JXB Advance Access originally published online on March 2, 2006
Journal of Experimental Botany 2006 57(5):1201-1210; doi:10.1093/jxb/erj092
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RESEARCH PAPER |
Functional analysis of CHX21: a putative sodium transporter in Arabidopsis
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT
* To whom correspondence should be addressed. E-mail: J.Pritchard{at}bham.ac.uk
Received 14 November 2005; Accepted 9 December 2005
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Abstract |
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The functional role of CHX21, a member of the Arabidopsis thaliana CHX cation transporter family, has been investigated in plants growing under ‘ideal’ conditions and in the presence of elevated NaCl levels. In public databases, AtCHX21 (At2g31910) is annotated as a putative Na+/H+ antiporter. In this study, Southern analysis was used to identify a genotype that contained a single transposon insertion within its genome; using PCR, this insertion was shown to be within the CHX21 locus. No CHX21 transcript was detectable in Atchx21 (mutant) plants using RT-PCR. In the absence of salt stress, Atchx21 showed significant quantitative differences from the wild type (AtCHX21) in development with respect to characters such as rosette width and flowering time. In the presence of 50 mM NaCl, (i) roots of Atchx21 elongated more slowly than the wild type, (ii) the leaf sap Na+ concentration was significantly lower in Atchx21 compared with the wild type, and (iii) the concentration of Na+ in the xylem was lower compared with the wild type. The concentration of Na+ exported from the leaf in the phloem was unchanged. Thus, loading of Na+ into the root xylem could explain changes in leaf concentration of Na+. This hypothesis was supported by immunolocalization which demonstrated that the AtCHX21 transporter could only be detected in root endodermal cells. Immunogold labelling of ultra-thin sections, followed by transmission electron microscopy, demonstrated the localization of the protein in the plasma membrane. The data demonstrate that the CHX21 transporter may play a role in regulation of xylem Na+ concentration and, consequently, Na+ accumulation in the leaf.
Key words: Cation transport, CHX transporter, endodermis, gene knockout, sodium, xylem
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Introduction |
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All organisms need to regulate ionic concentrations within their cells in order to survive. A range of membrane proteins have evolved to control the movement of ions in and out of cells and of their subcellular compartments. In plants, understanding of the processes by which different membrane transporters, pumps and channels serve to regulate cation levels in different tissues, cells, and cell compartments is patchy. However, the acquisition of the whole genome sequence of the model plant Arabidopsis has allowed the identification of large numbers of genes encoding putative cation transporters. A major effort is now required to define the precise role of each transporter in plant growth and development both in ‘ideal’ conditions and under conditions of cation stress (e.g. high salinity).
Through analysing the Arabidopsis whole genome sequence, Mäser et al. (2001)
reported that approximately 5% of Arabidopsis genes appear to encode membrane transport proteins. To date, 150 putative cation transporters have been identified and phylogenetic relationships between these have been proposed. However, the lack of physiological knowledge about Arabidopsis membrane-bound transporters poses a limitation on the analysis of ion transport mechanisms, ion homeostasis, and the ways in which plants cope with changing ionic environments (Yamaguchi et al., 2003). Na+ transporters are integral membrane proteins that play major roles in pH and Na+ homeostasis of cells throughout the biological kingdom (Padan et al., 2001; Putney et al., 2002) and are therefore obvious targets for functional analysis. Mäser et al. (2001) identified a large subfamily of genes encoding putative Na+ transporters within the Arabidopsis genome that they called the CPA2 (cation/proton antiporter) subfamily. This family contains the CHX (cation proton exchanger) family and genes have been named AtCHX1–28. There have been two publications on members of the AtCHX gene family. Cellier et al. (2004) provided evidence that AtCHX17 has a role in K+ acquisition and homeostasis. It is also reported that AtCHX23 is localized to the chloroplast envelope where it is thought to be involved in pH homeostasis and chloroplast development (Song et al., 2004). All members of the CPA2 subfamily share the same conserved regions and hence it is not possible to subclassify members on the basis of existing sequence information. There are no obvious indications of known functional regions and very little similarity to the KEA or NHX transporter families (no overlap in conserved regions). Hence, the sequence data do not currently allow any predictions with respect to substrate or mode of function.
Some other Arabidopsis transporters have been shown to transport sodium, including members of the NHX group. Since it appears that Na+ is not essential for plants, some of these putative Na+ transporters may have other functions, such as K+ transport. For example, SOS1 (AtNHX7) is a plasma membrane sodium transporter that controls Na+ entry into xylem in roots (Shi et al., 2002). However, the K+ uptake of sos1 mutants is lower than wild types at low K+ concentrations, suggesting that sos1 mutants have an impaired K+ uptake system (Wu et al., 1996). There is strong evidence that AtNHX1 is a tonoplast-located Na+/H+ antiporter in Arabidopsis (Apse et al., 1999; Gaxiola et al., 1999; Darley et al., 2000). However, use of tomato plants over-expressing AtNHX1 has shown that the transporter can also mediate the transport of K+ (Zhang and Blumwald, 2001); topological analysis of this transporter revealed that the ratio of Na+/K+ transport could be altered by deletion of the hydrophilic C terminus (Yamaguchi et al., 2003). Yeast NHX1 has recently been shown to function in proton homeostasis of a prevacuolar compartment (PVC) necessary for trafficking (PVC–Golgi pathway; Venema et al., 2003). Using an AtNHX1 knockout line in Arabidopsis, Apse et al. (2003) reported changes in leaf development in the absence of Na+ application, supporting the proposal that the gene is required for K+ homeostasis in plants. By contrast HKT1 (=SAS2) has been shown to have a role in remobilizing Na+ for export from the leaf in the phloem (Berthomieu et al., 2003).
