JXB Advance Access originally published online on January 31, 2006
Journal of Experimental Botany 2006 57(4):791-800; doi:10.1093/jxb/erj064
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RESEARCH PAPER |
Arabidopsis thaliana Cyclic Nucleotide Gated Channel 3 forms a non-selective ion transporter involved in germination and cation transport
1Department of Biology, University of York, York YO10 5DD, UK
2Plant Sciences Group, Institute for Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
* To whom correspondence should be addressed. E-mail: fjm3{at}york.ac.uk
Received 13 August 2005; Accepted 9 November 2005
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Abstract |
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The Arabidopsis thaliana genome contains 20 cyclic nucleotide gated channel (CNGC) genes encoding putative non-selective ion channels. Classical and reverse genetic approaches have revealed that two members of this family (CNGC2 and CNGC4) play a role in plant defence responses whereas CNGC1 and CNGC10 may participate in heavy metal and cation transport. Yet, it remains to be resolved how the ion transport attributes of CNGCs are integrated into their physiological function. In this study, CNGC3 is characterized through heterologous expression, GUS- and GFP-reporter gene fusions, and by adopting a reverse genetics approach. A CNGC3–GFP fusion protein shows that it is mainly targeted to the plasma membrane. Promoter GUS studies demonstrate CNGC3 expression predominantly in the cortical and epidermal root cells, but also a ubiquitous presence in shoot tissues. Expression of CNGC3 in yeast indicates it can function as a Na+ uptake and a K+ uptake mechanism. cngc3 null mutations decreased seed germination in the presence of NaCl but not KCl. Relative to the wild type, mutant seedling growth is more resistant to the presence of toxic concentrations of NaCl and KCl. The ionic composition and ion uptake characteristics of wild-type and mutant seedlings suggests that the growth advantage in these conditions may be due to restricted ion influx in mutant plants, and that CNGC3 functions in the non-selective uptake of monovalent cations in Arabidopsis root tissue.
Key words: Arabidopsis, cation, CNGC3, Cyclic Nucleotide Gated Channel, germination
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Introduction |
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Plants need an adequate supply of mineral nutrients to sustain growth and development. However, the availability of nutrients is subject to large fluctuations in both space and time. As sessile organisms, plants had to develop a significant degree of plasticity in their uptake capacity in the form of multiple transport pathways whose activities can be adjusted to specific environmental conditions and tissue requirements.
For cation uptake and translocation, electrophysiological studies have shown the presence of a large number of transport pathways including ion channels and ion carriers. A number of these transporters has also been identified at the gene level and revealed that uptake and distribution of particular ions can involve a number of genes and gene families. For example, K+ in Arabidopsis is transported by carriers of the KUP/HAK/KT family (Rodriguez-Navarro, 2000
), as well as by channels of the KAT/AKT family. Extrusion and vacuolar sequestration of Na+ involves activity of NHX type antiporters (Apse et al., 1999). In addition, members of other families such as KCOs, CHXs, and GLRs (Maeser et al., 2001) may be involved in the transport of cations such as K+, Na+, and Ca2+ giving a total of more than 100 putative gene products. This genetic diversity leads to a bewildering array of potential transport pathways whose physiological significance has yet to be understood.
An additional potential pathway for the uptake of monovalent ions consists in the ubiquitously distributed cyclic nucleotide gated channels (CNGCs) which comprise a large gene family in Arabidopsis (see Talke et al., 2003, for a review). The 20 members of this family are structurally related to Shaker type K+ channels with six putative trans-membrane spans and a pore region between spans 5 and 6. A domain in the 4th transmembrane span shows similarity to the Shaker type voltage sensor and a C-terminal domain is believed to bind both cyclic nucleotides and calmodulin (Koehler and Neuhaus, 2000).
