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Running head
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NPF2.4 loads chloride into Arabidopsis xylem
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Corresponding authors
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Mathew Gilliham
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ARC Centre of Excellence in Plant Energy Biology and The University of Adelaide,
PMB1, Glen Osmond, SA 5064, Australia. Tel: +61 8 8313 0485; Email:
[email protected].
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Mark Tester
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Centre for Desert Agriculture, King Abdullah University of Science and Technology,
Thuwal 23955-6900, Kingdom of Saudi Arabia. Tel.: +966 2 8082922; E-mail:
[email protected]
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Research area
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Membranes, Transport and Biogenetics
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Title of Article
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Identification of a stelar-localised transport protein that facilitates root-to-shoot transfer
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of chloride in Arabidopsis
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Authors and Institutions
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Bo Li1,2,3, Caitlin Byrt2,4, Jiaen Qiu1,2,4, Ute Baumann1,2, Maria Hrmova1,2, Aurelie
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Evrard1,2, Alexander A.T Johnson5, Kenneth D Birnbaum6, Gwenda M Mayo2, Deepa
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Jha1,2, Sam W Henderson2,4, Mark Tester*1,2,3, Mathew Gilliham*2,4, Stuart J Roy1,2
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Australia; 2School of Agriculture, Food, and Wine, University of Adelaide, SA 5064,
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Australia; 3Centre for Desert Agriculture, King Abdullah University of Science and
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Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia ; 4ARC Centre of
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Excellence in Plant Energy Biology, University of Adelaide, SA 5064, Australia; 5School
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of BioSciences, University of Melbourne, Parkville, Vic 3010, Australia ,6Centre for
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Genomics and Systems Biology, New York University, NY 10003, USA
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Author contributions
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B.L. performed the majority of the experiments and analysis and drafted the manuscript.
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C.B. conducted experiments for Figures 7A, 7E, 7F 7G and 7H. J.Q. conducted
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experiments for Figures 7H and 7I. K.D.B. advised and assisted with the FACS. A.E.,
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A.A., T.J. and U.B. analyzed the microarray data. G.M.M. developed the assays for the
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cross sections of GUS plant roots. M.H. constructed 3D protein models. D.J.
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characterised putative npf2.4 knockout lines. S.W.H. assisted with the 36Cl– flux assays.
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M.T. conceived the research and identified the candidate gene. M.G and S.J.R.
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supervised the research and co-wrote the manuscript. All authors commented on the
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manuscript.
Australian Centre for Plant Functional Genomics, University of Adelaide, SA 5064,
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One-sentence summary
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Identification and functional analysis of a gene encoding a Cl– transporter responsible for
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loading Cl– into root xylem of Arabidopsis
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Financial source
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This work was funded by Australian Research Council (ARC), Grains Research and
Development Corporation (GRDC) and the King Abdullah University for Science and
Technology funding to M.T., GRDC funding (UA00145) to S.J.R. and M.G., and ARC
Centre of Excellence (CE140100008) and ARC Future Fellowship (FT130100709)
funding to M.G..
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Corresponding authors
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Mark Tester
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[email protected]
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Mathew Gilliham
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[email protected]
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Abstract
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Under saline conditions higher plants restrict the accumulation of chloride ions (Cl–) in the
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shoot by regulating their transfer from the root symplast into the xylem-associated apoplast.
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To identify molecular mechanisms underpinning this phenomenon, we undertook a
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transcriptional screen of salt stressed Arabidopsis thaliana roots. Microarrays, quantitative
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RT-PCR and promoter-GUS fusions identified a candidate gene involved in Cl– xylem
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loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was
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highly expressed in the root stele compared to the cortex and its expression decreased after
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exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either
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transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of
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NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalysed passive Cl– efflux out of
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cells, and was much less permeable to NO3-. Shoot Cl– accumulation was decreased
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following NPF2.4 amiRNA knockdown whereas it was increased by over-expression of
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NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance
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transport of Cl– in plants, playing a role in the loading and the regulation of Cl– loading into
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the xylem of Arabidopsis roots during salinity stress.
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Introduction
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Soil salinity is a significant threat to world agriculture as it restricts plant growth and
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decreases the yield of crops (Munns, 2002; Munns and Tester, 2008; Roy et al., 2014). To
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feed a rapidly growing global population, world food production needs to increase by 70-110 %
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before 2050 (Tilman et al., 2011). Compounding matters, the area of salt affected farmland is
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expanding as a consequence of less frequent rainfall, irrigation with suboptimal water and
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rising water tables (Rengasamy, 2002, 2006). To meet the food demand of future generations,
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improving crop salinity tolerance is a high priority. One approach to enhance plant salinity
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tolerance is to minimise salt (NaCl) accumulation in the cytoplasm of the shoot, a primary
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site of salt damage, whilst maintaining the uptake of beneficial ions such as nitrate (NO3–)
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and potassium (K+) (Munns, 2002; Tester and Davenport, 2003; Teakle and Tyerman, 2010).
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Improving salt exclusion from the shoot is one means of enhancing plant salinity tolerance
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(Apse and Blumwald, 2007; Munns and Tester, 2008; Horie et al., 2009; Munns et al., 2012;
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Guan et al., 2014). Significant progress has been made in characterising the genes encoding
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proteins that are central to regulating shoot Na+ accumulation during salt stress (Roy et al.,
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2014; Munns and Gilliham, 2015). In particular, the function of the high affinity K+
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transporter HKT gene family (Uozumi et al., 2000; Mäser et al., 2002; Berthomieu et al.,
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2003; Davenport et al., 2007; Waters et al., 2013; Byrt et al., 2014), and the Na+/H+ antiporter
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(NHX) and salt overly sensitive SOS gene families (Wu et al., 1996; Shi et al., 2000; Qiu et
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al., 2002; Shi et al., 2002; Bassil and Blumwald, 2014) have been areas of intensive research.
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Manipulation of these pathways has led to improvements in the salt tolerance of the model
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plant Arabidopsis (Sunarpi et al., 2005; Møller et al., 2009) and crops (Ren et al., 2005;
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James et al., 2006; Byrt et al., 2007; Plett et al., 2010; Qiu et al., 2011; Munns et al., 2012).
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Excessive chloride ion (Cl–) accumulation in the cytoplasm of plant cells, particularly in the
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shoot, is also toxic to plants (Tyerman, 1992; Xu et al., 1999; Munns and Tester, 2008;
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Teakle and Tyerman, 2010; Geilfus et al., 2015), resulting in a reduction in plant growth, and
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symptoms such as leaf burn and leaf abscission for Cl– sensitive species (Abel, 1969; Parker
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et al., 1983; Cole, 1985). Chloride (Cl–), rather than sodium ions (Na+), is considered to be
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the more toxic ion for woody perennial species such as grapevine (Tregeagle et al., 2006;
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Gong et al., 2011), citrus (Storey and Walker, 1999) and legumes such as soybean (Luo et al.,
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2005) and lotus (Teakle et al., 2007). For cereal crops such as wheat (Martin and Koebner,
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1995) and barley (Tavakkoli et al., 2011), the toxic effects of Cl– and Na+ are additive and
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can often be overcome by restricting excess accumulation of both ions in the shoot. It has
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also been shown that the xylem-sap Cl– content of salt tolerant genotypes of wheat (Lauchli
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et al., 2008), citrus (Moya et al., 2003) and lotus (Teakle et al., 2007) is lower when
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compared with salt sensitive genotypes, suggesting that the control of Cl– concentration in the
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transpiration stream is a contributing factor to plant salinity tolerance. However, the
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molecular determinants of long distance Cl– transport in plants and how it is regulated in
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response to salinity stress are still largely unknown (Teakle and Tyerman, 2010; Henderson et
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al., 2014). While some candidate proteins, such as cation-Cl– co-transporters (CCC) in
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Arabidopsis (Colmenero-Flores et al., 2007) and rice (Kong et al., 2011) have been proposed
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to retrieve Cl– from the xylem, unresolved questions in regard to their localisation and mode
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of action remain (Teakle and Tyerman, 2010).
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Chloride is a micronutrient required by all plants, functioning as a key regulator of turgor
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(and stomatal movement), membrane potential and cytosolic pH (Xu et al., 1999; White and
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Broadley, 2001; Teakle and Tyerman, 2010). Cl– is acquired by plants from the soil at low
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concentrations by active symport with H+ (Sanders, 1980; Beilby and Walker, 1981; Felle,
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1994) and passively enters the plant at high concentrations due to a reversal in the
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electrochemical potential (Skerrett and Tyerman, 1994). Casparian bands in the endodermis
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limit the transfer of Cl– through an apoplastic pathway, thus most Cl– ions move from cell to
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cell towards xylem vessels following the symplastic pathway (Pitman, 1982). This means Cl–
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must cross root plasma membranes at least twice for it to be loaded into the xylem vessels,
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prior to being transferred to the shoot via the transpiration stream. Due to the measured ion
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concentrations in root cytoplasm and the xylem, the loading of Cl– into the root xylem is
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highly likely to be electrochemically passive, and it is thus likely to be facilitated by plasma
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membrane localised anion channels or carriers that are not yet identified (White and Broadley,
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2001; Munns and Tester, 2008; Teakle and Tyerman, 2010; Kollist et al., 2011; Henderson et
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al., 2014).
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While little is known about the genes and proteins responsible for Cl– loading into the root
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xylem apoplast, more is known about the biological processes involved. ABA was shown to
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significantly inhibit xylem loading of Cl–, as well as K+ (Rb+), whilst having a limited effect
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on the initial uptake of these ions from the external medium. This leads to an accumulation of
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both Cl– and K+ ions in the root, and results in a reduced delivery to the shoot (Cram and
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Pitman, 1972; Pitman et al., 1974; Pitman, 1977). The discovery of the gene underlying K+
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xylem loading, the stelar outwardly rectifying K+ channel (SKOR) illustrated that ABA
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down-regulates the K+ flux to the xylem vessels at both a transcriptional and a post-
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translational level (Gaymard et al., 1998; Roberts, 1998; Roberts and Snowman, 2000).
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Electrophysiological studies of maize and barley root xylem parenchyma cells identified
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three conductances that could facilitate the passive xylem loading of anions such as Cl– and
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NO3– into the xylem apoplast: xylem-parenchyma quickly-activating anion conductance (X-
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QUAC), xylem-parenchyma slowly-activating anion conductance (X-SLAC) and xylem-
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parenchyma inwardly-rectifying anion conductance (X-IRAC) (Kohler and Raschke, 2000;
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Kohler et al., 2002; Gilliham and Tester, 2005). X-QUAC, the anion conductance most likely
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to be responsible for the majority of Cl– xylem loading, was significantly down-regulated
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when plants were treated with ABA (Gilliham and Tester, 2005).
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Here, we have identified a gene encoding an anion transport protein with properties that are
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consistent with a role in Cl– loading into the root xylem apoplast in Arabidopsis. The
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knowledge gained here opens new avenues for manipulation of these pathways in Cl–
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sensitive species for improving salinity tolerance.
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Results
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Identification of candidate genes encoding transporters for Cl– xylem loading in
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Arabidopsis
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Based on accumulated physiological evidence, it was hypothesised that the rate-limiting step
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of Cl– loading to the shoot is in the root stele and is ultimately regulated by transcription of
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genes encoding Cl– transporters (Gilliham and Tester, 2005; Teakle and Tyerman, 2010;
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Henderson et al., 2014). Previously, a comparative transcriptomic analysis of pericycle and
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cortical cells in the root of Arabidopsis in response to salt (50 mM NaCl for 2 d) had been
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conducted using fluorescent-activated cell sorting (FACS) and Affymetrix microarrays
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(Evrard et al., 2012; Evrard, 2013). The data was subsequently re-analysed to identify stelar-
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expressed genes that were likely to encode membrane bound anion permeable transport
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proteins that had their expression reduced by salt stress. Two candidates were identified that
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satisfied these criteria from this preliminary screen: the known H+/NO3– symporter
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(AtNRT1.5/NPF7.3, At1g32450), which regulates NO3– transfer to Arabidopsis shoots (Lin et
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al., 2008) and an uncharacterised gene, At3g45700/NPF2.4, which was annotated as a
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proton-dependent oligo-peptide transporter. We prioritised the uncharacterised gene for
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further study. NPF2.4 was preferentially expressed in the stele compared to the cortex (log2
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fold change (pericycle vs. cortex) = 1.76) and was down regulated by salt (log2 fold change 0
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mM vs 50 mM = -1.37). A search of the GEO Profiles database (Barrett et al., 2006) revealed
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that NPF2.4 was rapidly down regulated in the root after exposure to salt in other studies
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(GEO accession GDS3216).
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Candidate gene At3g45700, also known as NPF2.4, is a member of the NAXT subfamily,
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a 7-gene clade belonging to the NPF in Arabidopsis
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NPF2.4 belongs to the NITRATE EXCRETION TRANSPORTER (NAXT) subfamily of the
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Nitrate Transporter 1/Peptide Transporter (NRT1/PTR) Family (NPF) in Arabidopsis
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(Segonzac et al., 2007; Tsay et al., 2007; Léran et al., 2014). The NAXT subfamily consists of
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seven genes (At3g45650, At3g45660, At3g45680, At3g45690, At3g45700, At3g45710 and
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At3g45720), which are clustered on chromosome 3 (Figure 1) (Tsay et al., 2007). The NAXT
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subfamily was so named after the properties of NAXT1/NPF2.7 (At3g45650), a NO3–
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transporter in root cortical cells involved in NO3– excretion from the root (Segonzac et al.,
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2007).