The aim of the current study was to start to elucidate the functional diversification among individuals of the unstudied CHX gene family of putative Na+/H+ antiporters in Arabidopsis. Membrane transporters act to alter many aspects of plant growth and development. At the whole plant level, accumulation of ions in the leaf is the result of the action of transporters in individual membranes. Solute concentrations in the leaf are the consequence of import from the root through the xylem and re-export to the root through the phloem. Disruption to a membrane transporter at any point along the root–leaf–phloem–root pathway could result in an alteration in ion levels in the root or leaf. Such changes in root solute relations may alter root growth or development yielding important clues about gene function. A range of techniques have been applied to analyse the variables of this simple model. Xylem ion concentration is a major component of ion flux to the leaf. However, extraction of xylem sap is confounded by the negative tensions in the xylem. This difficulty was overcome by using the xylem-feeding insect, Philaneus spumaris, as a xylem sampler (Watson et al., 2001). In addition, phloem-feeding aphids were used to obtain pure samples from sieve tubes using stylectomy (Pritchard, 1996).
In this study, detailed physiological analyses were applied to an insertional mutant of CHX21 (At2g31910) and have demonstrated that the gene plays a role in the regulation of xylem and so leaf Na+ content.
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Materials and methods |
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Plant genotyping
CHX21 mutant
Seeds of the SLAT line 01–19 (Sainsbury Laboratory Arabidopsis Transposants) produced in a Col-0 background were obtained from the Nottingham Arabidopsis Stock Collection (NASC) following interrogation of the Sequenced Insertion Site (SINS) database with the CHX21 gene sequence. The SLAT line comprised seed pooled from 50 plants, one of which was predicted to carry a copy of the non-autonomous transposon dSpm within the target gene. DNA was extracted, using the technique described by Gawel and Jarret (1991)
, as 20 pools each from 12 seedlings in order to identify a pool bearing a copy of dSpm in CHX21 using a PCR protocol. The individual bearing the mutant allele was then identified following DNA extractions from individual plants. PCR was carried out using 1 unit BIOTAQTM DNA Polymerase (Bioline), and 1.5 mM MgCl2 in a Hybaid Omn-E thermal cycler. Locus-specific primers NaT1F2 (5'-CCGGTGCTGGCTTATACTACCTTC-3') and NaT1R2 (5'-GTTAGCTCGACTAGGTGGATAGCA-3') along with transposon-specific primers dSpm1 (5'-CTTATTTCAGTAAGAGTGTGGGGTTTTGG-3') and dSpm11 (5'-GGTGCAGCAAAACCCACACTTTTACTTC-3') were used to amplify wild-type and mutant alleles. PCR amplification products were visualized by agarose gel electrophoresis. Products were purified using a QIAquick PCR Purification Kit (Qiagen) and sequence determined using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Version 2) and the ABI 3700 DNA sequencer. Individuals homozygous for the mutant allele were identified following the consistent failure to amplify a product specific to the wild-type allele using template DNA extracted from the original test plants and from any of their progeny following selfing. For the mutant lines, wild-type genotypes (bearing two copies of the wild-type allele at the target locus) were selected from each segregating population being studied. For subsequent study of the effects of the mutations, seed was bulked by selfing both mutants and their ‘paired’ wild-type genotypes and collecting seed batches produced under the same environmental conditions.
Southern blot analysis
Genomic DNA was extracted from wild-type and chx21 leaves as above and purified by a phenol/chloroform extraction (Sambrook et al., 1989). 2 µg DNA was digested overnight with HindIII, EcoRV, or SpeI, fractionated on a 0.8% agarose gel and blotted onto a Hybond N+ membrane (Amersham) by capillary transfer. The probe was a 362 bp fragment of the BASTA gene that exists in the transposon and was amplified using bastaF (5'-CATGAGCCCAGAACGACGCC-3') and bastaR (5'-TCTTGAAGCCCTGTGCCTCC-3') primers. This was cloned into pGEM-T (Promega), sequenced, and then reamplified. Blots were prehybridized overnight at 60 °C in modified Church and Gilbert's solution (Church and Gilbert, 1984) and incubated overnight at 60 °C with 32P radiolabelled probe (Feinberg and Vogelstein, 1983, 1984). Blots were washed at high stringency (0.2x SSC, 0.1% SDS) and the radioactivity was detected by autoradiography.