Understanding of CNGC function is fragmented. Heterologous expression appears frequently problematic, but the limited data available indicate that gating of plant CNGCs critically depends on the presence of cAMP and/or cGMP (Leng et al., 1999, 2002; Balague et al., 2003). Recent work further suggests that calmodulin reduced CNGC mediated current, possibly by interfering with cyclic nucleotide binding (Hua et al., 2003b). Voltage dependence in the form of inward rectification was observed for CNGC1 and CNGC2 (Leng et al., 1999) but not CNGC4 (Balague et al., 2003). Functional analysis in heterologous systems further showed that all CNGCs characterized freely conduct K+ and Na+ apart from CNGC2 which discriminates against Na+ (Leng et al., 2002; Balague et al., 2003; Hua et al., 2003a), a feature that is unknown in animal CNGCs and shown to be related to an unusual selectivity motif in the pore region (Hua et al., 2003a).
The role of CNGCs in mammalian physiology is well documented with respect to transduction of visual and olfactory stimuli via CNGC-mediated modulation of the membrane potential and Ca2+ signals. In addition, CNGCs are increasingly being implied in Ca2+ signalling in many other cell types (Kaupp and Seifert, 2002). In plants, the role of CNGCs is less clear. CNGCs have been shown to be present in many species (Talke et al., 2003). Some evidence suggests a role of CNGCs in heavy metal homeostasis, for example, in tobacco, overexpression of NtCBP4 led to hypersensitivity to Pb2+ (Arazi et al., 1999). By contrast, expression of a truncated version of NtCBP4 led to increased tolerance to Pb2+ and reduced uptake of this metal ion (Sunkar et al., 2000). A detailed characterization of null mutations in AtCNGC2 and AtCNGC4 revealed that both mutants exhibit altered responses to pathogen attack. cngc2 null mutations lead to a ‘defence no death’ phenotype after exposure to pathogens and fail to generate a typical hypersensitivity response or programmed cell death (Clough et al., 2000). cngc4 mutants show a lesion mimic phenotype where a partial hypersensitive response occurs in the absence of a pathogen (Balague et al., 2003). In addition, a loss of function in CNGC2 affects Ca2+ and monovalent cation homeostasis (Chan et al., 2003) whereas CNGC10 was recently shown to be involved in plant K+ uptake (Li et al., 2005).
Ca2+, K+ and cyclic nucleotides have all been shown to be involved in early signalling during defence responses (Clough et al., 2000; Jabs et al., 1997). For example, the production of reactive oxygen species (ROS) (which have antimicrobial activity) requires cAMP and Ca2+ in French bean cells (Bindschedler et al., 2001) whereas cGMP induces defence gene expression in tobacco suspension cells (Durner et al., 1998). Thus, in both cngc2 and cngc4 mutants, the presence of non-functional CNGCs could interfere with cyclic nucleotide signalling and/or ion fluxes that form part of the response to pathogen attack.
These studies provide some insights into the role of plant CNGCs suggesting their involvement in pathogen responses and possibly heavy metal homeostasis. In addition, functional data show that CNGCs act as monovalent cation transporters in heterologous systems. The latter prompted us to test whether CNGCs might be involved in the uptake and translocation of monovalent cations in planta. Three independent CNGC3 mutant alleles that showed a consistent phenotype associated with cation stress affected seed germination and distribution of monovalent cations.
