in response to stress (Xiong et al., 2002). A change in [Ca2+](cyt) has been observed in yeast as well as in plants under abiotic stresses. Therefore both organisms share some of the components in the stress signal transduction pathway (Xiong et al., 2002). It has been reported that a yeast strain lacking CCH1, a gene encoding a polypeptide of 2039 residues and sharing high homology with the 1-subunit of a voltage-gated l-type Ca2+ channel from mammalian cells, exhibits a biochemical defect in calcium uptake and consequently grows slower (Paidhungat and Garrett, 1997). This delay in growth of the cch1 mutant could be explained by the necessity for extracelluar calcium as a mediator in responding to high salt concentrations (Matheos et al., 1997). In yeast, the same transport system is mediating the uptake of sodium and lithium across the plasma membrane (Borst-Pauwels, 1991). Lithium chloride used with millimolar concentrations showed the same effect as sodium chloride in molar concentrations. Since salt stress implies ionic as well as osmotic stress conditions, the osmotic stress conferred by lithium is lower because of its effectiveness at lower concentrations (Nakamura et al., 1993). In order to confer the salt stress condition, LiCl was used as an analogue of sodium chloride in this study. The sensitivity of the cch1 mutant to a high-salt condition (Paidhungat and Garrett, 1997) provided the basis for establishing a yeast system to test the function of TaTPC1 in response to salt stress in relation to calcium. Therefore, the cch1 mutant was generated in the present study and used to identify the function of the present TaTPC1 gene from wheat. The TaTPC1 protein can recover the growth of the mutant cch1 yeast cells under LiCl stress, indicating that the TaTPC1 gene has a similar function to CCH1. The Ca2+ channel gene AtTPC1 from Arabidopsis also rescues the growth rate and the Ca2+ uptake activity of the yeast cch1 mutation (Furuichi et al., 2001). The direct evidence for the Ca2+ uptake activity of the present wheat TaTPC1 protein should be investigated further.
TaTPC1 transgenic plants have a higher proportion of stomata with an aperture smaller than that of the control plants. This fact suggests that the guard cells of the TaTPC1 transgenic plants may take up more Ca2+ via the TaTPC1 channel from the apoplast or the surrounding cells and make the stomata more closed. This result is consistent with the report that a rise in Ca2+ concentration in the guard cell is useful for closing stomatal apertures (Hamilton et al., 2000). When the Ca2+ concentration was increased to 1 mM, the stomatal aperture was reduced to 81% in transgenic plants and 68% in control plants. However, when the Ca2+ concentration was further increased to 10 mM, the stomatal aperture of the transgenic plants was further reduced to 62% of the original apertures, whereas the control plants maintained a reduction of 68%. This fact probably indicates that the Ca2+ effect on stomatal closing is non-linear in the transgenic and the control plants, and the guard cells of the transgenic plants are relatively less sensitive to the Ca2+ than those of the wild-type plants. The reason for this difference in sensitivity is not known. It is possible that the transgenic plants already had more TaTPC1 proteins and adsorbed the Ca2+ in guard cells, thus making the stomatal closing less sensitive at the 1 mM Ca2+ concentration. The regulation of the stomatal movement by the present TaTPC1 is consistent with the observations made by Peiter et al. (2005). They reported that an Arabidopsis tpc1 knockout mutant was defective in the response of stomata to extracellular calcium.
Although TaTPC1 is responsible for Ca2+ permeation, significantly more Ca2+ was not found to have accumulated in the transgenic plants than in the control plants when using the atomic absorbance spectrometer method (data not shown). This phenomenon may result from the fact that whole aerial parts of the plants were used for the Ca2+ measurement. The uneven distribution of Ca2+ in different cells, tissues, or organs may thus be averaged in the TaTPC1 transgenic plants. Alternatively, this phenomenon may be explained by the fact that, as a second messenger, Ca2+ can only have a transient increase that may lead to the initiation of a signal transduction. Duration of this increase for a long time would be harmful for plant survival and thus must be dismissed. Therefore, a more sensitive approach to Ca2+ probing should be used to reveal the transient change in Ca2+ concentration in transgenic plants (Furuichi et al., 2001). Further research should disclose more about the function of TaTPC1 in plants.
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Acknowledgements |
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This work was supported by the National Key Basic Research Project (2002CB111303, 2006CB100102), Chinese Academy of Sciences (KSCXZ-SW-327) and the National Science Foundation of China (30370130).
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Footnotes |
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* Present address: College of Life Sciences, Shanxi Normal University, Xian 710062, China.
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References |
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