RT-PCR analysis
Seeds were surface-sterilized in hypochlorite solution (1.16% (w/v) sodium hypochlorite containing 0.0005% (v/v) Tween-20) for 5 min, washed for 5 min in 70% ethanol then 3x5 min washes in distilled water. Surface-sterilized wild-type and chx21 seed was germinated on MS salts (Murashige and Skoog, 1962) with 1% sucrose and 1% agar (technical no. 3, Oxoid) in square Petri dishes. This formulation contained 20 mM K+ and 0.1 mM Na+. Seeds were vernalized for 2 d at 4 °C then transferred to 24 °C (16/8 h light/darkness) for 11 d and Petri dishes were placed upright inside a clear plastic box. Total RNA was isolated from 100 mg Arabidopsis root tissue using the RNeasy Plant mini kit (Qiagen), treated with RQ1 RNase-free DNase (Promega) and RT-PCR analysis was carried out using one-step RT-PCR kit (Qiagen) with primers NaT1F5 (5'-GGTCTCGGCTTGGACTTGAGGATG-3') and NaT1R6 (5'-AATGGCTAAACCATCACGCAAAGG-3') with actin as an internal control (ActinF 5'-CAGCATCATCACAAGCATCC-3' and ActinR 5'-TGCTGACCGTATGAGCAAAG-3'). The RT-PCR product was reamplified with nested primers NaT1F6 /(5'-GGGACATACTGCAATGTGTGCAGC-3') and NaT1R5 (5'-TACGTGAGTGTCTCCCACTTGACC-3') and the products resolved on an agarose gel. The NaT1F6/NaT1R5 cDNA fragment was cloned into pGEM-T, sequenced, reamplified, and the product was used as a probe. The gel was blotted onto a Hybond N+ membrane (Amersham) and probed as described above.
General growth characteristics in unstressed conditions
Thirty chx21 and 30 ‘paired’ (see above) wild-type plants were grown in compost in a controlled environment room under stress-free conditions at 24 °C (16/8 h light/dark) in a randomized pot design with guard plants at the edges. The day of sowing was designated as day 0. The following characteristics were measured: time to germination; leaf number on day 20; rosette width on day 20 (measured at widest point); time to flowering; height at flowering; rosette leaf number at flowering; cauline leaf number at flowering; rosette width at flowering. Statistical analysis was carried out by Student's t test.
Root extension on vertical agar slopes
Surface-sterilized seeds were germinated on agar (as above) in square Petri dishes containing nutrient agar supplemented with either 0 mM, 50 mM or 100 mM NaCl or KCl. They were vernalized and grown vertically as described above. In separate experiments, seeds were either germinated directly on agar containing elevated NaCl, or germinated on control agar and then transferred to high salt agar after 4 d. Root length measurements were made daily. Statistical analysis and comparison of the slopes was carried out by Analysis of Covariance.
Bulk leaf sap relations
Seeds were germinated and grown in vermiculite at 22 °C (18/6 h light/dark). Seedlings were watered with half-strength Long Ashton solution (Watson et al., 2001) until 28 d. To impose salt stress, plants were watered with half-strength Long Ashton solution supplemented with 50 mM NaCl for the 48 h prior to sampling. Rosette leaves from stressed and non-stressed were excised from mutant and wild-type plants and frozen at –20 °C. Leaf tissue was crushed and centrifuged at 13 000 rpm for 10 min and the supernatant re-centrifuged for 5 min. One µl of supernatant was removed and diluted with 9 µl deionized water and analysed by ion chromatography (see below).
Xylem ion flux analysis
Plants were grown under the same conditions as described for bulk leaf sap collection and treated for 48 h prior to sap collection. Xylem sap from transpiring plants was collected using the spittlebug Philaenus spumarius (Malone et al., 1999; Watson et al., 2001; Ponder et al., 2002) collected from grassland near the University of Birmingham campus. Insects were kept in a temperature-controlled room (20 °C) on bean plants (Vicia faba L. cv. The Sutton). Prior to sampling, insects were starved overnight at 4 °C to encourage subsequent feeding. Insects were caged onto stems of test Arabidopsis plants in parafilm sachets and held in a growth cabinet at 22 °C. After 4–6 h, droplets of excreta were collected by piercing the parafilm cage with a Hamilton syringe. Samples were analysed by ion chromatography (see below). In order to calculate transpiration rates, plants were grown in vermiculite (see above). They were then salt-stressed for 18 h and the tops of the pots were covered in parafilm at soil level leaving only the leaves uncovered. Immediately prior to taking the first measurement, the bottoms of the pots were also sealed. Pots and plants were subsequently weighed at appropriate intervals over 36 h. The total leaf area of each plant was determined to allow the calculation of transpiration rate per unit area. Statistical analysis was carried out for data pairs using Student's t test or for all data using ANOVA.