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Materials and methods |
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Plant growth and treatments
Arabidopsis thaliana (L.) ecotype Columbia (0) wild type and insertional mutant seeds were surface-sterilized and placed on agar plates or suspended in 8 ml of growth medium contained in 6-well plates. Growth medium composition was as described previously (Maathuis et al., 2003
) and contained 1.25 mM KNO3, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 0.625 mM KH2PO4 as macronutrients. After vernalization at 4 °C for 2 d, seeds were transferred to a growth cabinet or growth room with the following conditions (12 h, 200 µmol m–2 s–1 light intensity, 24/20 °C day/night temperature, RH 70–80%). T3 segregating T-DNA mutants for CNGC3 were obtained from the SALK institute (http://signal.salk.edu/cgi-bin/tdnaexpress) and genotypically tested through PCR using genomic primers designed using the SIGnAL toolbox (http://signal.salk.edu/tdnaprimers.html) in combination with T-DNA border primers (see (http://signal.salk.edu/cgi-bin/tdnaexpress for details) to identify homozygous mutants. The following gene-specific, intron-spanning, sets of primers were used to carry out RT-PCR on each line. Line 3-1: forward 5'-ATCAGAACCTTTAAGCGGCCC-3' and reverse 5'-CGATCAACGGACAAGAACCGT-3'; Line 3-2 forward 5'-GAAGCCCGAGCGATTTTGTCT-3' and reverse 5'-TAAGATTGCGGAGACCCCACC-3'. Line 3-3: forward 5'-TGTGCTCGTCTCAAAACGGTTC-3' and reverse 5'-CACATAAAGCTCTTTACTGGTTACA-3'. Ribosomal 18S and actin specific primers were used as the positive controls in all reactions. No CNGC3 transcript was detected in any of the T-DNA lines.
Treatments for phenotypic characterization consisted of the additions of ionic salts to the growth medium in the form of chloride salts, except Pb2+ which was added as Pb(NO3)2. At least three different concentrations were tested for each ionic treatment. Mean plant fresh weights were determined from 30–40 seedlings per plate and at least three independent experiments were carried out per treatment.
Germination assays were performed by placing around 100 seeds of wild type and mutant in six-well plates containing 8 ml of control growth medium or growth medium supplemented with increasing concentrations of monovalent cations. After incubation at 4 °C for 48 h, plates were transferred to growth rooms and germination was scored on day 5 for radicle emergence.
GFP-reporter gene studies
The mGFP5 gene was inserted into a pART7 plasmid (Gleave, 1992) behind the 35S promoter at the BamHI site using forward primer mGFP5f (BamHI): 5'-AAAGGATCCATGAAAGGAGAAGAACTTTTCACT-3' and reverse primer mGFP5r (BamHI): 5'-AAGGATCCTTATTTGTATAGTTCATCCAT-3') introducing a start and stop codon, respectively. CNGC3 was then amplified by PCR using forward primer CNGC3f: 5'-CCCCCCGGGATGAATCCCCAAAGAAACAAATTCGTAA-3' and reverse primer CNGC3r: 5'-TCCCCCCGGGGGTTTCATCCATAGGAAACTCA-3', to introduce an XmaI cloning site. Both pART7-GFP and CNGC3 were digested with XmaI and gel purified. Ligation was performed at 16 °C overnight using T4 DNA ligase. The ligation products were transformed by electroporation into electro-competent E. coli (Invitrogen) and plated on LB ampicillin plates. Colonies were confirmed by colony PCR using pART7f primer (5'-CAATCCCACTATCCTTCGTAAGA-3') and CNGC3r primer and subsequently through sequencing.
Either the CNGC3-pART7-GFP or the pART7-GFP construct were introduced into onion epidermal slices placed on an agar supportive medium through a biolistics approach as described by Helenius et al. (2000). Alternatively, the same constructs were used for transient expression of Arabidopsis leaf protoplasts according to Abel and Theologis (1994). CNGC3-GFP expression and pART7-GFP (control) expression were visualized 24–48 h post-transformation using a Zeiss LSM 510 meta confocal microscope.
Tissue ion analysis
Seedlings and mature plants for ICP analysis were grown hydroponically in the conditions described above. After various treatments, tissues were washed twice for 10 min with cold 20 mM CaCl2 or LaCl3 solution to exchange ions bound to the cell wall matrix. Harvested tissues were frozen in liquid nitrogen and ground prior to acid digestion in concentrated nitric acid in a CEM MARS5 microwave (CEM, Buckingham, UK) and analysed on a Ciros Inductively Coupled Plasma Optical Emission Spectrometer (Ciros. Kleve, Germany).