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In silica modelling suggests NPF2.4 is a membrane embedded transporter that can bind
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Cl–
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The candidate gene NPF2.4 harbours four exons and three introns, and is predicted to encode
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a 548 amino acid protein, consisting of 12 trans-membrane α-helices with a hydrophilic loop
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between trans-membrane α-helices 6 and 7 (Figure 2). The protein sequence of NPF2.4 was
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found to have a 64% identity and a 78% similarity to NAXT1/NPF2.7 and a 76% identity and
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a 85% similarity to NPF2.3, a NO3– selective xylem loader in Arabidopsis root (Taochy et al.,
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2015)(Supplemental Figure 1).
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A three-dimensional (3D) molecular model of NPF2.4 was constructed using crystal
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structures of a proton-dependent oligo-peptide transporter from Shewanella oneidensis (PDB
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accession 2XUT) (Figure 2A) (Newstead et al., 2011) and a structure of the NRT1.1/NPF6.3
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H+/NO3– transporter from Arabidopsis in complex with a NO3– ion (Figure 2B) (Sun et al.,
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2014). The putative 3D structure of NPF2.4 derived through use of both structural templates
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indicated the presence of a central cavity, which was common to both structural templates
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(Figure 2C). The presence of Cl– ions could be simulated, and several residues including
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Tyr37, Va141, Asn67 and Met334 were identified in NPF2.4 as important in forming a
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potential cavity essential for anion transport activity (Figure 2D). This provides an additional
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basis for further study of NPF2.4 as a candidate for transporting Cl– between roots and shoots.
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NPF2.4 expression is down-regulated by both salt and ABA
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Expression profiling by qRT-PCR indicated that both NaCl and ABA treatment significantly
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reduced the transcript abundance of NPF2.4. The reduction in gene expression was found to
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be concentration dependent, with 100 mM NaCl treatment reducing NPF2.4 transcript
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abundance more than 50 mM NaCl (Figure 3A). When treated with 75 mM NaCl for 5 days,
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the abundance of NPF2.4 mRNA in the roots was significantly reduced by almost 90% when
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compared with untreated plants (2 mM NaCl) (Figure 3B). Exposure to 20 µM ABA for 4 h
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or 16 h significantly reduced NPF2.4 transcripts in the roots, with the reduction increasing
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over the time of the assay (Figure 3C).
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NPF2.4 is preferentially expressed in root stelar cells
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To examine the localisation of NPF2.4 expression, a 1.5 kb of the putative promoter
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sequence of NPF2.4 (proNPF2.4) was used to drive the expression of the UidA reporter gene
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(Figure 4). Compared to non-transformed Col-0 plants (Figure 4A), proNPF2.4 driven GUS
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activity was detected in the cells associated with the root vascular bundle in both primary and
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lateral roots of plants transformed with proNPF2.4:UidA (Figure 4B, 4C and 4D). GUS
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activity was also detectible in the vascular cells of both cotyledons and true leaves (Figure 4E
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and 4F). Transverse sections of 10-d old proNPF2.4:UidA expressing roots further
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demonstrated GUS activity in the root stelar cells (Figures 4G). ProNPF2.4 driven GUS
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activity was also detected in flower and developing siliques (Figure 4H and 4I).
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GUS activity in proNPF2.4:UidA expressing plants is regulated by salt, indicating the
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existence of salt responsive regulatory elements in the putative NPF2.4 promoter
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To investigate the responsiveness of the NPF2.4 promoter to salt stress, proNPF2.4:UidA
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plants were treated with 75 mM or 150 mM NaCl for 5 d on MS plates. A reduction in the
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intensity of proNPF2.4 associated GUS activity was observed in response to salt treatments.
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A fluorescence based MUG assay quantified that proNPF2.4:UidA plants treated with 75
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mM or 150 mM NaCl for 5 d had approximately 70% and 30% GUS activity, respectively,
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when compared with 0 mM NaCl grown plants (Figure 5).
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The 1.5 kb putative promoter region of NPF2.4 was compared with the entries of the plant
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cis-acting regulatory DNA elements (PLACE). Multiple copies of binding sites that are
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targets of transcription factors belonging to ABA signal transduction pathways were
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identified (Supplemental Table 1).
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NPF2.4 is localised at the plasma membrane
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To determine the sub-cellular localisation of NPF2.4, Col-0 plants were stably transformed
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with Green Fluorescent Protein (GFP) fused to the 3′- end of NPF2.4. GFP was detected on
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the periphery of root cells in the 10-d old transgenic plants (Figure 6A). After plasmolysis
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was performed to detach the plasma membrane from the cell wall, fluorescence was detected
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on Hechtian strands (Figures 6B). To further verify the NPF2.4 localisation, yellow
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fluorescent protein (YFP) was fused to the 5′ end of NPF2.4 and transiently expressed in
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Arabidopsis mesophyll protoplasts. Figures 6C to 6F show that NPF2.4-associated YFP
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fluorescence overlapped with the CFP fluorescence of the plasma membrane marker
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ECFP::Rop11 (Munns et al., 2012).
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NPF2.4 facilitates Cl– movement across the cell membrane when expressed in Xenopus
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laevis oocytes
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To test whether NPF2.4 was able to facilitate Cl– transport, two-electrode voltage clamping
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(TEVC) of Xenopus laevis oocytes injected with NPF2.4-cRNA was performed. When the
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plasma membrane of NPF2.4-cRNA injected oocytes was clamped at a negative membrane
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potential (i.e. that common to plant cells), an inward current was induced in the presence of
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external Cl–, which was consistent with the efflux of anions from the oocytes into the
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extracellular medium through NPF2.4 (Figure 7A; Supplemental Figure 2A). To examine
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whether Cl– transport activity of NPF2.4 was affected by H+ concentration (as suggested by
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the original annotation of NPF2.4 as a proton-dependent oligo-peptide transporter), oocytes
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were incubated in 50 mM Cl– at pH values of either 7.5 or 5.5. No pH dependency of the
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inward current was observed (Figure 7B; Supplemental Figure 2B and 2C). The magnitude of
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inward current carried by NO3– and Cl– was compared in NPF2.4-injected oocytes and
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controls, it was shown that negative currents were much larger and conductance was higher
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when Cl– was the external anion, rather than NO3– (Figures 7C and 7D; Supplemental Figure
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2D – 2G); the PCl–/PNO3– permeability ratio of NPF2.4 was calculated to be 4.4 ± 0.8 (with
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either 50 mM NaCl or NaNO3 in the bathing solution). To distinguish whether the observed
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inward currents at negative membrane potentials were associated with movements of Cl– or
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Na+, oocytes pre-injected with NPF2.4-cRNA or water were incubated in the solutions
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having a constant concentration of Na+ (45 mM) with increasing concentrations of Cl– (0, 5,
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10, 25, 50 mM), the inward currents observed were larger when increased Cl– concentration
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in the external solution (Figure 7E; Supplemental Figure 2H and 2I). When used solutions
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that have a constant concentration of Cl– (45 mM) with increasing concentrations of Na+ (0, 5,
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10, 25, 50 mM), similar association between external Na+ concentrations and the size of
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inward currents was observed (Figure 7F; Supplemental Figure 2J and 2K). The effect of
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different cations on the stimulation of Cl–-carrying currents was examined, and found not to
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be Na+ specific, K+ also stimulated Cl–-carrying currents, whereas NMDG+, Ca2+ and Mg2+
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did not (Figure 7G; Supplemental Figure 2L). When Na+ was present in the absence of Cl–,
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no detectible currents were observed (Figure 7A, Figure 7G; Supplemental Figure 2L). To
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test if Na+ is only activating the transport of Cl– by NPF2.4, not being co-transported with Cl–,
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Elemental content of Cl–, Na+ and K+ was measured in both NPF2.4-cRNA- and water-
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injected oocytes incubated in Ca2+ ringer solution for 2 d with and without a 2 h low NaCl
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treatment. NPF2.4-cRNA injected oocytes had a significantly lower (P < 0.001) Cl– level
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compared with water-injected controls. No significant difference was observed in Na+ content
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or K+ content between NPF2.4-cRNA injected oocytes and water-injected controls (Figure
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7H). To test whether the observed outward currents at positive membrane potentials (Figure
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7C) were due to Cl– entering the cell through NPF2.4, a unidirectional 36Cl– influx assay for
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NPF2.4- and water-injected oocytes was performed by incubation in solutions containing 100
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mM NaCl or 100 mM NMDG-Cl spiked with low activities of 36Cl– for 60 mins (Figure 7I).
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Oocytes injected with NPF2.4-cRNA exhibited significantly greater uptake of 36Cl– than the
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water-injected oocytes in presence of NaCl. This stimulated influx was not affected by
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changes in pH values of 7.5 and 5.5 (Figure 7I). When NaCl was replaced with NMDG-Cl,
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there was not a significant increase in the uptake of 36Cl– in NPF2.4-cRNA injected oocytes
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compared to water-injected controls at both pH values tested (Figure 7I). A unidirectional
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Na+ uptake assay, using oocytes incubated in 100 mM NaCl solution spiked with
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confirmed that there was no trans-membrane movement of Na+ through NPF2.4
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(Supplemental Figure 2M).
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Na+,
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Knockdown of NPF2.4 resulted in reduced shoot Cl– accumulation
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Putative Atnpf2.4 T-DNA knockouts were ordered from stock centres, but no plants
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containing an insert within NPF2.4, and/or lacking NPF2.4 expression could be identified
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(Supplemental Figure 3). Therefore, to elucidate the function of NPF2.4 in planta, artificial
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microRNAs (amiRNAs) were designed to knockdown NPF2.4 expression. These were
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transformed into Col-0 plants. Quantitative RT-PCR showed that T2 amiRNA plants had 50%
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to 80% lower transcript abundance of NPF2.4 compared with the null segregants (Figure 8A).
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These amiRNA plants were shown to have significantly less Cl– in the shoot when grown
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under a low salt conditions (2 mM NaCl) (21 – 32% lower Cl–) (Figure 8B). Meanwhile,
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shoot NO3– content (Figure 8C), as well as Na+ and K+ (Supplemental Figure 4A and 4B),
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remained similar between knockdown lines and null segregants. Under a high salt load (75
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mM NaCl), when the endogenous NPF2.4 expression ordinarily decreased in such conditions
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(Figure 3B), the transcript abundance of NPF2.4 in the amiRNA knockdown lines and null
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segregants was at a similar low level (Supplemental Figure 4C), and no significant difference
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in shoot Cl– concentration was found (Supplemental Figure 4D). Transcript levels of NPF2.5,
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the closest homolog of NPF2.4, were found to be unaffected in the root of NPF2.4
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knockdown lines when compared with null segregants (Supplemental Figure 4E).
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Constitutive over-expression of NPF2.4 resulted in increased Cl– accumulation in the
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shoot
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To investigate the effect of constitutive over-expression of NPF2.4 on shoot Cl–
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accumulation, NPF2.4 was constitutively over-expressed in Col-0 plants under the control of
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a 35S promoter. Two independent T3 NPF2.4 over-expression lines were grown for 4 weeks
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in hydroponics before being exposed to 2 mM or 75 mM NaCl for 5 d. Greater NPF2.4
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mRNA levels were observed in both NPF2.4 over-expression lines as detected by qRT-PCR
326
(Supplemental Figure 5). After the 75 mM NaCl treatment, Cl– accumulation in the shoot was
327
higher in both over-expression lines compared to the null segregants, with OEX-NPF2.4-2
328
exhibiting a significant increase of 23 % (Figure 9A). At the same time, no significant
329
difference in shoot content of NO3– was found between the transgenic lines and null
330
segregants (Figure 9B). Small but non-significant increases in shoot Cl– accumulation were
331
observed in NPF2.4 over-expression lines grown in 2 mM NaCl (Figure 9C), while no
332
alternation in shoot content of NO3– was observed (Figure 9D).
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333
Discussion
334
NPF2.4 meets many of the predicted characteristics for a gene encoding an anion channel
335
involved in loading Cl– to the root xylem. It encodes a protein targeted to the plasma
336
membrane of root stelar cells in Arabidopsis (Figures 4 and 6) and its expression is strongly
337
down-regulated by salt and ABA treatment (Figure 3 and 5). NPF2.4 was found to facilitate
338
Cl–-dependent currents across the cell membrane at negative membrane potentials when
339
expressed in Xenopus laevis oocytes (Figure 7A), and Cl– transport was not affected by
340
changes in external pH (Figures 7B and 7I). This is consistent with the predicted mechanism
341
of xylem loading for Cl–, where the difference in electrochemical potential for Cl– across the
342
plasma membrane of cells in the stele favours passive Cl– efflux into the xylem, without the
343
need for direct expenditure of energy (Teakle and Tyerman, 2010).