Leaf and xylem sap ion analysis
Inorganic ions were analysed by ion chromatography using a Dionex DX-120 Ion Chromatograph (Dionex corporation, CA, USA). For cation analysis, an IonPac CS12a 4x250 mm Column and a CSRS-Ultra 4 mm Suppressor were used; for anion analysis an IonPac AS14 4 mm Column, and an ASRS-Ultra 4 mm Suppressor were employed. Cations were eluted using 1.4 ml concentrated sulphuric acid in 2.0 l of deionized water, while anions were eluted using 11 ml 1 M Na2CO3 and 1.4 ml 0.5 M NaHCO3 in 2.0 l of deionized water. Each 2 µl prepared leaf sap sample was mixed with 200 µl of the appropriate eluent for analysis. Each 5 µl xylem sap sample (undiluted) was mixed with 300 µl of appropriate eluent for analysis. The cation standard solution contained NaCl, NH4Cl, KCl, MgCl2, and CaCl2 (at 1 mM) and the anion standard solution contained KCl, KNO3, K2PO4, and 
(at 1 mM). Statistical analysis was carried out for data pairs using Student's t test or for all data using ANOVA.
Sieve element (SE) ion analysis
Approximately 10 aphids of Myzus persicae were caged on the stem of each experimental plant for 24 h prior to stylectomy. The stylets of feeding aphids were cut by microcautery (Pritchard, 1996). Following a successful cut, cages were immediately filled with water-saturated paraffin oil to prevent evaporation. SE sap exuding from the stylet tube was collected into microcapillaries and frozen at –20 °C until analysed. Osmotic pressure was measured using a modified nanolitre osmometer by melting point-depression (Tomos et al., 1994). Inorganic ion concentrations in SE sap were analysed by energy-dispersive X-ray (EDX) microanalysis (Hinde et al., 1998) using rubidium fluoride (RbF) as an internal standard. SE samples were expelled onto a pioloform-coated copper electron microscope grid (Agar Scientific, Essex, UK) under paraffin oil using a micro-constriction pipette. Grids were stored in a tissue culture dish in a desiccator until analysed using a Philips XL30 FEG ESEM electron microscope. Statistical analysis was carried out by ANOVA.
Immunolocalization
A peptide (SKSGVRNEMLPYSL) was designed to a unique region of the predicted protein sequence of CHX21 and submitted to ISL (Immune Systems Limited, UK) for synthesis and antibody production. Mutant and wild-type Arabidopsis plants were grown hydroponically in half-strength Long Ashton solution. Membrane proteins were extracted and the protein concentrations quantified using the Bradford Bio-Rad assay. The 1° antibody concentration was optimized using Dot Blots using donkey anti-rabbit alkaline phosphatase-conjugated 2° antibody and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as the substrate. Preimmune serum was used as a control.
Surface-sterilized seed was germinated on MS salts (Murashige and Skoog, 1962) with 3% sucrose and 0.8% agar. Seeds were vernalized for 48 h at 4 °C then transferred to 24 °C (16/8 light/dark) for 14 d. The apical 1 mm was removed and the next 10 mm of root was cut into 5 mm sections and grouped to form bundles. Root bundles were fixed overnight in 2% paraformaldehyde, 0.1% glutareldehyde in 0.1 M sodium cacodylate, before being dehydrated in a series of alcohol concentrations to 90% ethanol, incubated for 2x2 h in 1:1 90% ethanol:LR White hard grade resin (Agar Scientific) then 2x12 h in 100% LR White resin. Bundles were transferred to gelatin capsules and polymerized under UV light for 2 d. Two µm sections were cut using an ultramicrotome.
Fluorescence immunolocalization was as previously described (Armstrong et al., 2002) with 1/1000 diluted 1° antibody (in blocking buffer: 1% BSA, 0.1% Triton X-100 in PBS) and diluted 1/30 (in blocking buffer) 2° antibody (anti-rabbit FITC-conjugate, Sigma). Slides were examined by a Nikon Eclipse TE300 fluorescence microscope and images were captured with smartcapture® software.
Immunogold labelling
Root tissue was grown and embedded as mentioned above. Ultra-thin (90–150 nm) sections were collected on formvar-coated nickel grids. Immunogold labelling was completed as previously described (Armstrong et al., 2002) with 1° antibody diluted 1/1000 (in blocking buffer) and goat anti-rabbit IgG conjugated to 5 nm gold particles (BB International) diluted 1/50 (in blocking buffer). Grids were stained with 2% uranyl acetate(aq) for 45 min at 37 °C before washing (3x5 min) with double distilled water. Grids were examined on a Jeol 1200Ex electron microscope.
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Results |
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Molecular characterization of a CHX21 gene knock-out
A putative knockout of gene AtCHX21 (At2g31910) was identified in the SLAT collection using the Sequence Insertion Site database. Locus- and transposon-specific primers were used in PCR experiments to identify, from the pooled material supplied, individuals containing a copy of the transposon within the target locus. Sequencing of the PCR products indicated the site of insertion of the transposon dSpm into CHX21 (Fig. 1). Individuals homozygous for the mutant allele were identified following consistent failure of their DNA to support amplification of a portion of the wild-type allele; this was confirmed by the absence of the wild-type allele from any progeny produced by selfing these homozygotes.