CNGC3 promoter-GUS-reporter gene construction
The 5'-upstream region of AtCNGC3 was identified by screening a genomic library of Arabidopsis. A DNA sequence was isolated by PCR (Expand Long Template PCR System, Roche) on genomic DNA with a forward primer containing a SalI site (5'-ATGTCGACGTAACATTGTGTTAGTAACTTACC-3') and a reverse primer introducing a BamHI site just upstream of the ATG codon (5'-TTGGATCCCGCTGATTGTTTTCTTCCACAGAT-3'). This 3 kb fragment was introduced into pGEM-T-Easy vector (Promega) and multiplied after transformation of E. coli (XL2 Blue Ultracomp Cells, Stratagene). The construct was digested by HindIII and BamHI to produce a 1.6 kb long promoter region and inserted into the vector pBI101.3 (Jefferson, 1987) containing the ß-glucuronidase (GUS) gene. The chimeric construct was transformed into Agrobacterium tumefaciens strain LBA4404 (Invitrogen) and selected colonies were used to transform Arabidopsis plants by the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on agar plates containing 50 mg l–1 kanamycin. Tissues for histochemical GUS localization were stained with 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) as described by Jefferson (1987) and cleared in ethanol.
CNGC3 heterologous expression in yeast
A full-length cDNA for AtCNGC3 was subcloned into the expression vector pKT11 or pYES2 under the control of the Saccharomyces cerevisiae GPD or GAL promoters, respectively. The following yeast strains were used for heterologous expression: mid1 cch1 (MATa, mid1
KanMX, cch1KanMX, leu2-3, 112, his4, trp1, ura3-52, rme; Fischer et al., 1997), G19 (ena1::HIS3::ena4; Bañuelos et al., 1998), and a K+ uptake deficient strain (MATa, his3d200, trk1d51, trk2d50::lox-kanMX-lox, tok1d1::HIS3, leu2-3, 112trp1d901, ura3-52, suc2d9). Competent yeast cell preparation and transformation were carried out using the polyethyleneglycol method as described by Dohmen et al. (1991). The mutant yeast strains were transformed with empty vector (pKT11 or pYES2) (EV) or CNGC3 (pKT11-AtCNGC3 or pYES2-AtCNGC3). Transformants were selected for and maintained on plates containing synthetic deficient (SD) medium with the required nutrients minus uracil (Sherman et al., 1986).
G19 yeast growth experiments:
Transformants were grown at 30 °C in liquid arginine phosphate (AP) medium (Rodriguez-Navarro and Ramos, 1984) containing 1 mM KCl, 2 mM MgSO4, 0.2 mM CaCl2, and 3% (w/v) glucose or galactose. 100 mM NaCl was added for the salt treatment. Numbers of cells were deduced from the OD600 of liquid cultures according to a calibration curve based on representative cell counts.
Measurements of yeast ion contents:
For the determination of intracellular Na+ contents, cultures were grown in Na+-free liquid AP medium at 30 °C to the early stationary phase. 100 mM NaCl was added and 5 ml samples were taken from the culture after various incubation times. Cells were collected on a nitrocellulose filter, washed twice with ice-cold 100 mM MgCl2, and rinsed onto a new filter. Ions were acid-extracted in 5 ml 100 mM HCl and 100 mM MgCl2 and the concentrations of Na+ were determined by atomic absorption spectroscopy. Intracellular ion contents are expressed in nmol 10–6 cells. For K+ contents assays, cells of the EV and CNGC3 expressing yeast strains were grown on medium containing 100 mM KCl to an OD600 of around 1.5, concentrated by centrifugation (2 min, 2000 rpm) and washed in K+-free buffer. Cells were starved in K+-free medium for 24 h and subsequently resuspended in medium containing 20 mM K+. After 24 h growth on the low K+ medium, cells were harvested, washed twice, and acid extracted K+ contents were determined using a K+-selective electrode.