344
At more positive membrane potentials, the presence of outward currents in NPF2.4-injected
345
oocytes indicates that NPF2.4 can also mediate Cl– influx into cells (Figure 7C). This was
346
confirmed in NPF2.4 expressing oocytes using a
347
inwardly directly Cl– fluxes were dependent, in the conditions tested, upon the presence of
348
Na+ and were absent when NMDG+ was the balancing cation (Figure 7I). However, it appears
349
Cl– transport was not exclusively dependent upon the presence of Na+, as when K+ was
350
present but not H+, Ca2+, Mg2+ or NMDG+, there were also significant Cl– dependent currents
351
(Figures 7F and G). Only Cl– concentration differences and not Na+ or K+ were detected in
352
NPF2.4-injected oocytes (Figure 7H), and no inwardly directed Na+ fluxes were detected
353
(Supplementary Figure 2M). These data indicate that both monovalent cations K+ and Na+ are
354
likely to activate Cl– transport but not be carried by NPF2.4, and it is likely that the in vivo
355
activating cation is K+ in low salt conditions. Under high salt (NaCl) conditions, currents
356
carried by NPF2.4 are likely to be minimised by the lower expression levels of NPF2.4
357
despite a higher concentration of another activating cation. Interestingly, the transport
358
properties of NPF2.4 appear to be specific to Cl–, with no indication of the protein being
359
involved in NO3– transport (Figures 7C and 7D), which is unique when compared with other
360
published NPF2s that are generally permeable to NO3– (Segonzac et al., 2007; Tsay et al.,
361
2007; Léran et al., 2014). Combining all evidence, it is suggested that, at negative membrane
362
potentials, NPF2.4 facilitates Cl– efflux across cell membranes, consistent with its proposed
363
role in facilitating a component of the loading of Cl– into the xylem in Arabidopsis.
36
Cl– uptake assay (Figure 7I). These
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364
The use of transgenic plants demonstrated that altering levels of NPF2.4 expression could
365
affect shoot Cl– accumulation but not that of NO3–, Na+ and K+ (Figure 8F, 9B, 9D and
366
Supplemental Figure 4A, 4B). This ion profile provides further evidence that Na+ and K+ are
367
not transported by NPF2.4. Reduction in expression of NPF2.4 in Arabidopsis using
368
amiRNA resulted in decreased Cl– accumulation in the shoot (Figures 8A and 8B;
369
Supplemental Figure 4A), while NPF2.4 over-expression resulted in increased shoot Cl–
370
content (under high salt conditions) (Figures 9A and 9C), thus indicating a role for NPF2.4 in
371
the accumulation of Cl– in Arabidopsis shoots. The increased Cl– accumulation in NPF2.4
372
overexpression lines was not significant in low salt conditions (Figure 9C). Møller et al.
373
(2009) previously observed that the loss of cell specificity when constitutively
374
overexpressing AtHKT1;1 may not in all conditions lead to an expected change in ion profile,
375
with the use of gene knockouts or cell specific complementation being more reliable. In the
376
present study, amiRNA knockdown was performed to lend a greater cell type specificity
377
when manipulating expression of NPF2.4. Reducing expression of NPF2.4 by 80% under a
378
low salt condition (2 mM NaCl), without affecting the expression of genes that are
379
homologous to NPF2.4, resulted in a decrease in the shoot accumulation of Cl– by up to 31%
380
when compared with the null segregants (Figure 8A, 8B and Supplemental Figure 4E),
381
however, this was insufficient to account for a measurable difference in growth
382
(Supplemental Figure 6). Due to the nature of the amiRNA system, knocking down the
383
expression of NPF2.4 does not completely remove its transcript, thus still leaving small
384
amounts of NPF2.4 mRNA that can be translated into a functioning protein. Unfortunately no
385
lines with NPF2.4 completely knocked-out could be identified to test whether a lack of
386
NPF2.4 expression resulted in a greater reduction of shoot Cl–. Under high salt conditions (75
387
mM NaCl), the endogenous down-regulation of NPF2.4 expression (Figure 3) resulted in
388
similar levels of NPF2.4 expression in both knockdowns and null segregants (Supplemental
389
Figure 4C), thus as expected, no difference in shoot Cl– concentration was found
390
(Supplemental Figure 4D).
391
Cl– xylem loading is a complex process, as revealed by at least 3 anion conductances
392
identified in stelar protoplasts (Kohler and Raschke, 2000; Gilliham and Tester, 2005). The
393
electrophysiological profiles of NPF2.4 in Xenopus oocytes did not resemble QUAC, the
394
likely major conductance for Cl– loading into the xylem. Firstly, there was a lack of
395
rectification (Figure 7C). This suggests the involvement of other transport proteins and/or
396
regulators in the loading of Cl– into the xylem (Roy et al., 2008). Furthermore, NPF2.4-
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397
induced currents in oocytes were considerably smaller in the presence of NO3– compared to
398
Cl– (Figure 7C, Figure 7D), whereas X-QUAC type currents were much more permeable to
399
NO3– compared to Cl– (Kohler and Raschke, 2000; Gilliham and Tester, 2005). From the
400
plant data it is clear that NPF2.4 is not the only transporter that facilitates the loading of Cl–
401
into the transpiration stream. Shoot Cl– is reduced by 31% relative to controls, and shoot
402
NO3– remains unchanged when there is an 80% reduction in NPF2.4 expression in the
403
amiRNA lines indicating that other mechanisms contribute to the remaining Cl– loading
404
(Figure 8A and 8B). There are other anion transporters in Arabidopsis that may contribute to
405
Cl– xylem loading, many of which contribute to NO3– loading, such as NRT1.5/NPF7.3 and
406
NRT1.8/NPF7.2 (Lin et al., 2008; Li et al., 2010), SLAH1 and SLAH3, a root-stelar specific
407
homolog of SLAC1 (a protein responsible for Cl- efflux from guard cells) (Negi et al., 2008;
408
Vahisalu et al., 2008) and CCC (Colmenero-Flores et al., 2007). Interestingly,
409
NRT1.5/NPF7.3 and NRT1.8/NPF7.2 were shown to be down- and up- regulated respectively,
410
by salt stress, with nrt1.5/npf7.3 knockout plants exhibiting improved salinity tolerance (Lin
411
et al., 2008; Li et al., 2010; Chen et al., 2012). The involvement of these other proteins in Cl–
412
transport has not yet been reported.
413
The reduction in NPF2.4 expression after the application of ABA and the suggestion of
414
ABA’s role in regulating Cl– transport from the root to the shoot warrants further study. ABA
415
is a signal known to down-regulate Cl– loading of the shoot and the conductances that are
416
responsible for this flux (Cram and Pitman, 1972; Gilliham and Tester, 2005), whereas in
417
stomatal guard cells ABA activates Cl– efflux from cells (Negi et al., 2008; Vahisalu et al.,
418
2008; Geiger et al., 2009; Lee et al., 2009; Vahisalu et al., 2010). Therefore, it will be
419
interesting to explore the differences in ABA signalling pathways for these processes that
420
differentially regulate anion efflux from root and shoot cells.
421
The Cl– transport activity of NPF2.4 observed in this study adds to the list of solutes
422
transported by members of the NPF. To date, NO3–, dipeptides, histidine, malate,
423
glucosinolate, auxin and ABA have been reported as substrates transported by various NPFs
424
(Yamashita et al., 1997; Zhou et al., 1998; Jeong et al., 2004; Krouk et al., 2010; Kanno et al.,
425
2012; Nour-Eldin et al., 2012). The increasing volume of genomic and functional evidence on
426
the roles of the NPF will assist in addressing the question posed by Tsay et al. as to why
427
plants have so many NPF genes (53 in Arabidopsis and 80 in rice) while yeast, drosophila
428
and humans have only one, three, and six members, respectively (Tsay et al., 2007). NPF
429
transporters identified from organisms other than higher plants are di-/tri- peptide transporters,
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
430
it was therefore speculated that the NO3– transport activity of this family in Arabidopsis has
431
evolved from an ancient peptide transporter (Stacey et al., 2002; Tsay et al., 2007). Since then,
432
the NPF has expanded, presumably to fill specific transport roles in specific environments,
433
making this family increasingly diverse.
434
In conclusion, NPF2.4 is a protein involved in Cl– xylem loading in Arabidopsis, regulating
435
Cl– accumulation in the shoot in response to salt stress. To our knowledge, the identification
436
and functional characterisation of NPF2.4 has, for the first time, revealed the molecular
437
properties of a protein that loads Cl– into the xylem. As such, NPF2.4 may be a target for
438
improving plant salinity tolerance in crops that are sensitive to Cl– accumulation. Further
439
research is needed to investigate whether NPF2.4 orthologs are involved in salinity tolerance
440
in other plant species and whether manipulation of NPF2.4-like genes, and other genes
441
involved in Cl– transport to the shoot of crop plants is important for alleviating Cl– toxicity.
442
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443
Methods
444
Plant material and growth conditions
445
Plant material:
446
T-DNA knockout lines (SALK_111056 and SALK_111071), which were annotated with the
447
T-DNA site of insertion being in the NPF2.4 gene, as well as Col-0 Arabidopsis seeds were
448
obtained from the European Arabidopsis Stock Centre (Nottingham, UK).
449
Soil grown Arabidopsis:
450
Arabidopsis was grown in soil prepared following the methods described in Møller et al.
451
(2009). Plants were kept with light/dark photo periods of either 16 h/8 h (long-day) or 10
452
h/14 h (short-day). Temperature was maintained between 21°C to 23°C; humidity was
453
optimized between 60% and 75%; photon irradiance at the level of plant the leaves was
454
approximately 120 μmol m-2 s-1.
455
Hydroponics grown Arabidopsis:
456
Arabidopsis plants were germinated and grown in hydroponics following the method
457
described in Conn et al. (2013). Growth conditions were similar to those for soil grown plants.
458
At time of salt application, NaCl solutions of different concentrations as indicated in the text
459
were applied to the growth solution four weeks after germination with supplemental Ca2+
460
added.
461
Expression analysis
462
Gene expression analysis by qRT-PCR was performed following the method described in
463
Burton et al. (2008). The primers for determining NPF2.4 expression were 5ʹ
464
CAGAAGCTAATCCGCAAACC 3ʹ (Forward) and 5ʹ AGGAACCAGCCATAGCACTG 3ʹ
465
(Reverse).
466
described in Jha et al. (2010).
467
Phylogenetic analysis
468
Multiple amino acid sequence alignment was performed with ClustalW2. Proteins used in the
469
alignment included characterised Arabidopsis NPFs and seven members of the NAXT
470
subfamily as shown in the table below. Protein sequences were retrieved from the
471
Arabidopsis genome annotation database release 9 (http://www.arabidopsis.org/). Neighbour-
The selected control genes and data normalisations followed the protocols
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472
joining phylogenetic tree was generated using Mega5, with 5000 bootstrap iterations (Tamura
473
et al., 2011).
474
Protein 3D structural modelling
475
3D models of NPF2.4 were constructed and evaluated as described in Cotsaftis et al. (2012)
476
using two structural templates: (i) a proton dependent oligopeptide transporter from
477
Shewanella oneidensis (Newstead et al., 2011) (PDB accession 2XUT), and (ii) a NO3–
478
transporter from Arabidopsis in complex with the NO3– ion (Sun et al., 2014) (PDB accession
479
4OH3). The amino acid sequences of 2XUT (488 residues) and 4OH3 (553 residues, NO3–
480
ion replaced by Cl– ion) were aligned with NPF2.4 (503 and 528 residues) using Local Meta-
481
Threading Server (LOMETS) (Wu and Zhang, 2007), Alignment Annotator (Gille et al.,
482
2014), followed by manual alignment adjustments. The aligned sequences were used as input
483
parameters to generate 3D models of NPF2.4 using Modeller 9v8 (Sali and Blundell, 1994)
484
on a Linux station, running the Fedora 12 operating system. Three types of 3D models (using
485
2XUT or 4OH3 as the templates, the latter with and without Cl–) were selected from 40
486
models, based on their low value of the Modeller 9v8 objective function and the most
487
favourable DOPE energy scoring parameters (Shen and Sali, 2006). Images of structural
488
models were generated in the PyMol Molecular Graphics System, Version 1.3 Schrödinger,
489
LLC.
490
Generation of NPF2.4 over-expression lines and NPF2.4 amiRNA knockdown lines
491
492
amiRNA NPF2.4:
Web MicroRNA Designer (http://wmd2.weigelworld.org/cgi-bin/mirnatools.pl) (Schwab et
493
al., 2006) was used to design two amiRNA constructs to knockdown NPF2.4 gene expression.
494
Two 21 bp target sequences were identified from the NPF2.4 CDS, allowing the generation
495
of two independent amiRNA constructs. A set of primers (Supplemental Table 2) was used to
496
incorporate the 21 bp amiRNA sequence into the MIR319a vector (Schwab et al., 2006). The
497
amiRNA constructs then were cloned into pCR8 and transferred to pTOOL2 (Roy et al., 2013)
498
by a LR reaction (Invitrogen) and transformed into Arabidopsis Col-0 plants using
499
Agrobacterium-mediated transformation (Clough and Bent, 1998; Weigel and Glazebrook,
500
2002).
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501
35S:NPF2.4:
502
cDNA of NPF2.4 was amplified using Platinum Taq (Invitrogen, CA) and cloned into
503
Gateway® enabled pCR8 entry vector (Invitrogen, CA). NPF2.4 was transferred from pCR8
504
to the pTOOL2 destination vector (Roy et al., 2013) using LR Clonase II (Invitrogen, CA)
505
and transformed into Arabidopsis Col-0 plants using Agrobacterium-mediated floral dip
506
transformation (Clough and Bent, 1998; Weigel and Glazebrook, 2002).
507
508
Promoter GUS fusions.