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780 bp.Wild-type and chx21 DNA was digested with HindIII, EcoRV, or SpeI. This was hybridized to a probe designed to a region of the BASTA gene within the transposon construct. Single bands were obtained with chx21 DNA which confirms that there is a single insertion in the genome (Fig. 2a).
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Expression of CHX21 in different genotypes was analysed by RT-PCR to see whether the transposon insertion in chx21 prevents a transcript from being produced. RNA was extracted from root tissue from wild-type and chx21 plants, reverse transcribed, and then amplified using gene-specific primers. A cDNA transcript of the predicted size was evident in the wild type, but no detectable cDNA was evident for chx21. The identity of the cDNA was confirmed by hybridization to a chx21 cDNA radiolabelled probe (Fig. 2b).
Growth characteristics of unstressed mutant
Growth and developmental characteristics of the chx21 mutant were scored under unstressed conditions in a controlled environment room under long days (Table 1). In order to ensure the use of appropriate control genotypes, chx21 was compared to a genotype homozygous for the wild-type allele under study (CHX21) and produced, by segregation, from genotypes heterozygous at the locus under study. Chx21 seed germinated after 2.9 d, 0.7 d later than wild-type seed (P <0.001). There was no significant difference between rosette leaf numbers at day 20 for chx21 and wild type (P >0.05). Rosette width of chx21 seedlings was significantly reduced (P <0.05) compared with the wild-type plants. Despite there being no significant difference in height at flowering, chx21 took longer to flower compared with the wild type (P <0.001) and had significantly wider rosettes (P <0.05) with more rosette leaves (P <0.05) at flowering. Chx21 plants also had more cauline leaves (P <0.05) at flowering.
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Root extension
Under unstressed conditions, analysis of covariance of the slopes indicated that there was no significant difference in root elongation rate over 300 h from germination between the chx21 and wild-type plants (Fig. 3; P >0.1). There was a reduction in root extension rates of both wild type (P <0.05) and chx21 (P <0.001) in the presence of 50 mM NaCl. However, 50 mM NaCl reduced root growth of chx21 to a greater extent than the wild type (P <0.001). Very little extension occurred in either chx21 or the wild type when exposed to 100 mM NaCl (data not shown). In the presence of 50 mM KCl, only chx21 root growth rate was reduced (P <0.001).
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Ionic relations of bulk leaf sap
The concentrations of cations and anions were measured in the bulk leaf sap of chx21 and wild-type plants (Fig. 4a, b) following growth in the presence and absence of 50 mM NaCl. In the absence of elevated external NaCl, bulk leaf sap Na+ concentrations were lower in chx21 (15.0±7.6 mM) than wild-type plants (34.8±7.9 mM. In addition, chx21 plants had lower K+ concentrations (77.0±3.1 mM, than wild type (105.5±2.0 mM, Fig. 4a). Exposure of plants to 50 mM NaCl increased the leaf sap Na+ concentration of wild-type plants from 34.8 mM to 67 mM. However, following exposure to 50 mM NaCl, leaf sap of chx21 plants contained less Na+ than the wild type (19.1±7.6 mM; Fig. 4b). NaCl treatment of wild-type plants decreased the K+ leaf concentration from 105.5±2.0 mM to 84.8±3 mM, but there was no alteration in the K+ levels in the chx21. Analysis of variance demonstrated that all these differences were significant at at least P <0.05. In contrast to K+ and Na+, there were no significant differences in leaf concentration of Ca2+ or Mg2+.
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Xylem ion analysis
The xylem transports ions from roots to leaves; it is possible to collect samples that accurately reflect xylem sap ion compositions of transpiring plants using the xylem-feeding insect Philaenus spumarius. Following growth in the absence and presence of elevated NaCl, the concentrations of anions and cations in the xylem sap of chx21 plants were measured in intact, transpiring plants using the insect sampling system (Fig. 4c, d). Absence of the CHX21 gene was significantly associated with reduced xylem concentration of sodium from 0.45 mM to 0.11 mM in unstressed plants, and from 2.45 mM to 1.45 mM in salt-stressed plants. Analysis of variance revealed that the difference caused by the 50 mM salt treatments was significant at P >0.001 and the absence of the CHX21 gene was significant at P >0.05.
An assessment of the amount of Na+ delivered to leaves by the xylem must take account of the volume of water moving through it. Exposure of wild-type and chx21 plants to 50 mM NaCl did not significantly alter transpiration rates from unstressed plants (P >0.05). There was no difference in transpiration rates measured over 32 h between the wild type (0.024±0.006 ml–1 cm2 h–1) and chx21 plants (0.020±0.003 ml–1cm2 h–1) regardless of stress. Thus, the concentration of ions in the xylem are assumed to reflect changes in the flux of the ion to the leaves.