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Results |
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Phenotypic characterization of CNGC3 T-DNA insertional mutants
Three independent CNGC3 insertion mutants, lines salk_056832, salk_066634, and salk_151673 (named cngc3-1, 3-2, and 3-3, respectively) were derived from the SALK Institute (Alonso et al., 2003
). Lines 3-1, 3-2, and 3-3 carry insertions in the 3rd, 3rd, and 7th exon, respectively, at approximately 850, 1020, and 2200 bp of the genomic sequence of At2g46430 (Fig. 1). On the basis of kanamycin segregation, all three lines appeared to contain a single T-DNA insert. RT-PCR analysis showed none of the three lines expressed full length CNGC3 transcript (data not shown).
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Germination of cngc3 mutants is affected by cation stress:
When cngc3 seeds were placed on plates or suspended in growth medium containing increasing concentrations of monovalent salts, germination rates were progressively lower (Fig. 2). However, there were clear distinctions between mutant and wild-type seed germination. For all three independent lines (3-1, 3-2, and 3-3) germination in the presence of 100–140 mM NaCl was significantly lower than for the wild type. This effect was specific for NaCl and did not occur with either comparable concentrations of KCl or NH4Cl (data not shown). Iso-osmolar concentrations of sorbitol (up to 300 mM) only had a relatively small impact on seed germination (data not shown) that was similar for wild-type and mutant seeds.
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Thus, the data suggest cngc3 germination is not altered in its osmotic sensitivity but exhibits a specific sensitivity to toxicity of Na+, but not K+ or
Growth of cngc3 is affected by cations:
In control growth conditions, no or very little difference could be observed between growth characteristics of mutant and wild-type plants. When plants were germinated on control plates (containing 1.85 mM K+ and 0.5 mM Ca2+) and transferred to plates with increasing concentrations of monovalent salts no substantial differences in growth rates between mutant and wild-type plants were observed in the presence of NH4Cl or LiCl (data not shown). However, mutant plants grew better when exposed to NaCl stress, but this difference was significant only at intermediate concentrations of NaCl (40–80 mM, Fig. 3A; for clarity, only data for lines 3-1 and 3-2 are shown). In equimolar concentrations, KCl produced comparable growth inhibition in Arabidopsis to that observed with NaCl. However, the presence of high concentration of KCl, which had no discernible effect on cngc3 germination, inhibited wild-type growth more than growth of mutant lines. The relative growth advantage of the mutant lines became more pronounced at high KCl concentrations (Fig. 3B).
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No significant differences in phenotype of cngc3 mutants and wild-type plants were detected when plants were grown in media containing various amounts of Zn2+ (0.8–1.2 mM), Pb2+ (0.3–0.6 mM), Cd2+ (0.1–0.2 mM), low (0 mM) or high (30 or 50 mM) Ca2+, and 200 mM sorbitol. Mutant plants did not show altered phenotypes regarding their gravitropic response and in response to pathogens (data not shown).
Ion contents and composition in CNGC3 mutants
The germination and growth data suggest that in planta, CNGC3 may be involved in uptake and/or translocation of alkali cations such as K+ and Na+. Thus, the lack of CNGC3 function in the mutant lines may have an impact on homeostasis of these ions. Tissue concentrations and net uptake were therefore determined for a number of different elements using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). Since the three independent mutants showed similar phenotypes the subsequent analyses were carried out for lines 3-1 and 3-2 only. ICP data revealed that wild-type and cngc3 seedlings grown in control conditions contain similar levels of macro and micronutrients (data not shown). Growth for several days on high levels of KCl (100–120 mM) or NaCl (80–120 mM) did not lead to any significant differences between mutant and wild-type seedlings for Na+ content. However, these conditions did cause a significantly (P <0.05) lower level of K+ accumulation in mutant lines where K+ content increased by about 40% compared with wild-type seedlings where K+ rose by around 80% (Table 1). These results suggest that mutant plants are more capable of preventing K+ accumulation.