The primers 5ʹ TAGAGAAGAAGACTATCATGGG
509
GGAGGTACTTTGACTTGTGTTTGAG 3ʹ (Reverse) were used to amplify 1.5 kb of the
510
sequence upstream from the start codon of NPF2.4. This fragment was cloned into pCR8 and
511
transferred by LR reaction into pMDC162 to drive UidA expression. The destination vector
512
was transformed into Arabidopsis Col-0 plants using the Agrobacterium-mediated method
513
(Clough and Bent, 1998; Weigel and Glazebrook, 2002). Two-week old T2 proNPF2.4:UidA
514
plants were stained for GUS activity for 1 h following the protocol described in Weigel and
515
Glazebrook (2002). GUS activity was visualised using a Leica MZ16FA stereo microscope
516
(Leica microsystem, Wetzlar, Germany). To prepare transverse sections of roots of
517
proNPF2.4:UidA plants, GUS stained material was washed with milliQ water and fixed at
518
4 °C overnight using 0.1 M phosphate buffer with 5 % glutaraldehyde. Samples were washed
519
twice with 1 × PBS for 10 min each and embedded in 1 % agarose. Tissue was dehydrated by
520
15 min incubations in an ethanol series of 50%, 70%, 90%, 100% (v/v). Samples were
521
transferred to Technovit 7100 hydroxyethyl methacrylate resin solution with 2.5% (v/v)
522
PEG400 and polymerised in thin-walled PCR tubes. 8 µm thick transverse root sections were
523
cover slipped in DPX mountant, and imaged on a Leica ASLMD compound microscope
524
equipped with a DFC480 CCD camera.
525
To test the effect of the salt treatments on GUS activity, proNPF2.4:UidA plants were grown
526
vertically on ½ MS media for 7 d before transferred onto ½ MS media containing 0 mM, 75
527
mM or 150 mM NaCl for a 5-d salt treatment. Salt treated plants were GUS-stained for 1 h
528
and examined under a Leica MZ16FA stereo microscope (Leica microsystem, Wetzlar,
529
Germany). A fluorescent 4-methylumelliferyl-β-Galactopyranoside (MUG) assay was
530
performed to quantify the GUS activity of proNPF2.4:UidA plants after the salt treatments
531
following the protocol as described in Peña (2004).
3ʹ
(Forward)
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
and
5ʹ
532
NPF2.4::GFP fusion constructs and visualization of fluorescence in planta
533
To determine the subcellular localisation of NPF2.4 in planta, GFP6 was fused to either the 5′
534
and 3′ end of NPF2.4 and used for constructing GFP::NPF2.4 and NPF2.4::GFP. The
535
coding sequence of NPF2.4 was inserted into the destination vectors pMDC44 and pMDC83
536
(Curtis and Grossniklaus, 2003) to generate the necessary vectors. The fusion vectors were
537
transformed into Arabidopsis Col-0 plants using an Agrobacterium-mediated method (Clough
538
and Bent, 1998; Weigel and Glazebrook, 2002). T2 transgenic plants were geminated on ½
539
MS media under hygromycin selection. 10 d after the germination, plants were stained with
540
10 µg/ml propidium iodide for 5 min and rinsed with deionised water. Plasmolysis was
541
conducted on a slide by applying 10 % (w/v) sucrose solution on to the roots of GFP6
542
expressing plants. GFP (excitation 488 nm, emission 505-530 nm) and PI (excitation 543 nm,
543
emission longpass 560 nm) fluorescence were visualised using a LSM5 PASCAL laser
544
scanning microscope (Carl Zeiss, Jena, Germany) running PASCAL imaging software
545
(version 3.2 SP2, Carl Zeiss).
546
Arabidopsis mesophyll cell transient transformation
547
To determine the subcellular localisation of NPF2.4 in Arabidopsis protoplasts, YFP was
548
fused to the 5ʹ end of NPF2.4 by cloning the coding sequence of NPF2.4 into the destination
549
vector pattR-YFP (Subramanian et al., 2006). A protein known to be targeted to the plasma
550
membrane (ROP11) was used as a positive control (eCFP::ROP11; Molendijk et al., 2008).
551
Protoplast isolation from Arabidopsis mesophyll cells and PEG-mediated transient
552
transformation were conducted using the protocols described in Yoo et al. (2007) to introduce
553
the fusion vector. CFP (excitation 436nm, emission 470-535) and YFP (excitation 514,
554
emission 525-610 nm) fluorescence were visualised using a LSM5 PASCAL laser scanning
555
microscope (Carl Zeiss, Jena, Germany) running PASCAL imaging software (version 3.2
556
SP2, Carl Zeiss).
557
Electrophysiology
558
TEVC
559
A pGEMHE-DEST destination vector (Shelden et al., 2009) was developed for heterologous
560
expression of NPF2.4 in Xenopus laevis oocytes. cRNA was transcribed from this vector
561
using mMESSAGE mMACHINE kit (Ambion, Austin, TX) following the manufacturer’s
562
instructions, and injected into the following Munns et al. (2012). Two-electrode voltage
563
clamping was performed following Roy et al. (2008). Osmolarities of all solutions were
Downloaded from on June 14, 201722
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
564
adjusted using mannitol (Sigma, USA) to 240-260 mOsmol/kg (Fiske® 210 Micro-Sample
565
Osmometer, Advanced Instruments, USA). Membrane currents were recorded in solutions
566
containing 5 mM MES, 2 mM Ca-gluconate ± NMDG-Cl (or NaCl) or NMDG-NO3 (or
567
NaNO3) as indicated. Data were acquired and analysed using pClamp 8.2 (Axon Instruments,
568
Union City, CA). Experiments were repeated using oocytes harvested from different Xenopus
569
laevis surgeries.
570
Isotope uptake assay (36Cl– and 22Na+)
571
Xenopus laevis oocytes microinjected with either NPF2.4 cRNA or water were prepared for
572
36
573
isethionate, 2 mM potassium gluconate, 1.8 mM calcium gluconate, 1 mM magnesium
574
gluconate, 5 mM HEPES, 2.5 mM sodium pyruvate and 5 % gentamicin, pH 7.4) for 120 min
575
2 d after injection. Oocytes were then incubated in uptake buffer (100 mM NaCl, 2 mM Ca-
576
gluconate, 2 mM K-gluconate and 5 mM MES, 240 mOsm kg-1 H2O, pH 7.5) supplemented
577
with 1 µCi/mL of Na36Cl (Radiochemical Centre Limited, Amersham, England.) or 2 µCi/mL
578
22
579
cold uptake buffer, which did not contain
580
oocyte transferred into 1.5 ml microcentrifuge tubes containing 20 µL of 0.1 M nitric acid
581
and macerated by pipetting or transferred to scintillation vials containing 200 µL of 10% SDS
582
and left overnight. 4 mL of scintillation fluid was added to each scintillation vial.
583
Radioactivity, counts per minute, of
584
scintillation counter (Perkin Elmer, USA) or a Beckman Coulter LS6500 scintillation counter
585
(Gladesville, Australia).
586
Elemental analysis of Cl–, Na+, K+ and NO3–
587
Concentration of Cl–, Na+, K+ and NO3– was determined in the whole shoot of Arabidopsis
588
seedlings. The shoot was weighed, freeze-dried and ground into a powder. To determine
589
Arabidopsis shoot and Xenopus laevis oocyte Cl– concentrations, approximately 10-20 mg of
590
material was digested in 2 mL of 1% nitric acid at 80°C overnight and 10 oocytes were
591
digested in 1 mL of 1% nitric acid at 75°C for 2 h. Cl– content was analysed using a Chloride
592
Analyser (Sherwood Scientific model 926, Cambridge, UK) following the manufacturer’s
593
instructions. The NO3– assay described in Kamphake et al. (1967) was used for the
594
determination of the NO3– concentration in plant tissue. To examine Na+ and K+ in plants or
595
oocytes the youngest fully expanded leaf or incubated oocytes were harvested and digested in
596
1% nitric acid at 80°C overnight for plant tissues or 75°C for 2 h for oocytes following Byrt
Cl– and
22
Na+ uptake. Oocytes were incubated in Cl– free ND96 media (96 mM sodium
NaCl (PerkinElmer, Massachusetts, USA) for 1 h. The oocytes were washed twice with ice-
36
36
Cl– or
Cl– or
22
22
Na+, respectively and each individual
Na+ was measured using a Tri-Carb liquid
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
597
et al. 2014. Samples were diluted and measured relative to appropriate standard solutions
598
using a model 420 Flame Photometer (Sherwood Scientific Ltd, Cambridge, United
599
Kingdom).
600
Acknowledgements
601
The authors thank: Andrew Jacobs for the destination vectors used in this study; Yuan Li for
602
performing the qRT-PCRs (Australian Centre for Plant Function Genomics, Adelaide,
603
Australia); Steven Tyerman and Sunita Ramesh (University of Adelaide, Adelaide, Australia)
604
for the aid with the electrophysiology experiments; Detlef Weigel (Max Planck Institute for
605
Developmental Biology, Tübingen, Germany) for providing pRS300 plasmid for the
606
generation of amiRNA constructs and Christina Morris for editing English language of this
607
manuscript.
608
609
610
Conflict of interests
611
The authors declare that they have no conflict of interests.
612
613
614
Supplemental Figure 1 Multiple sequence alignment of NPF2.4, NPF2.3 and NPF2.1/
615
NAXT1.
616
Supplemental Figure 2 Raw and control data for expression of NPF2.4 in Xenopus laevis
617
oocytes.
618
Supplemental Figure 3 NPF2.4 transcript levels of lines SALK_111056 and SALK_111071
619
annotated as T-DNA knockouts for NPF2.4 showed the failed disruption of NPF2.4
620
expression in both lines
621
Supplemental Figure 4 Under low salt condition, shoot concentration of both Na+ and K+
622
were similar between NPF2.4 knockdowns and null segregants.
Downloaded from on June 14, 201724
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
623
Supplemental Figure 5 Expression validation of NPF2.4 in the root of NPF2.4 over-
624
expression lines under both low salt and high salt conditions.
625
Supplemental Figure 6 Biomass of NPF2.4 knockdowns was unaffected by low salt
626
condition compared to null segregants.
627
Supplemental Table 1 The ABA signaling pathway associated cis-acting elements in the
628
putative promoter region of NPF2.4.
629
Supplemental Table 2 Primers used for the generation of NPF2.4-amiRNA constructs.
630
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631
References
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671
672
673
Abel GH (1969) Inheritance of the capacity for chloride inclusion and chloride exclusion by
soybeans. Crop Sciences 9: 697-698
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis
AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in
abscisic acid signaling. Plant Cell 15: 63-78
Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K (1997)
Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acidregulated gene expression. Plant Cell 9: 1859-1868
Apse MP, Blumwald E (2007) Na+ transport in plants. FEBS Letters 581: 2247-2254
Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA,
Phillippy KH, Sherman PM, Holko M, Yefanov A, Lee H, Zhang N, Robertson
CL, Serova N, Davis S, Soboleva A. (2013) NCBI GEO: archive for functional
genomics data sets--update. Nucleic Acids Research 41, D991-D995
Bassil E, Blumwald E (2014) The ins and outs of intracellular ion homeostasis: NHX-type
cation/H+ transporters. Current Opinion in Plant Biology 22: 1-6
Beilby MJ, Walker NA (1981) Chloride transport in Chara: I. Kinetics and current-voltage
curves for a probable proton symport. Journal of Experimental Botany 32: 43-54
Berthomieu P, Conejero G, Nublat A, Brackenbury WJ, Lambert C, Savio C, Uozumi N,
Oiki S, Yamada K, Cellier F, Gosti F, Simonneau T, Essah PA, Tester M, Very
A-A, Sentenac H, Casse F (2003) Functional analysis of AtHKT1 in Arabidopsis
shows that Na+ recirculation by the phloem is crucial for salt tolerance. The EMBO
Journal 22: 2004-2014
Burton RA, Jobling SA, Harvey AJ, Shirley NJ, Mather DE, Bacic A, Fincher GB (2008)
The genetics and transcriptional profiles of the cellulose synthase-like HvCslF gene
family in barley. Plant Physiology 146: 1821-1833
Byrt CS, Platten JD, Spielmeyer W, James RA, Lagudah ES, Dennis ES, Tester M,
Munns R (2007) HKT1;5-like cation transporters linked to Na+ exclusion loci in
wheat, nax2 and kna1. Plant Physiology 143: 1918-1928
Chen C-Z, Lv X-F, Li J-Y, Yi H-Y, Gong J-M (2012) Arabidopsis NRT1.5 is another
essential component in the regulation of nitrate reallocation and stress tolerance. Plant
Physiology 159: 1582-1590
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. The Plant Journal 16: 735-743.