Sieve element (SE) ion relations
In order to determine whether recirculation between the leaves and roots through the phloem contributed to the amount of K+ and Na+ accumulated in bulk leaf sap, analyses were performed using SE sap collected using aphid stylectomy. There was no significant difference in osmotic pressure (OP) between non-stressed chx21 (0.98 MPa) and wild type (1.26 MPa) SE sap (P >0.05; Fig. 5a). Following exposure to 50 mM NaCl, stressed wild-type plants had a significantly higher SE sap OP (1.41 MPa) compared with stressed chx21 SE sap (0.94 MPa; P <0.05; Fig. 5b). Surprisingly, 50 mM NaCl stress, representing a water stress of about 0.25 MPa, did not significantly alter the SE sap OP of either chx21 (P >0.05) or wild-type (P >0.05) Arabidopsis plants. With the exception of an increase in SE K+ following salt treatment, there was no significant difference in the concentrations of any ions tested following 50 mM NaCl treatment or absence of the CHX21 gene.
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Immunolocalization of AtCHX21 protein
The peptide SKSGVRNEMLPYSL was predicted to be uniquely encoded by AtCHX21 and was used to raise an antiserum in rabbits. Initial dot blot assays using wild-type Arabidopsis membrane preparations showed that the antiserum produced a strong signal (compared with preimmune serum controls) when used in combination with a donkey anti-rabbit second antibody conjugated to alkaline phosphatase (not shown). The same primary antibody was then incubated with transverse sections of wild-type and chx21 root, leaf, and stem before using FITC-conjugated second antibody to visualize localization of the antigen using fluorescence microscopy. The results showed a clear signal from the endodermal cells in the wild-type Arabidopsis roots (Fig. 6) that was not seen in chx21 root sections or wild-type root sections treated with preimmune serum. Furthermore, no signal was detected in leaf and stem sections (data not shown). In addition, this observation confirms that chx21 is a genuine knockout on the basis that no protein containing this epitope was detected, which is in accordance with the lack of a detectable transcript by RT-PCR. This signal was absent in all stem and leaf sections of wild-type and mutant.
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Membrane localization of CHX21 was investigated by immunogold labelling. Ultra-thin root sections were incubated with primary antibody and then localized using goat anti-rabbit IgG conjugated to 5 nm gold particles and viewed under a transmission electron microscope. Gold particles were localized to the plasmalemma when sections were incubated with primary antibody, but these were not detected with sections treated with preimmune serum.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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The aim of this study was to investigate the functions of the AtCHX21 gene belonging to the CHX cation transporter gene family in Arabidopsis thaliana. The gene putatively encodes a Na+/H+ antiporter. Detailed, quantitative physiological analyses of individuals homozygous for mutation in this gene have been made in order to establish whether the current annotation is correct.
PCR using gene- and transposon-specific primers mapped the location of the transposon insertion within the CHX21 locus (Fig. 1). Southern blot analysis confirmed that this is the only insertion in the chx21 genome and, furthermore, there is only a single copy of the inserted transposon at this locus. RT-PCR showed that there is no detectable CHX21 transcript in chx21. In addition, immunolocalization failed to detect a signal in chx21 providing further evidence that chx21 is a gene knockout. Therefore, the only difference between the wild type and chx21 genomes is a mutation in the CHX21 locus in the mutant chx21.
In the absence of elevated NaCl levels, chx21 showed quantitative differences from the wild type in its growth and development. Individuals homozygous for the mutation had significantly narrower rosettes at day 20 and at flowering and flowered significantly later than the wild-type genotype.
External Na+ inhibited root elongation to a greater extent in chx21 compared with the wild type, however, roots from chx21 had the same elongation rate as wild-type roots in the absence of elevated cations. In addition, they elongated significantly slower than the wild type in the presence of 50 mM KCl (although the effect was less marked than 50 mM NaCl) suggesting that the encoded transporter is not completely Na+-specific.
A reduced capacity of the mature region of the root (where ions are taken up from the soil) to transport cations in the xylem to the leaves would result in the accumulation of these cations in this tissue. As a consequence, one would expect changes in the levels of these cations delivered to the root growing zone through the phloem sieve elements (Pritchard et al., 2000). While root SE cation levels were not measured in this study, such a perturbation of the system would be expected to alter the biophysical parameters of turgor pressure and cell wall rheology that regulate root cell extension (Pritchard, 1994).
Leaf concentrations of K+ and Na+ were altered in the chx21 mutant compared with the wild-type plants. For example, in unstressed plants, leaf K+ was lower in the mutant, but this difference disappeared when plants were salt stressed. In plants stressed with 50 mM NaCl, the leaf sap Na+ concentration was significantly lower in chx21 than in the wild type. No effects on calcium or magnesium could be detected. Other studies have noted changes in leaf inorganic solute relations following inactivation of endodermal transporters. A knockout of the root SAS1 transporter increased the leaf accumulation of Na+ relative to the wild type, but did not affect K+, Ca2+ or Mg2+ (Nublat et al., 2001). Inactivation of AHA4, an endodermal H+-ATPase altered the proportion of Na+ accumulated in salt-stressed leaves relative to ions including K+, Ca2+, and Mg2+ (Vitart et al., 2001). Regulation of solute levels in whole plants is complex and inactivation of individual solute transporters is likely to alter overall composition through a combination of both direct and indirect effects.