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It was also tested whether short-term uptake of Na+ was altered in the mutants. When net uptake of Na+ was measured over periods of 1 h and 3 h, a consistently lower influx was observed in mutant lines compared to wild-type plants (Table 2). Further uptake experiments during an intermediate period of 24 h showed that this difference was no longer present (Table 2). These data indicate that, during the initial stages of salt stress, CNGC3 makes a considerable contribution to Na+ influx but after prolonged periods net uptake fluxes not only diminish but also converge to similar values for wild-type and mutant lines, suggesting a limited participation of CNGC3 in ion uptake in salinity-adapted plants.
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Heterologous expression of CNGC3 in yeast
The observed phenotype and ICP data for cngc3 mutants suggest CNGC3 involvement in monovalent cation transport. Nevertheless, altered responses to ionic stress can originate from many factors and the loss of function in CNGC3 may not affect ionic fluxes per se but could be due to related phenomena such as signalling, membrane potential homeostasis or osmotic adjustment. In order to establish whether CNGC3 can function as an actual cation pathway and might, therefore, directly alter ion fluxes in planta, the CNGC3 gene was expressed in yeast strains that were defective in various aspects of cation transport.
The yeast strain ‘G-19’ carries deletions in the major Na+-extruding pumps ENA1-4, thereby greatly enhancing its salt sensitivity. Expression of AtCNGC3 in this yeast background did not lead to any significant difference in growth between the CNGC3 expressing and empty vector (EV) expressing strains in control medium. However, after a few h of growth it becomes apparent that a rise in NaCl concentration in the medium to 100 mM leads to a significant reduction in growth for the CNGC3-expressing strain compared with the EV control (Fig. 4A). In addition, ion contents analyses showed that the CNGC3 expressing yeast accumulates significantly more Na+ compared with the EV strain. The difference in cellular Na+ content was already apparent after 1 h (Fig. 4B) and suggests that CNGC3 functions as a Na+ permeable uptake mechanism in these yeast cells.
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To assess CNGC3 K+ permeability, a K+-uptake-deficient yeast deleted in the K+ transporters, Trk1, Trk2, and Tok1 was used that requires K+ concentrations of around 10 mM or higher for growth. Whereas complementation of this strain with K+ channels such as AKT1 restores growth at micromolar external K+, CNGC3 expression in this yeast background leads to partial complementation only in the millimolar range of external K+ (Fig. 4C). In addition, CNGC3 results in increased K+ accumulation (Fig. 4D). Combined, the yeast expression data suggest that CNGC3 can function as a pathway for Na+ and K+ transport.
Heterologous expression of CNGC3 in a yeast strain that lacks high affinity Ca2+ uptake did not affect yeast growth characteristics.
CNGC3 expression patterns
Data concerning possible CNGC3 substrates can give helpful clues towards putative roles of this transporter. In addition, the physiological function of gene products is often associated with their location both at the cellular and subcellular level. The latter issue may be of particular relevance in providing a rationale for the existence of large gene families such as the CNGC family. Therefore, expression localization studies were carried out for CNGC3 using GUS–promoter constructs and GFP–CNGC3 fusion constructs.
GUS reporter gene expression shows ubiquitous CNGC3 expression:
A 1.6 kb 5' upstream region of CNGC3 was cloned from genomic DNA using a PCR approach and fused to the GUS reporter gene to determine where CNGC3 expression is most prevalent. Figure 5A and B show that, in plate-grown plants, CNGC3 expression occurs in seeds prior to germination. In 2-d-old (Fig. 5C) and 1-week-old (Fig. 5D) seedlings, CNGC3 expression is prevalent along elongated root tissue including root hairs and this pattern remains similar in mature roots (Fig. 5E). Root cross-sections (Fig. 5F) indicate that expression is predominantly in epidermal and cortical tissue and absent or limited in stelar regions.