Cole PJ (1985) Chloride toxicity in citrus. Irrigation Science 6: 63-71
Colmenero-Flores JM, Martinez G, Gamba G, Vazquez N, Iglesias DJ, Brumos J, Talon
M (2007) Identification and functional characterization of cation-chloride
cotransporters in plants. The Plant Journal 50: 278-292
Conn SJ, Hocking B, Dayod M, Xu B, Athman A, Henderson S, Aukett L, Conn V,
Shearer MK, Fuentes S (2013) Protocol: optimising hydroponic growth systems for
nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant
methods 9: 4
Downloaded from on June 14, 201726
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
Cotsaftis O, Plett D, Shirley N, Tester M, Hrmova M (2012) A two-staged model of Na+
exclusion in rice explained by 3D modeling of HKT transporters and alternative
splicing. PLoS ONE 7: e39865
Cram W, Pitman M (1972) The action of abscisic acid on ion uptake and water flow in plant
roots. Australian Journal of Biological Sciences 25: 1125-1132
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput
functional analysis of genes in planta. Plant Physiology 133: 462-469
Davenport RJ, Munoz-Mayor A, Jha D, Essah PA, Rus ANA, Tester M (2007) The Na+
transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis. Plant,
Cell & Environment 30: 497-507
Evrard A (2013) Cell type-specific transcriptional responses of plants to salinity. PhD thesis,
The University of Adelaide, Adelaide, Australia
Evrard A, Bargmann BR, Birnbaum K, Tester M, Baumann U, Johnson AT (2012)
Fluorescence-activated cell sorting for analysis of cell type-specific responses to
salinity stress in Arabidopsis and rice. In S Shabala, TA Cuin, eds, Plant Salt
Tolerance, Vol 913. Humana Press, pp 265-276
Felle HH (1994) The H+/Cl– symporter in root-hair cells of Sinapis alba (an
electrophysiological study using ion-selective microelectrodes). Plant Physiology 106:
1131-1136
Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, MichauxFerriere N, Thibaud JB, Sentenac H (1998) Identification and disruption of a plant
shaker-like outward channel involved in K+ release into the xylem sap. Cell 94: 647655
Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann C, AlRasheid KAS, Grill E, Romeis T, Hedrich R (2010) Guard cell anion channel
SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities.
Proceedings of the National Academy of Sciences 107: 8023-8028
Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese
A, Al-Rasheid KAS, Romeis T, Hedrich R (2009) Activity of guard cell anion
channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.
Proceedings of the National Academy of Sciences 106: 21425-21430
Geilfus C-M, Mithöfer A, Ludwig-Müller J, Zörb C, Muehling KH (2015) Chlorideinducible transient apoplastic alkalinizations induce stomata closure by controlling
abscisic acid distribution between leaf apoplast and guard cells in salt-stressed Vicia
faba. New Phytologist DOI: 10.1111/nph.13507
Gille C, Birgit W, Gille A (2014) Sequence alignment visualization in HTML5 without Java.
Bioinformatics 30: 121-122
Gilliham M, Tester M (2005) The regulation of anion loading to the maize root xylem. Plant
Physiology 137: 819-828
Gong H, Blackmore D, Clingeleffer P, Sykes S, Jha D, Tester M, Walker R (2011)
Contrast in chloride exclusion between two grapevine genotypes and its variation in
their hybrid progeny. Journal of Experimental Botany 62: 989-999
Guan R, Qu Y, Guo Y, Yu L, Liu Y, Jiang J, Chen J, Ren Y, Liu G, Tian L, Jin L, Liu Z,
Hong H, Chang R, Gilliham M, Qiu L (2014) Salinity tolerance in soybean is
modulated by natural variation in GmSALT3. The Plant Journal 80: 937-950
Henderson SW, Baumann U, Blackmore DH, Walker AR, Walker RR, Gilliham M
(2014) Shoot chloride exclusion and salt tolerance in grapevine is associated with
differential ion transporter expression in roots. BMC Plant Biology 14: 273
Downloaded from on June 14, 201727
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
Horie T, Hauser F, Schroeder JI (2009) HKT transporter-mediated salinity resistance
mechanisms in Arabidopsis and monocot crop plants. Trends in Plant Science 14:
660-668
James RA, Davenport RJ, Munns R (2006) Physiological characterization of two genes for
Na+ exclusion in durum wheat, Nax1 and Nax2. Plant Physiology 142: 1537-1547
Jeong J, Suh S, Guan C, Tsay Y-F, Moran N, Oh CJ, An CS, Demchenko KN,
Pawlowski K, Lee Y (2004) A nodule-specific dicarboxylate transporter from alder is
a member of the peptide transporter family. Plant Physiology 134: 969-978
Jha D, Shirley N, Tester M, Roy SJ (2010) Variation in salinity tolerance and shoot sodium
accumulation in Arabidopsis ecotypes linked to differences in the natural expression
levels of transporters involved in sodium transport. Plant, Cell & Environment 33:
793-804
Kamphake LJ, Hannah SA, Cohen JM (1967) Automated analysis for nitrate by hydrazine
reduction. Water Research 1: 205-216
Kanno Y, Hanada A, Chiba Y, Ichikawa T, Nakazawa M, Matsui M, Koshiba T,
Kamiya Y, Seo M (2012) Identification of an abscisic acid transporter by functional
screening using the receptor complex as a sensor. Proceedings of the National
Academy of Sciences 109: 9653-9658
Kaplan B, Davydov O, Knight H, Galon Y, Knight MR, Fluhr R, Fromm H (2006)
Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRErelated sequences as Ca2+-responsive cis elements in Arabidopsis. Plant Cell 18:
2733-2748
Kohler B, Raschke K (2000) The delivery of salts to the xylem. Three types of anion
conductance in the plasmalemma of the xylem parenchyma of roots of barley. Plant
Physiology 122: 243-254
Kohler B, Wegner LH, Osipov V, Raschke K (2002) Loading of nitrate into the xylem:
apoplastic nitrate controls the voltage dependence of X-QUAC, the main anion
conductance in xylem-parenchyma cells of barley roots. The Plant Journal 30: 133142
Kollist H, Jossier M, Laanemets K, Thomine S (2011) Anion channels in plant cells. FEBS
Journal 278: 4277-4292
Kong X-Q, Gao X-H, Sun W, An J, Zhao Y-X, Zhang H (2011) Cloning and functional
characterization of a cation–chloride cotransporter gene OsCCC1. Plant Molecular
Biology 75: 567-578
Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova
K, Tillard P, Leon S, Ljung K, Zazimalova E, Benkova E, Nacry P, Gojon A
(2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient
sensing in plants. Developmental Cell 18: 927-937
Lauchli A, James RA, Huang CX, McCully M, Munns R (2008) Cell-specific localization
of Na+ in roots of durum wheat and possible control points for salt exclusion. Plant,
Cell & Environment 31: 1565-1574
Lee SC, Lan W, Buchanan BB, Luan S (2009) A protein kinase-phosphatase pair interacts
with an ion channel to regulate ABA signaling in plant guard cells. Proceedings of the
National Academy of Sciences 106: 21419-21424
Léran S, Varala K, Boyer J-C, Chiurazzi M, Crawford N, Daniel-Vedele F, David L,
Dickstein R, Fernandez E, Forde B, Gassmann W, Geiger D, Gojon A, Gong J-M,
Halkier BA, Harris JM, Hedrich R, Limami AM, Rentsch D, Seo M, Tsay Y-F,
Zhang M, Coruzzi G, Lacombe B (2014) A unified nomenclature of NITRATE
TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends in
Plant Science 19: 5-9
Downloaded from on June 14, 201728
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
Li J-Y, Fu Y-L, Pike SM, Bao J, Tian W, Zhang Y, Chen C-Z, Zhang Y, Li H-M, Huang
J, Li L-G, Schroeder JI, Gassmann W, Gong J-M (2010) The Arabidopsis nitrate
transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates
cadmium tolerance. Plant Cell 22: 1633-1646
Lin SH, Kuo HF, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HL, Wang YY,
Tsai CB, Gojon A, Tsay YF (2008) Mutation of the Arabidopsis NRT1.5 nitrate
transporter causes defective root-to-shoot nitrate transport. Plant Cell 20: 2514 - 2528
Luo GZ, Wang HW, Huang J, Tian AG, Wang YJ, Zhang JS, Chen SY (2005) A
putative plasma membrane cation/proton antiporter from soybean confers salt
tolerance in Arabidopsis. Plant Molecular Biology 59: 809-820
Marcotte WR, Russell SH, Quatrano RS (1989) Abscisic acid-responsive sequences from
the em gene of wheat. Plant Cell 1: 969-976
Martin PK, Koebner RMD (1995) Sodium and chloride ions contribute synergistically to
salt toxicity in wheat. Biologia Plantarum 37: 265-271
Mäser P, Hosoo Y, Goshima S, Horie T, Eckelman B, Yamada K, Yoshida K, Bakker
EP, Shinmyo A, Oiki S, Schroeder JI, Uozumi N (2002) Glycine residues in
potassium channel-like selectivity filters determine potassium selectivity in four-loopper-subunit HKT transporters from plants. Proceedings of the National Academy of
Sciences 99: 6428-6433
Molendijk AJ, Ruperti B, Singh MK, Dovzhenko FA, Milia M, Westphal L, Rosahl S,
Soellick TR, Uhrig J, Weingarten L, Huber M, Palme K (2008) A cysteine-rich
receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop
GTPase interactors. Plant Journal 53: 909-923.
Møller IS, Gilliham M, Jha D, Mayo GM, Roy SJ, Coates JC, Haseloff J, Tester M
(2009) Shoot Na+ exclusion and increased salinity tolerance engineered by cell typespecific alteration of Na+ transport in Arabidopsis. Plant Cell 21: 2163-2178
Møller IS, Tester M (2007) Salinity tolerance of Arabidopsis: a good model for cereals?
Trends in Plant Science 12: 534-540
Moya JL, Gomez-Cadenas A, Primo-Millo E, Talon M (2003) Chloride absorption in saltsensitive Carrizo citrange and salt-tolerant Cleopatra mandarin citrus rootstocks is
linked to water use. Journal of Experimental Botany 54: 825-833
Munns R (2002) Comparative physiology of salt and water stress. Plant, Cell & Environment
25: 239-250
Munns R, Gilliham M (2015) Salinity tolerance of crops – what is the cost? New
Phytologist: DOI: 10.1111/nph.13519
Munns R, James RA, Xu B, Athman A, Conn SJ, Jordans C, Byrt CS, Hare RA,
Tyerman SD, Tester M, Plett D, Gilliham M (2012) Wheat grain yield on saline
soils is improved by an ancestral Na+ transporter gene. Nature Biotechnology 30:
360-364
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual Review of Plant
Biology 59: 651-681
Nakashima K, Fujita Y, Katsura K, Maruyama K, Narusaka Y, Seki M, Shinozaki K,
Yamaguchi-Shinozaki K (2006) Transcriptional regulation of ABI3- and ABAresponsive genes including RD29B and RD29A in seeds, germinating embryos, and
seedlings of Arabidopsis. Plant Molecular Biology 60: 51-68
Marcotte WR, Russell SH, Quatrano RS (1989) Abscisic acid-responsive sequences from
the em gene of wheat. Plant Cell 1: 969-976
Narusaka Y, Nakashima K, Shinwari ZK, Sakuma Y, Furihata T, Abe H, Narusaka M,
Shinozaki K, Yamaguchi-Shinozaki K (2003) Interaction between two cis-acting
Downloaded from on June 14, 201729
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis RD29A gene
in response to dehydration and high-salinity stresses. The Plant Journal 34: 137-148
Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M, Uchimiya H,
Hashimoto M, Iba K (2008) CO2 regulator SLAC1 and its homologues are essential
for anion homeostasis in plant cells. Nature 452: 483-486
Newstead S, Drew D, Cameron AD, Postis VLG, Xia X, Fowler PW, Ingram JC,
Carpenter EP, Sansom MSP, McPherson MJ, Baldwin SA, Iwata S (2011)
Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton
symporters, PepT1 and PepT2. The EMBO Journal 30: 417-426
Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jorgensen ME, Olsen CE,
Dreyer I, Hedrich R, Geiger D, Halkier BA (2012) NRT/PTR transporters are
essential for translocation of glucosinolate defence compounds to seeds. Nature 488:
531-534
Parker MB, Gascho GJ, Gaines TP (1983) Chloride toxicity of soybeans grown on atlantic
coast flatwoods soils. Agronomy Journal 75: 439-443
Peña L (2004) Transgenic Plants: Methods and Protocols Humana Press, Totowa, New
Jersey
Pitman M, Luttge U, Lauchli A, Ball E (1974) Effect of previous water stress on ion uptake
and transport in barley seedlings. Functional Plant Biology 1: 377-385
Pitman MG (1977) Ion transport into the xylem. Annual Review of Plant Physiology 28: 7188
Pitman MG (1982) Transport across plant roots. Quarterly Reviews of Biophysics 15: 481554
Plett D, Safwat G, Gilliham M, Skrumsager Møller I, Roy S, Shirley N, Jacobs A,
Johnson A, Tester M (2010) Improved salinity tolerance of rice through cell typespecific expression of AtHKT1;1. PLoS ONE 5: e12571
Plett DC, Møller IS (2010) Na+ transport in glycophytic plants: what we know and would
like to know. Plant, Cell & Environment 33: 612-626
Qiu L, Wu D, Ali S, Cai S, Dai F, Jin X, Wu F, Zhang G (2011) Evaluation of salinity
tolerance and analysis of allelic function of HvHKT1 and HvHKT2 in Tibetan wild
barley. TAG Theoretical and Applied Genetics 122: 695-703
Qiu Q-S, Guo Y, Dietrich MA, Schumaker KS, Zhu J-K (2002) Regulation of SOS1, a
plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3.
Proceedings of the National Academy of Sciences 99: 8436-8441
Ren Z-H, Gao J-P, Li L-G, Cai X-L, Huang W, Chao D-Y, Zhu M-Z, Wang Z-Y, Luan
S, Lin H-X (2005) A rice quantitative trait locus for salt tolerance encodes a sodium
transporter. Nature Genetics 37: 1141-1146
Rengasamy P (2002) Transient salinity and subsoil constraints to dryland farming in
Australian sodic soils: an overview. Australian Journal of Experimental Agriculture
42: 351-361
Rengasamy P (2006) World salinization with emphasis on Australia. Journal of
Experimental Botany 57: 1017-1023
Roberts SK (1998) Regulation of K+ channels in maize roots by water stress and abscisic
acid. Plant Physiology 116: 145-153
Roberts SK, Snowman BN (2000) The effects of ABA on channel-mediated K+ transport
across higher plant roots. Journal of Experimental Botany 51: 1585-1594
Roy SJ, Gilliham M, Berger B, Essah PA, Cheffings C, Miller AJ, Davenport RJ, Liu
LH, Skynner MJ, Davies JM, Richardson P, Leigh RA, Tester M (2008)
Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana.