The concentration of Na+ delivered to shoots in xylem sap was significantly lower in chx21 compared with the wild type. Leaf accumulation of Na+ was correlated with xylem Na+ concentration in wheat (Watson et al., 2001). Similarly, in the present study, the differences in patterns of accumulation of Na+ in the leaf were accompanied by qualitatively similar patterns of ion concentration in the xylem that supplies the leaf. Transpiration and xylem ion concentration data demonstrated that less Na+ was delivered to leaves through the xylem in the chx1 mutant than in the wild-type plants. This simple analysis assumes that the amount of water lost from the plant by transpiration represents the volume moving in the xylem. Care must be taken in interpreting the xylem ion concentrations since these were measured using xylem in the flowering stalk. The morphology of Arabidopsis makes it difficult routinely to sample xylem between root and rosette leaves and it has been assumed that the ion profile in the xylem of the infloresence stem is representative of xylem in the leaf. Both are supplied by the same root system although differences in import/export along the pathway could result in different xylem ion concentrations in these two tissues.
The difference in the amount of sodium delivered to the leaves through the xylem in chx21 and the wild type is consistent with the observed differences in sodium concentration in the leaves of the two genotypes. However, it is possible that leaf ion concentrations are, in part, controlled by retranslocation of material out of the leaves by the phloem as has been reported (Zhong et al., 1998; Lohaus et al., 2000; Berthomieu et al., 2003). To test the hypothesis that ions were exported from the phloem, ion levels in the sieve elements were measured using aphids to obtain pure SE samples. There was no difference in the SE Na+ concentration between chx21 and wild type. In wild-type plants, an increase in sieve-element K+ concentration was consistent with the elevated xylem flux loading the leaf and the reduced leaf K+ levels. However, unlike the xylem, there is no indication of the volume flux through the sieve element and it was not possible to relate SE concentration to SE flux. The unchanged osmotic pressure measurements suggest that the driving forces between source and sink are not significantly different; however, lack of information on phloem resistances and the absence of direct measurements of flux preclude further speculation. It is interesting to note a significantly lower SE sap osmotic pressure in salt-stressed mutant plants, despite a concomitant decrease in SE inorganic ions. The difference may reflect a decrease in SE organic solutes. In this regard, it is interesting to note that absence of the AKT2 K+ channel resulted in a decrease in SE sucrose concentration (Deeken et al., 2002).
On the basis of the changes in root, leaf, and xylem physiology observed, it is hypothesized that gene AtCHX21 is expressed in root tissue and that the protein product plays a role in the entry of sodium ions into the xylem. To test this, a rabbit antiserum was produced using an AtCHX21-specific oligopeptide and this was used for immunolocalization studies. The transporter could only be detected in root endodermal cells and the use of immunogold labelling demonstrated the presence of the protein product only at the plasma membrane of these cells.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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The absence of this transporter in chx21 correlated with a lower concentration of Na+ in the xylem and a lower accumulation of Na+ in the leaves of plants grown in elevated NaCl conditions. This is consistent with a role in the regulation of ion accumulation in the leaf. Currently, the model for the function of the AtCHX21 gene is that it is expressed in the root endodermis where the wild-type protein product has a role in moving Na+ ions across the plasmalemma out of the endodermal cells into the apoplast of the stele from which they are carried to the leaves in the transpiration stream.
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This work was supported by BBSRC studentships to DH and AE. We would like to acknowledge Fay Hughes for technical support, the provision of Arabidopsis seed material from the Nottingham Arabidopsis Stock Centre, UK, and antiserum production by ISL (Immune Systems Limited).
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Apse MP, Aharon GS, Snedden WA, Blumwald E. 1999. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258.
Apse MP, Sottosanto JB, Blumwald E. 2003. Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter. The Plant Journal 36, 229–239.[CrossRef][Web of Science][Medline]
Armstrong SJ, Caryl AP, Jones GH, Franklin FCH. 2002. Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. Journal of Cell Science 115, 3645–3655.
Berthomieu P, Conejero G, Nublat A, et al. 2003 Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance EMBO Journal 22, 2004–2014.[CrossRef][Web of Science][Medline]
Cellier F, Conéjéro G, Ricaud L, Luu DT, Lepetit M, Gosti F, Casse F. 2004. Characterization of AtCHX17, a member of the cation/H+ exchangers, CHX family, from Arabidopsis thaliana suggests a role in K+ homeostasis. The Plant Journal 39, 834–846.[CrossRef][Web of Science][Medline]
Church GM, Gilbert W. 1984. Genomic sequencing. Proceedings of the National Academy of Sciences, USA 81, 1991–1995.