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Expression in shoot tissues is virtually absent in very young seedlings (Fig. 5C), but becomes more pronounced 6–7 d after germination (Fig. 5D). Initially, expression occurs in small clusters of cells that tend to concentrate near the leaf vasculature and cotyledon periphery, creating a spotty pattern (Fig. 5D, and inset). During leaf development, expression gradually increases and becomes ubiquitous around the vascular bundles, particularly those of mature leaves (Fig. 5G, H). Leaves of comparable age from soil-grown plants generally showed much lower levels of expression (Fig. 5H, inset) possibly due to a different nutrient composition of this medium.
GFP–reporter gene expression indicates CNGC3 is predominantly located at the plasma membrane:
A C-terminal fusion between the CNGC3 coding region and GFP was constructed in the pART vector. This construct was used to express CNGC3 transiently in epidermal onion cells using gold particle biolistics. Figure 6A shows GFP expression under the control of the constitutive 35S promoter only, which generates diffuse and delocalized GFP expression. Expression of the CNGC3-GFP fusion protein is restricted to a defined pattern that predominantly comprises the peripheral membrane (Fig. 6B) although some expression in ER/Golgi type membrane structures is also present (Fig. 6C) which may be due to the use of a strong promoter. However, transient homologous expression in protoplasts showed the GFP signal exclusively in the cell periphery confirming a plasma membrane location (Fig. 6D, E) which was distinctly different from Golgi and ER marker lines (data not shown). After protoplast lysis which releases intact vacuoles, no observable tonoplast GFP signal was observed (data not shown).
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Discussion |
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Non-selective ion channels have been extensively characterized in planta (Demidchik et al., 2002
). Yet, it generally remains to be resolved what physiological purpose these transporters fulfil, a task that is greatly frustrated by the uncertainty about which genes encode non-selective channels. Completion of Arabidopsis genome sequencing revealed a large number of putative non-selective ion channels, notably the cyclic nucleotide gated channel (CNGC) gene family, consisting of 20 genes. In animal cells, CNGCs have been shown to serve specific goals such as transducing olfactory and visual stimuli (Kaupp and Seifert, 2002), but their functions in plants are mostly obscure.
As potential non-selective cation transporters, CNGCs can be envisaged to participate in the uptake and/or translocation of monovalent and possibly divalent cations (Chan et al., 2003; Li et al., 2005). Such processes have physiological relevance since they may contribute to plant nutrition and cell signalling but also since they provide a potential mechanism for the entry and distribution of toxic ions like Na+ and heavy metals. To test how CNGCs might function in these processes, CNGC3 expression was studied using a reverse genetics approach. GUS-reporter studies show expression in embryo and high levels of CNGC3 expression in epidermal and cortical root cells, but not in stelar tissue. In shoots, CNGC3 expression is less pronounced and mainly limited to vasculature surrounding tissue. GFP-reporter analyses show plasma membrane localization but also signal in endomembrane structures similar to ER. This is particularly the case when CNGC3 is heterologously expressed in onion cells. By contrast, transient homologous expression shows a clear CNGC3 plasma membrane location and no or very little overlap with any other structures. cngc3 null mutants did not show any visible phenotype when plants were grown in control conditions. Nor did a loss of function in CNGC3 alter the response to gravitropism or pathogen attack (results not shown). Furthermore, plant exposure to heavy metals Pb2+, Zn2+, and Ni2+ and high or low Ca2+ failed to reveal a significant difference between wild-type and mutant plants. However, a loss of function in CNGC3 generated changes in germination and ionic composition when plants were exposed to monovalent cation stress.