Plant, Cell & Environment 31: 861-871
Downloaded from on June 14, 201730
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
Roy, SJ, Huang W, Wang XJ, Evrard E, Schmöckel SM, Zafar ZU, Tester M (2013) A
novel protien kinase involved in Na+ exclusion revealed from positional cloning. Plant,
Cell & Environment 36: 553-568
Roy SJ, Negrão S, Tester M (2014) Salt resistant crop plants. Current Opinion in
Biotechnology 26: 115-124
Sali A, Blundell T (1994) Comparative protein modelling by satisfaction of spatial restraints.
Protein Structure by Distance Analysis 64: C86
Sanders D (1980) The mechanism of Cl− transport at the plasma membrane of Chara
corallina I. Cotransport with H+. The Journal of Membrane Biology 53: 129-141
Schachtman DP, Tyerman SD, Terry BR (1991) The K+/Na+ selectivity of a cation channel
in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive
wheat species. Plant Physiology 97: 598-605
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene
silencing by artificial microRNAs in Arabidopsis. Plant Cell 18: 1121-1133
Segonzac C, Boyer JC, Ipotesi E, Szponarski W, Tillard P, Touraine B, Sommerer N,
Rossignol M, Gibrat R (2007) Nitrate efflux at the root plasma membrane:
Identification of an Arabidopsis excretion transporter. Plant Cell 19: 3760-3777
Shelden MC, Howitt SM, Kaiser BN, Tyerman SD (2009) Identification and functional
characterisation of aquaporins in the grapevine, Vitis vinifera. Functional Plant
Biology 36: 1065-1078
Shen M-y, Sali A (2006) Statistical potential for assessment and prediction of protein
structures. Protein Science 15: 2507-2524
Shi H, Quintero FJ, Pardo JM, Zhu J-K (2002) The putative plasma membrane Na+/H+
antiporter SOS1 controls long-distance Na+ transport in plants. Plant Cell 14: 465-477
Shi HZ, Ishitani M, Kim CS, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene
SOS1 encodes a putative Na+/H+ antiporter. Proceedings of the National Academy of
Sciences 97: 6896-6901
Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K
(2003) Two different novel cis-acting elements of ERD1, a CLPA homologous
Arabidopsis gene function in induction by dehydration stress and dark-induced
senescence. The Plant Journal 33: 259-270
Skerrett M, Tyerman SD (1994) A channel that allows inwardly directed fluxes of anions in
protoplasts derived from wheat roots. Planta 192: 295-305
Stacey G, Koh S, Granger C, Becker JM (2002) Peptide transport in plants. Trends in Plant
Science 7: 257-263
Storey R, Walker RR (1999) Citrus and salinity. Scientia Horticulturae 78: 39-81
Subramanian C, Woo J, Cai X, Xu X, Servick S, Johnson CH, Nebenführ A, Von Arnim
AG (2006) A suite of tools and application notes for in vivo protein interaction assays
using bioluminescence resonance energy transfer (BRET). The Plant Journal 48: 138152
Sun J, Bankston JR, Payandeh J, Hinds TR, Zagotta WN, Zheng N (2014) Crystal
structure of the plant dual-affinity nitrate transporter NRT1.1. Nature 507: 73-77
Sunarpi, Horie T, Motoda J, Kubo M, Yang H, Yoda K, Horie R, Chan W-Y, Leung HY, Hattori K, Konomi M, Osumi M, Yamagami M, Schroeder JI, Uozumi N
(2005) Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na+
unloading from xylem vessels to xylem parenchyma cells. The Plant Journal 44: 928938
Takano J, Noguchi K, Yasumori M, Kobayashi M, Gajdos Z, Miwa K, Hayashi H,
Yoneyama T, Fujiwara T (2002) Arabidopsis boron transporter for xylem loading.
Nature 420: 337-340
Downloaded from on June 14, 201731
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S (2011) MEGA5:
Molecular evolutionary genetics analysis using maximum likelihood, evolutionary
distance, and maximum parsimony methods. Molecular Biology and Evolution 28:
2731-2739.
Taochy C, Gaillard I, Ipotesi E, Oomen R, Leonhardt N, Zimmermann S, Peltier JB,
Szponarski W, Simonneau T, Sentenac H (2015) The Arabidopsis root stele
transporter NPF2. 3 contributes to nitrate translocation to shoots under salt stress. The
Plant Journal DOI:10.1111/tpj.12901
Tavakkoli E, Fatehi F, Coventry S, Rengasamy P, McDonald GK (2011) Additive effects
of Na+ and Cl– ions on barley growth under salinity stress. Journal of Experimental
Botany 62: 2189-2203
Teakle NL, Flowers TJ, Real D, Colmer TD (2007) Lotus tenuis tolerates the interactive
effects of salinity and waterlogging by 'excluding' Na+ and Cl– from the xylem.
Journal of Experimental Botany 58: 2169-2180
Teakle NL, Tyerman SD (2010) Mechanisms of Cl– transport contributing to salt tolerance.
Plant, Cell & Environment 33: 566-589
Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Annals of
Botany 91: 503-527
Tilman D, Balzer C, Hill J, Befort BL (2011) Global food demand and the sustainable
intensification of agriculture. Proceedings of the National Academy of Sciences 108:
20260-20264
Tregeagle JM, Tisdall JM, Blackmore DH, Walker RR (2006) A diminished capacity for
chloride exclusion by grapevine rootstocks following long-term saline irrigation in an
inland versus a coastal region of Australia. Australian Journal of Grape and Wine
Research 12: 178-191
Tsay YF, Chiu CC, Tsai CB, Ho CH, Hsu PK (2007) Nitrate transporters and peptide
transporters. FEBS Letters 581: 2290 - 2300
Tyerman SD (1992) Anion channels in plants. Annual Review of Plant Physiology and Plant
Molecular Biology 43: 351-373
Uozumi N, Kim EJ, Rubio F, Yamaguchi T, Muto S, Tsuboi A, Bakker EP, Nakamura
T, Schroeder JI (2000) The Arabidopsis HKT1 gene homolog mediates inward Na+
currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant
Physiology 122: 1249-1260
Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb
homolog is induced by dehydration stress and its gene product binds to the conserved
MYB recognition sequence. Plant Cell 5: 1529-1539
Vahisalu T, Kollist H, Wang YF, Nishimura N, Chan WY, Valerio G, Lamminmaki A,
Brosche M, Moldau H, Desikan R, Schroeder JI, Kangasjarvi J (2008) SLAC1 is
required for plant guard cell S-type anion channel function in stomatal signalling.
Nature 452: 487-491
Vahisalu T, Puzõrjova I, Brosché M, Valk E, Lepiku M, Moldau H, Pechter P, Wang YS, Lindgren O, Salojärvi J, Loog M, Kangasjärvi J, Kollist H (2010) Ozonetriggered rapid stomatal response involves the production of reactive oxygen species,
and is controlled by SLAC1 and OST1. The Plant Journal 62: 442-453
Waters S, Gilliham M, Hrmova M (2013) Plant high-affinity potassium (HKT) transporters
involved in salinity tolerance: structural insights to probe differences in ion selectivity.
International Journal of Molecular Sciences 14: 7660-7680
Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY
Downloaded from on June 14, 201732
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
White PJ, Broadley MR (2001) Chloride in soils and its uptake and movement within the
plant: A review. Annals of Botany 88: 967-988
Wu S, Zhang Y (2007) LOMETS: A local meta-threading-server for protein structure
prediction. Nucleic Acids Research 35: 3375-3382
Wu SJ, Ding L, Zhu JK (1996) SOS1, a genetic locus essential for salt tolerance and
potassium acquisition. Plant Cell 8: 617-627
Xu G, Magen H, Tarchitzky J, Kafkafi U (1999) Advances in chloride nutrition of plants.
In LS Donald, ed, Advances in Agronomy, Vol Volume 68. Academic Press, pp 97150
Yamashita T, Shimada S, Guo W, Sato K, Kohmura E, Hayakawa T, Takagi T,
Tohyama M (1997) Cloning and functional expression of a brain peptide/histidine
transporter. Journal of Biological Chemistry 272: 10205-10211
Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell
system for transient gene expression analysis. Nature Protocols 2: 1565-1572
Zhou J-J, Theodoulou FL, Muldin I, Ingemarsson B, Miller AJ (1998) Cloning and
functional characterization of a Brassica napus transporter that is able to transport
nitrate and histidine. Journal of Biological Chemistry 273: 12017-12023
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
987
Figure legends and Tables
988
989
Figure 1 Bioinformatic analysis of NPF2.4. Phylogenetic tree shows relationships between
990
the 7 members of the NAXT subfamily and 7 other NPF2s. Multiple amino acid sequence
991
alignment was performed with ClustalW2 with a gap open of 10 and a gap extension of 0.1.
992
Phylogenetic tree was generated using program MEGA package 5.0 using default settings.
993
Names are shown for proteins that are available in literature. Scale bar indicates the
994
maximum like-hood distance.
995
996
Figure 2 NPF2.4 is likely to form a membrane embedded ion transporter. A 3D homology
997
model of NPF2.4
998
oneidensis (PDB accession 2XUT) and (B) a H+/NO3– transporter from Arabidopsis (PDB
999
accession 4OH3) as structural templates. Putative binding site for Cl– ions is enclosed in a
1000
pocket (arrow), and is delineated by four amino acid residues Tyr37, Val41, Asn67 and
1001
Met334 coloured in atomic colours. Left and right arrowheads indicate NH2- and COOH-
1002
termini that are predicted to face intracellular sides. (C) Superposition of both models
1003
showing very similar folds and positions of residues participating in binding of Cl–. (D) A
1004
detail of the putative Cl– binding pocket, signifying residues that are likely to bind Cl–, and
1005
their predicted structural shift upon binding Cl–. Sticks in magenta and atomic colours
1006
indicate residues in the NPF2.5 apo- and Cl–-complexed models, respectively. Dashed lines
1007
specify distances between the four binding residues and the Cl– ion.
(A) a proton dependent oligo-peptide transporter from Shewanella
1008
1009
Figure 3 NPF2.4 expression is down-regulated by both salt and ABA. Four-week old Col-0
1010
Arabidopsis plants were treated with NaCl or ABA as indicated before their whole roots were
1011
harvested for qRT-PCR analysis. (A) NPF2.4 transcripts detected in the root of plants treated
1012
with 2 mM (control), 50 mM or 100 mM NaCl for 24 h. (B) NPF2.4 transcripts detected in
1013
the root of plants treated with 2 mM (control) or 75 mM NaCl for 5 days before harvest (C)
1014
NPF2.4 transcripts detected in the root of plants treated with 20 µM +/– cis, trans ABA for 4
1015
h or 16 h. Results are presented as mean ± SEM (n = 4 or 5), expression levels were
Downloaded from on June 14, 201734
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1016
normalized to controls. Significance is indicated by the asterisks (One way ANOVA and
1017
Tukey test, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).
1018
1019
Figure 4 NPF2.4 is predominantly expressed in the root stelar cells. (A) A non-transformed
1020
Col-0 plant showing no GUS activity. (B and C) Strong proNPF2.4 driven GUS activity was
1021
detected in the cells associated with mature root stele after 1-h of histochemical staining. (D)
1022
GUS activity was detected in root-shoot junction. (E and F) GUS activity was detected
1023
mainly in the root and the vascular cells in both cotyledon and true leaves. (G) Root cross
1024
sections prepared from 7-d old proNPF2.4:GUS plants showing GUS activity in the stelar
1025
cells. (H and I) GUS activity was detected in the flower and siliques.
1026
1027
Figure 5 proNPF2.4 driven GUS activity in roots is reduced by salt treatments. Two-week
1028
old T2 proNPF2.4:UidA plants were GUS-stained for 1 h after salt treatments as indicated for
1029
5 d. Roots were subjected to MUG assay. Absorbance measured using a spectrophotometer at
1030
366 mm to measure MU concentrations. Error bars represent mean ± SEM (n = 3).
1031
Significance is indicated by the asterisks (One-way ANOVA and Tukey test, *P ≤ 0.05; **P
1032
≤ 0.01).
1033
1034
Figure 6 NPF2.4 protein localizes to the plasma membrane. (A and B) Constitutive stable
1035
expression of GFP::NPF2.4 in the Arabidopsis root, scale bars = 20 µm. (A) Confocal image
1036
of root cells of 10-d old GFP::NPF2.4 expressing plants showing the plasma membrane
1037
localization of GFP tagged NPF2.4. (B) Plasmolysis performed on the GFP::NPF2.4
1038
expressing plants showing Hechtian strands as indicated by the white arrows. (C to E)
1039
Transient expression of YFP::NPF2.4 fusion construct in Arabidopsis mesophyll protoplasts,
1040
scale bars = 100 µm. (C) Confocal image of YFP::NPF2.4 fusion protein in Arabidopsis
1041
mesophyll protoplasts. (D) Cyan fluorescence of plasma membrane marker ECFP::ROP11.