Darley CP, van Wuytswinkel OCM, van der Woude K, Mager WH, de BoerAH. 2000. Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers. Biochemical Journal 351, 241–249.[CrossRef][Web of Science][Medline]
Deeken R, Geiger D, Fromm J, Koroleva O, Ache P, Langenfeld-Heyser R, Sauer N, May ST, Hedrich R. 2002. Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 216, 334–344.[CrossRef][Web of Science][Medline]
Feinberg AP, Vogelstein B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6–13.[CrossRef][Web of Science][Medline]
Feinberg AP, Vogelstein B. 1984. Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 137, 266.[CrossRef][Web of Science][Medline]
Gawel NJ, Jarret RL. 1991. A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Molecular Biology Reporter 9, 262–266.[CrossRef]
Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR. 1999. The Arabidopsis thaliana proton transporters AtNHX1 and Avp1 can function in cation detoxification in yeast. Proceedings of the National Academy of Sciences, USA 96, 1480–1485.
Hinde P, Richardson P, Koyro HW, Tomos AD. 1998. Quantitative X-ray microanalysis of solutes in individual plant cells: a comparison of microdroplet and in situ frozen-hydrated data. Journal of Microscopy 191, 303–310.[Web of Science][Medline]
Lohaus G, Hussmann M, Pennewiss K, Schneider H, Zhu JJ, Sattelmacher B. 2000. Solute balance of maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching. Journal of Experimental Botany 51, 1721–1732.
Malone M, Watson R, Pritchard J. 1999. The spittlebug Philaenus spumarius feeds from mature xylem at the full hydraulic tension of the transpiration stream. New Phytologist 143, 261–271.[CrossRef][Web of Science]
Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H. 2001. Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiology 126, 1646–1667.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologica Plantarum 15, 473–497.[CrossRef]
Nublat A, Desplans J, Casse F, Berthomieu P. 2001. sas1, an Arabidopsis mutant overaccumulating sodium in the shoot, shows deficiency in the control of the root radial transport of sodium. The Plant Cell 13, 125–137.
Padan E, Venturi M, Grechman Y, Dover N. 2001. Na+/H+ antiporters. Biochimica et Biophysica Acta 1505, 144–157.[Medline]
Ponder KL, Watson RJ, Malone M, Pritchard J. 2002. Mineral content of excreta from the spittlebug Philaenus spumarius closely matches that of xylem sap. New Phytologist 153, 237–242.[CrossRef][Web of Science]
Pritchard J. 1994. The control of cell expansion in roots. New Phytologist 127, 3–26.[CrossRef][Web of Science]
Pritchard J. 1996. Aphid stylectomy reveals an osmotic step between sieve tube and cortical cells in barley roots. Journal of Experimental Botany 47, 1519–1524.
Pritchard J, Winch S, Gould N. 2000. Phloem water relations and root growth. Australian Journal of Experimental Botany 27, 539–548.
Putney LK, Denker SP, Barber DL. 2002. The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annual Review of Pharmacoogy and Toxicology 42, 527–552.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Shi HZ, Quintero FJ, Pardo JM, Zhu JK. 2002. The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport in plants. The Plant Cell 14, 465–477.
Song C-P, Guo Y, Qiu Q, Lambert G, Galbraith DW, Jagendorf A, Zhi J-K. 2004. A probable Na+ (K+)/H+ exchanger on the chloroplast envelope function in pH homeostasis and chloroplast development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 101, 10211–10216.
Tomos AD, Hinde PSS, Richardson PBR, Pritchard J, Fricke W. 1994. Microsampling and measurements of solutes in single cells. In: Harris N, Oparka KJ, eds. Plant cell biology: a practical approach. Oxford: IRL Press, 297–314.
Venema K, Belver A, Marin-Manzano MC, Rodriguez-Rosales MP, Donaire JP. 2003. A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants. Journal of Biological Chemistry 278, 22453–22459.
Vitart V, Baxter I, Doerner P, Harper JF. 2001. Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis. The Plant Journal 27, 91–201.
Watson R, Pritchard J, Malone M. 2001. Direct measurement of sodium and potassium in the transpiration stream of salt-excluding and non-excluding varieties of wheat. Journal of Experimental Botany 52, 1873–1881.
Wu SJ, Ding L, Zhu JK. 1996. SOS1, a genetic locus essential for salt tolerance and potassium acquisition. The Plant Cell 8, 617–627.[Abstract]
Yamaguchi T, Apse MP, Shi HZ, Blumwald E. 2003. Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity. Proceedings of the National Academy of Sciences, USA 100, 12510–12515.
Zhang HX, Blumwald E. 2001. Transgenic salt-tolerant tomato plants accumulate salt in the foliage but not in fruit. Nature Biotechnology 19, 765–768.[CrossRef][Web of Science][Medline]
Zhong WJ, Kaiser W, Kohler J, Bauer-Ruckdeschel H, Komor E. 1998. Phloem loading of inorganic cations and anions by the seedling of Ricinus communis L. Journal of Plant Physiology 152, 328–335.[Web of Science]
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