The cngc3 null mutant shows altered germination rates in the presence of NaCl
Increasing levels of monovalents in the growth medium had a marked detrimental effect on germination rates in both wild type and mutant. This is probably the result of many factors such as water deficit, the build up of ion toxicity, and increased levels of the germination inhibiting hormone ABA However, clear differences between wild type and mutants were present. In the presence of high ambient levels of Na+, mutants were considerably more sensitive than wild-type plants, although this is more apparent for lines 3-1 and 3-3. Although it is not clear where this differential response originates, subtle and pleiotropic differences could be due to T-DNA positional effects.
The response to increasing concentrations of K+ and 
was mostly similar for both genotypes. The latter stages of germination require the uptake of large amounts of osmotica, particularly monovalent ions. Non-selective ion channels such as CNGCs could form a convenient pathway for the influx of cations to promote germination, however, they would also increase the risk of ion toxicity at elevated concentrations. According to this scenario, a cngc3 null mutation would be expected to show detrimental effects for germination at low external ion concentrations whereas it would be beneficial at high external Na+. In fact, our data suggest the opposite: mutant germination at low Na+ is not affected or slightly improved and at high Na+ it is significantly worse than for wild-type seeds (Fig. 1). These data suggest that, during germination, CNGC3 is unlikely to be involved in substantial ion uptake from the external medium but rather that CNGC3 might participate in avoiding ionic toxicity possibly by relaying Na+ from sensitive to tolerant tissues in the developing embryo. Since CNGC3 is likely to function as a non-rectifying transporter (Balague et al., 2003) it could potentially mediate both influx and efflux of Na+, depending on tissue-specific and local ionic gradients. Non-selective ion channels have indeed been shown to be present in the seed coat parenchyma cells where they function in releasing univalent ions into the seed apoplast (Zhang et al., 2002). The absence of such a pathway might cause an accumulation of toxic ions in parenchyma cells which are likely to be more sensitive than the seed apoplast. Thus, if CNGC3 were to participate in this or similar processes, its absence could be detrimental during germination.
Mutations in CNGC3 affect growth and ion composition in the presence of high levels of monovalent cations
When using control medium, growth of mutant and wild-type plants was generally comparable. In the presence of high concentrations of Na+, mutant lines grew marginally better than wild-type plants, a growth difference that became more pronounced when plants were exposed to high concentrations of K+. Elevated levels of K+ induce a growth reduction in Arabidopsis that is comparable to that caused by Na+ and thus K+ can be considered equally toxic (Liu and Zhu, 1997).
In shoots of older seedlings and more mature plants, considerable expression of CNGC3 was observed around the vasculature. Thus, in shoots, CNGC3 may contribute to the distribution and/or translocation of ions delivered by the xylem. An over-supply of these ions would be detrimental to shoot water relations and photosynthesis. Further experimentation will be necessary to show whether a lack of CNGC3 function might reduce shoot loading of these ions.
Growth tolerance in the presence of toxic concentrations of monovalent cations can be achieved in many ways, for example, by limiting ion influx, increased compartmentation into vacuoles or through relocation of ions from sensitive to less sensitive tissues. The results show that, in mutant seedlings, net Na+ uptake and K+ accumulation are reduced. In addition, there is clear evidence that in heterologous systems CNGC3 can function as a Na+ and K+ uptake pathway. GUS data show that in seedlings, CNGC3 is mainly expressed in epidermal and cortical root tissues. Thus, it is tempting to conclude that in these conditions CNGC3 may form part of the uptake pathway for Na+ and K+ and loss of its function therefore leads to decreased uptake of these ions. However, the reduction in mutant Na+ uptake, although substantial, is transient which may explain why no or only a slight growth advantage is displayed by mutant plants.
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Acknowledgements |
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We are grateful to Alonso Rodríguez-Navarro (Polytechnic University, Madrid) for providing the yeast G19 strain and to David Lindsey (Department of Chemistry) for assistance with the atomic absorption spectrometer. Part of the described research was supported by BBSRC funding to AG and GJP.
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References |
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