1042
(E) Bright image of the transformed protoplast. (F) Merged image showing the co-
1043
localization of YFP::NPF2.4 fusion protein and the plasma membrane marker.
1044
Figure 7 NPF2.4 generates Na+- and K+- dependent Cl– transport when expressed in Xenopus
1045
laevis oocytes. NPF2.4-cRNA injected oocytes were exposed to different solutions as
Downloaded from on June 14, 201735
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1046
indicated. (A) Currents over time for NPF2.4-cRNA injected oocytes, 2 d after injection,
1047
clamped at –60 mV, in the presence of 45 mM Na-glutamate (45 Na) and 45 mM Na-
1048
glutamate plus 50 mM NMDG-Cl (45 Na + 50 Cl). (B) Currents elicited by NPF2.4-cRNA
1049
when oocytes, held at –140 mV in 50 mM NaCl, were exposed to pH 7.5 or 5.5 (n = 4) (C).
1050
Currents elicited by NPF2.4-cRNA when oocytes were exposed to different concentrations of
1051
NaCl (n = 3). (D) Currents elicited by NPF2.4-cRNA when oocytes were exposed to different
1052
concentrations of NaNO3 (n = 3). (E) Currents elicited by NPF2.4-cRNA when oocytes were
1053
exposed to a constant concentration of Na+ (45 mM) with varying concentrations of Cl– (0, 5,
1054
10, 25, 50 mM). (F) Currents elicited by NPF2.4-cRNA when oocytes were exposed to a
1055
constant concentration of Cl– (45 mM) with varying concentrations of Na+ (0, 5, 10, 25, 50
1056
mM). (G) Conductance of NPF2.4-cRNA injected oocytes exposed to solutions containing
1057
various different ions; conductance was calculated for –80 mV to –100 mV, dots are means,
1058
maximum and minimum values ± SEM are represented by a vertical bar (n = 3) or box (n =
1059
3–8); horizontal bars indicate the median; columns with different letters indicate statistically
1060
significant differences (P < 0.05). (H) Concentrations of Cl–, Na+ and K+ measured in the
1061
NPF2.4-cRNA injected oocytes and water controls pre-incubated in Ca2+ ringer solution for 2
1062
d. n = 16 samples for NPF2.4-cRNA injected oocytes; n = 10 samples for water controls;
1063
each sample contained 10 oocytes; One way ANOVA and Tukey test, ***P ≤ 0.001. (I) 36Cl–
1064
uptake measured in oocytes injected with either NPF2.4-cRNA or water, in a background of
1065
100 mM Cl– for 60 min; results are presented as mean ± SEM (n = 20); columns with
1066
different letters indicate statistically significant differences (P < 0.05). (B-D, F) Data were
1067
plotted after subtraction of currents measured in water-injected oocytes; water-injected
1068
control data and total currents in NPF2.4-cRNA injected oocytes are included in
1069
Supplemental Figure 2.
1070
1071
Figure 8 Shoot Cl- concentration was significantly reduced in the NPF2.4 artificial
1072
microRNA knockdowns under low salt conditions (2 mM NaCl), whereas the shoot NO3–
1073
concentration of the NPF2.4 knockdowns was similar to null segregants. 4-week old
1074
hydroponically grown plants were treated with 2 mM NaCl for 5 days. (A) NPF2.4
1075
expression in the roots of NPF2.4 knockdowns. (B) Cl– concentrations in the shoot of
1076
NPF2.4 knockdowns and null segregants. (C) Shoot NO3– concentrations of NPF2.4
1077
knockdowns and null segregants. Results are presented as mean ± SEM, n = 4-7. Significance
Downloaded from on June 14, 201736
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1078
is indicated by the asterisks (One way ANOVA and Tukey test, * P ≤ 0.05; **P ≤ 0.005;
1079
***P ≤ 0.001).
1080
1081
Figure 9 Shoot accumulation of Cl– was increased in the NPF2.4 over expression lines, while
1082
NO3- content remained unaffected. (A) Cl– concentrations in the shoot of NPF2.4 over-
1083
expression lines and null segregants after high salt treatment (75 mM NaCl). (B) NO3–
1084
concentrations in the shoot of NPF2.4 over-expression lines and null segregants after high
1085
salt treatment. (C) Cl– concentrations in the shoot of NPF2.4 over-expression lines and null
1086
segregants after low salt treatment (2 mM NaCl). (D) NO3– concentrations in the shoot of
1087
NPF2.4 over-expression lines and null segregants after low salt treatment. Error bars
1088
represent mean ± SEM. n = 4. Significance is indicated by the asterisks (Single factor
1089
ANOVA and Tukey test, * P ≤ 0.05.
1090
1091
Supplemental Figure 1 Multiple sequence alignment of NPF2.4, NPF2.3 and NPF2.1/
1092
NAXT1, protein sequence alignment was performed using ClustalW2 program with a gap
1093
open of 10 and a gap extension of 0.1. The black and shaded regions represent identical
1094
residues and conservative substitutions, respectively.
1095
1096
Supplemental Figure 2 Raw and control data for expression of NPF2.4 in Xenopus laevis
1097
oocytes. (A) Currents over time for water injected control oocytes, 2 d after injection,
1098
clamped at –60 mV, in the presence of 45 mM Na-glutamate (45 Na) and 45 mM Na-
1099
glutamate plus 50 mM NMDG-Cl (45 Na + 50 Cl). (B) Total currents (i.e. without leak
1100
subtraction) in oocytes expressing NPF2.4-cRNA, held at -140 mV in 50 mM NaCl, when
1101
exposed to pH 7.5 or 5.5 (n = 4). (C) Currents for water injected control oocytes, held at -140
1102
mV in 50 mM NaCl, and exposed to pH 7.5 or 5.5 (n = 4). (D) Non-control subtracted
1103
currents elicited by NPF2.4-cRNA when oocytes were exposed to different concentrations of
1104
NaCl (n = 3). (E) Currents for water injected oocytes exposed to different concentrations of
1105
NaCl (n = 3). (F) Total currents (i.e. without leak subtraction) in oocytes expressing NPF2.4-
1106
cRNA when oocytes were exposed to different concentrations of NaNO3 (n = 3). (G)
1107
Currents for water injected oocytes exposed to different concentrations of NaNO3 (n = 3). (H)
1108
Total currents (i.e. without leak subtraction) in oocytes expressing NPF2.4-cRNA when
Downloaded from on June 14, 201737
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1109
oocytes were exposed to 45 mM Na-glutamate and varying concentrations of NMDG-Cl (n =
1110
3–6). (I) Currents for water injected oocytes exposed to 45 mM Na-glutamate and varying
1111
concentrations of NMDG-Cl (n = 3–4). (J) Total currents (i.e. without leak subtraction) in
1112
oocytes expressing NPF2.4-cRNA when oocytes were exposed to 45 mM NMDG-Cl and
1113
varying concentrations of Na-glutamate (n = 5). (K) Currents for water injected oocytes
1114
exposed to 45 mM NMDG-Cl and varying concentrations of Na-glutamate (n = 5). (L)
1115
Conductance for water injected oocytes exposed to solutions containing various different ions;
1116
conductance was calculated for –80 mV to –100 mV, dots are means, maximum and
1117
minimum values ± SEM are represented by a vertical bar (n = 3) or box (n = 4–6); horizontal
1118
bars indicate the median; columns with different letters indicate statistically significant
1119
differences (P ≤ 0.05). (M)
1120
cRNA or water, in a background of 100 mM Na+ for 60 min; results are scintillation counts
1121
per minute, presented as mean ± SEM (n = 22 to 30).
22
Na+ uptake measured in oocytes injected with either NPF2.4-
1122
1123
Supplemental Figure 3 NPF2.4 transcript levels of lines SALK_111056 and SALK_111071
1124
annotated as T-DNA knockouts for NPF2.4 showed the failed disruption of NPF2.4
1125
expression in both lines. (A) T-DNA insertions of both obtained SALK lines and results of
1126
genotyping PCRs showed that S_111056 line lost its T-DNA insertion within the ORF of
1127
NPF2.4. (B) Quantitative RT-PCR was performed on 11 and 14 progenies of T3
1128
SALK_111056 and SALK_111071, respectively and 4 Col-0 WT Arabidopsis plants. Plants
1129
were grown hydroponically for 4 weeks. Results are presented as mean ± SEM.
1130
1131
Supplemental Figure 4 Under low salt condition, shoot concentration of both Na+ and K+
1132
were similar between NPF2.4 knockdowns and null segregants. Expression of NPF2.5 was
1133
not affected in the roots of NPF2.4 knockdowns. Under high salt condition, shoot Cl-
1134
concentration of knockdowns remained similar to that of null segregants when NPF2.4
1135
expression in the knockdowns was down-regulated to similar low levels as null segregants. 4-
1136
week old hydroponically grown plants were treated with salt for 5 d. (A) concentrations of Cl-,
1137
Na+ and K+ in the shoot of NPF2.4 knockdowns and null segregants after low salt treatment,
1138
data was normalized relative to nulls. (B) Expression levels of NPF2.4 in knockdowns and
1139
null segregants after low salt treatment. (C) NPF2.4 expression in the roots of NPF2.4
1140
knockdown lines after high salt treatment. (D) Cl- concentrations in the shoot of NPF2.4
Downloaded from on June 14, 201738
- Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1141
knockdowns and null segregants after high salt treatment. (E) Expression levels of NPF2.5 in
1142
the roots of NPF2.4 knockdowns after low salt treatment. Results are presented as mean ±
1143
SEM, n = 3 or 4. Significance is indicated by the asterisks (Single factor ANOVA and Tukey
1144
test, * P ≤ 0.05).
1145
1146
Supplemental Figure 5 Expression validation of NPF2.4 in the root of NPF2.4 over-
1147
expression lines under both low salt and high salt conditions. (A) Quantitative RT-PCR on
1148
the cDNA samples prepared from roots of NPF2.4 over expression lines after high salt
1149
treatment (75 mM NaCl). (B) Quantitative RT-PCR on the cDNA samples prepared from
1150
roots of NPF2.4 over expression lines after low salt treatment (2 mM NaCl). Results are
1151
presented as mean ± SEM, n = 4. Significance is indicated by the asterisks (Single factor
1152
ANOVA and Tukey test, * P ≤ 0.05; ***P ≤ 0.001).
1153
Supplemental Figure 6 Biomass of NPF2.4 knockdowns was unaffected by low salt
1154
condition compared to null segregants. 4-week old hydroponically grown plants were treated
1155
with salt for 5 days. Result is presented as mean ± SEM, n = 3 or 4.
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1156
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
40
AT3G45720
AT3G45690
AT3G45680
AT3G45700
NPF2.4
AT3G45710
AT3G45660
AT3G45650
AT1G18880
AT3G47960
AT5G62680
NPF2.7/NAXT1
NPF2.9/NRT1.9
NPF2.10/GTR1
NPF2.11/GTR2/NRT1.10
AT5G28470
AT1G27080
AT1G69870
NPF2.12/NRT1.6
NPF2.13/NRT1.7
AT1G69860
0.2
Figure 1 Bioinformatic analysis of NPF2.4.
Phylogenetic tree shows relationships between the 7 members of the NAXT subfamily and 7 other
NPF2s. Multiple amino acid sequence alignment was performed with ClustalW2 with a gap open of 10
and a gap extension of 0.1. Phylogenetic tree was generated using program MEGA package 5.0 using
default settings. Names are shown for proteins that are available in literature. Scale bar indicates the
maximum like-hood distance.
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
A
B
C
D
Figure 2 NPF2.4 is likely to form a membrane embedded ion transporter.
A 3D homology model of NPF2.4 (A) a proton dependent oligo-peptide transporter from Shewanella
oneidensis (PDB accession 2XUT) and (B) a H+/NO3– transporter from Arabidopsis (PDB accession
4OH3) as structural templates. Putative binding site for Cl– ions is enclosed in a pocket (arrow), and is
delineated by four amino acid residues Tyr37, Val41, Asn67 and Met334 coloured in atomic colours.
Left and right arrowheads indicate NH2- and COOH-termini that are predicted to face intracellular
sides. (C) Superposition of both models showing very similar folds and positions of residues
participating in binding of Cl–. (D) A detail of the putative Cl– binding pocket, signifying residues that
are likely to bind Cl–, and their predicted structural shift upon binding Cl–. Sticks in magenta and atomic
colours indicate residues in the NPF2.5 apo- and Cl–-complexed models, respectively. Dashed lines
specify distances between the four binding residues and the Cl– ion.
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1.5
1.0
*
0.5
***
Relative expression of NPF2.4
1.5
1.5
10
0
1.0
0.5
**
d
0.0
75
m
M
N
aC
l5
C
on
tr
ol
B
Relative expression of NPF2.4
50
m
M
m
M
N
aC
l2
N
aC
l2
4h
4h
0.0
C
on
tr
ol
Relative expression of NPF2.4
A
1.0
*
0.5
***
16
h
A
B
M
20
20
M
A
B
A
A
4h
0.0
C
on
tr
ol
C
Figure 3 NPF2.4 expression is down-regulated by both salt and ABA.
Four-week old Col-0 Arabidopsis plants were treated with NaCl or ABA as indicated before their whole
roots were harvested for qRT-PCR analysis. (A) NPF2.4 transcripts detected in the root of plants treated
with 2 mM (control), 50 mM or 100 mM NaCl for 24 h. (B) NPF2.4 transcripts detected in the root of
plants treated with 2 mM (control) or 75 mM NaCl for 5 days before harvest (C) NPF2.4 transcripts
detected in the root of plants treated with 20 M +/– cis, trans ABA for 4 h or 16 h. Results are
presented as mean ± SEM (n = 4 or 5), expression levels were normalized to controls. Significance is
indicated by the asterisks (One way ANOVA and Tukey test, *P 0.05; **P 0.01; ***P 0.001).
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
A
D
G
B
C
E
F
H
I
Figure 4 NPF2.4 is predominantly expressed in the root stelar cells.
(A) A non-transformed Col-0 plant showing no GUS activity. (B and C) Strong proNPF2.4 driven GUS
activity was detected in the cells associated with mature root stele after 1-h of histochemical staining.
(D) GUS activity was detected in root-shoot junction. (E and F) GUS activity was detected mainly in
the root and the vascular cells in both cotyledon and true leaves. (G) Root cross sections prepared from
7-d old proNPF2.4:GUS plants showing GUS activity in the stelar cells. (H and I) GUS activity was
detected in the flower and siliques.
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Figure 5 proNPF2.4 driven GUS activity in roots is reduced by salt treatments.
Two-week old T2 proNPF2.4:UidA plants were GUS-stained for 1 h after salt treatments as indicated
for 5 d. Roots were subjected to MUG assay. Absorbance measured using a spectrophotometer at 366
mm to measure MU concentrations. Error bars represent mean ± SEM (n = 3). Significance is indicated
by the asterisks (One-way ANOVA and Tukey test, *P 0.05; **P 0.01).
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
A
AA
A
C
E
B
B
B
D
F
Figure 6 NPF2.4 protein localizes to the plasma membrane.
(A and B) Constitutive stable expression of GFP::NPF2.4 in the Arabidopsis root, scale bars = 20 m.
(A) Confocal image of root cells of 10-d old GFP::NPF2.4 expressing plants showing the plasma
membrane localization of GFP tagged NPF2.4. (B) Plasmolysis performed on the GFP::NPF2.4
expressing plants showing Hechtian strands as indicated by the white arrows. (C to E) Transient
expression of YFP::NPF2.4 fusion construct in Arabidopsis mesophyll protoplasts, scale bars = 100
m. (C) Confocal image of YFP::NPF2.4 fusion protein in Arabidopsis mesophyll protoplasts. (D)
Cyan fluorescence of plasma membrane marker ECFP::ROP11. (E) Bright image of the transformed
protoplast. (F) Merged image showing the co-localization of YFP::NPF2.4 fusion protein and the
plasma membrane marker.
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A
45 Na
45 Na
45 Na
45 Na
45 Na + 50 Cl 45 Na + 50 Cl
45 Na + 50 Cl
Currents (nA) at -140 mV
B
200 nA
1 min
C
0
-200
-400
-600
-800
-1000
pH 7.5
pH 5.5
D
500
100 mM NaCl pH 7.5
50 mM NaCl pH 7.5
5 mM NaCl pH 7.5
1000
100 mM NaNO3 pH 7.5
50 mM NaNO3 pH 7.5
Current (nA)
Current (nA)
5 mM NaNO3 pH 7.5
500
-150
-100
-50
50
Voltage (mV)
Voltage (mV)
-150
-100
-50
50
-500
-500
-1000
-1000
E
F
1000
45 mM Na + 0 mM NMDG-Cl
45 mM Na + 5 mM NMDG-Cl
45 mM Na + 10 mM NMDG-Cl
45 mM Na + 25 mM NMDG-Cl
45 mM Na + 50 mM NMDG-Cl
Voltage (mV)
-150
-100
-50
1000
45 mM NMDG-Cl + 0 mM Na
45 mM NMDG-Cl + 5 mM Na
45 mM NMDG-Cl + 10 mM Na
45 mM NMDG-Cl + 25 mM Na
45 mM NMDG-Cl + 50 mM Na
Current (nA)
50
-150
Voltage (mV)
-100
-50
50
-1000
-1000
-2000
-2000
H
20
bcd
15
abc
10
5
abc abc
a
50
50
m
M
N
agl
m uta
m
M
a
N
M te
50 DG
m -C
l
M
50 Na
C
25 mM l
K
m
C
M
25
M l
g
m
C
l2
M
C
10
a
C
0
l
10 mM 2
0
K
m
C
l
M
N
aC
l
0
100
Cl-
***
80
Na+
K+
60
40
20
0
4
d
N
PF
2.
d
2O
cd
H
Conductance (nS)
25
mM [Cl-], [Na+ ] or [K+ ] per oocyte
G
Current (nA)
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Uptake of 36Cl - (nmol/oocyte/h)
I
20
pH 5.5
pH 7.5
25
NaCl
NMDG
b
b
NaCl
NMDG
15
10
a
a
5
a
a
C
C
2O
H
2O
N
PF
2.
4
H
H
2O
N
PF
2.
4
2O
N
PF
2.
4
H
N
PF
2.
4
0
Figure 7 NPF2.4 generates Na+- and K+- dependent Cl– transport when expressed in Xenopus laevis
oocytes.
NPF2.4-cRNA injected oocytes were exposed to different solutions as indicated. (A) Currents over time
for NPF2.4-cRNA injected oocytes, 2 d after injection, clamped at –60 mV, in the presence of 45 mM
Na-glutamate (45 Na) and 45 mM Na-glutamate plus 50 mM NMDG-Cl (45 Na + 50 Cl). (B) Currents
elicited by NPF2.4-cRNA when oocytes, held at –140 mV in 50 mM NaCl, were exposed to pH 7.5 or
5.5 (n = 4) (C). Currents elicited by NPF2.4-cRNA when oocytes were exposed to different
concentrations of NaCl (n = 3). (D) Currents elicited by NPF2.4-cRNA when oocytes were exposed to
different concentrations of NaNO3 (n = 3). (E) Currents elicited by NPF2.4-cRNA when oocytes were
exposed to a constant concentration of Na+ (45 mM) with varying concentrations of Cl- (0, 5, 10, 25, 50
mM). (F) Currents elicited by NPF2.4-cRNA when oocytes were exposed to a constant concentration of
Cl- (45 mM) with varying concentrations of Na+ (0, 5, 10, 25, 50 mM). (G) Conductance of NPF2.4cRNA injected oocytes exposed to solutions containing various different ions; conductance was
calculated for –80 mV to –100 mV, dots are means, maximum and minimum values ± SEM are
represented by a vertical bar (n = 3) or box (n = 3–8); horizontal bars indicate the median; columns with
different letters indicate statistically significant differences (P < 0.05). (H) Concentrations of Cl-, Na+
and K+ measured in the NPF2.4-cRNA injected oocytes and water controls pre-incubated in Ca2+ ringer
solution for 2 d. n = 16 samples for NPF2.4-cRNA injected oocytes; n = 10 samples for water controls;
each sample contained 10 oocytes; One way ANOVA and Tukey test, ***P 0.001. (I) 36Cl– uptake
measured in oocytes injected with either NPF2.4-cRNA or water, in a background of 100 mM Cl– for
60 min; results are presented as mean ± SEM (n = 20); columns with different letters indicate
statistically significant differences (P < 0.05). (B-D, F) Data were plotted after subtraction of currents
measured in water-injected oocytes; water-injected control data and total currents in NPF2.4-cRNA
injected oocytes are included in Supplemental Figure 2.
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8000
6000
4000
***
2000
45
44
K
D
-N
PF
2.
43
K
D
-N
PF
2.
42
K
D
-N
PF
2.
41
-N
PF
2.
K
D
-N
PF
2.
N
ul
l
**
**
*
10
*
5
45
44
K
D
-N
PF
2.
43
-N
PF
2.
K
D
K
D
-N
PF
2.
K
D
42
41
-N
PF
2.
N
ul
l
0
-N
PF
2.
moles NO3 /g FW in the shoot
**
15
40
30
20
-
C
***
0
K
D
moles Cl /g FW in the shoot
-
B
**
***
***
K
D
Copy number per g NPF2.4
cDNA under normal condition
A
10
N
K
ul
D
l
-N
PF
2.
K
4D
1
-N
PF
2.
K
4D
2
-N
PF
2.
K
4D
3
-N
PF
2.
K
4D
4
-N
PF
2.
45
0
Figure 8 Shoot Cl- concentration was significantly reduced in the NPF2.4 artificial microRNA
knockdowns under low salt conditions (2 mM NaCl), whereas the shoot NO3– concentration of the
NPF2.4 knockdowns was similar to null segregants.
4-week old hydroponically grown plants were treated with 2 mM NaCl for 5 days. (A) NPF2.4
expression in the roots of NPF2.4 knockdowns. (B) Cl– concentrations in the shoot of NPF2.4
knockdowns and null segregants. (C) Shoot NO3– concentrations of NPF2.4 knockdowns and null
segregants. Results are presented as mean ± SEM, n = 4-7. Significance is indicated by the asterisks
**P
0.001).
(One way ANOVA and Tukey
test, *from
P on0.05;
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2017 - 0.005;
Published***P
by www.plantphysiol.org
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10
5
.4
-2
E
X
-N
P
F2
O
O
E
X
-N
P
F2
.4
-1
0
X
-N
P
O
E
80
60
40
20
0
N
ul
O
l
E
X
-N
P
F2
.4
-1
O
E
X
-N
P
F2
.4
-2
D
F2
.4
-2
.4
-2
.4
-1
O
E
X
-N
P
F2
15
N
ul
l
moles Cl-/g FW in the shoot
C
0
E
X
-N
P
F2
N
ul
l
O
0
10
X
-N
P
50
O
E
100
20
F2
.4
-1
*
150
30
N
ul
l
B
moles NO3-/g FW in the shoot
200
moles NO3-/g FW in the shoot
moles Cl-/g FW in the shoot
A
Figure 9 Shoot accumulation of Cl– was increased in the NPF2.4 over expression lines, while NO3content remained unaffected.
(A) Cl– concentrations in the shoot of NPF2.4 over-expression lines and null segregants after high salt
treatment (75 mM NaCl). (B) NO3– concentrations in the shoot of NPF2.4 over-expression lines and
null segregants after high salt treatment. (C) Cl– concentrations in the shoot of NPF2.4 over-expression
lines and null segregants after low salt treatment (2 mM NaCl). (D) NO3– concentrations in the shoot of
NPF2.4 over-expression lines and null segregants after low salt treatment. Error bars represent mean ±
SEM. n = 4. Significance is indicated by the asterisks (Single factor ANOVA and Tukey test, * P 0.05.
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Parsed Citations
Abel GH (1969) Inheritance of the capacity for chloride inclusion and chloride exclusion by soybeans. Crop Sciences 9: 697-698
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function
as transcriptional activators in abscisic acid signaling. Plant Cell 15: 63-78
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K (1997) Role of Arabidopsis MYC and MYB homologs in
drought- and abscisic acid-regulated gene expression. Plant Cell 9: 1859-1868
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Apse MP, Blumwald E (2007) Na+ transport in plants. FEBS Letters 581: 2247-2254
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Holko M, Yefanov
A, Lee H, Zhang N, Robertson CL, Serova N, Davis S, Soboleva A. (2013) NCBI GEO: archive for functional genomics data sets-update. Nucleic Acids Research 41, D991-D995
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bassil E, Blumwald E (2014) The ins and outs of intracellular ion homeostasis: NHX-type cation/H+ transporters. Current Opinion in
Plant Biology 22: 1-6
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Beilby MJ, Walker NA (1981) Chloride transport in Chara: I. Kinetics and current-voltage curves for a probable proton symport.
Journal of Experimental Botany 32: 43-54
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Berthomieu P, Conejero G, Nublat A, Brackenbury WJ, Lambert C, Savio C, Uozumi N, Oiki S, Yamada K, Cellier F, Gosti F,
Simonneau T, Essah PA, Tester M, Very A-A, Sentenac H, Casse F (2003) Functional analysis of AtHKT1 in Arabidopsis shows that
Na+ recirculation by the phloem is crucial for salt tolerance. The EMBO Journal 22: 2004-2014
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Burton RA, Jobling SA, Harvey AJ, Shirley NJ, Mather DE, Bacic A, Fincher GB (2008) The genetics and transcriptional profiles of
the cellulose synthase-like HvCslF gene family in barley. Plant Physiology 146: 1821-1833
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Byrt CS, Platten JD, Spielmeyer W, James RA, Lagudah ES, Dennis ES, Tester M, Munns R (2007) HKT1;5-like cation transporters
linked to Na+ exclusion loci in wheat, nax2 and kna1. Plant Physiology 143: 1918-1928
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Chen C-Z, Lv X-F, Li J-Y, Yi H-Y, Gong J-M (2012) Arabidopsis NRT1.5 is another essential component in the regulation of nitrate
reallocation and stress tolerance. Plant Physiology 159: 1582-1590
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The
Plant Journal 16: 735-743.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cole PJ (1985) Chloride toxicity in citrus. Irrigation Science 6: 63-71
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded
from on June
14, 2017
- Published
byTalon
www.plantphysiol.org
Colmenero-Flores JM, Martinez G, Gamba
G, Vazquez
N, Iglesias
DJ,
Brumos J,
M (2007) Identification and functional
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
characterization of cation-chloride cotransporters in plants. The Plant Journal 50: 278-292
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Conn SJ, Hocking B, Dayod M, Xu B, Athman A, Henderson S, Aukett L, Conn V, Shearer MK, Fuentes S (2013) Protocol: optimising
hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant methods 9: 4
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cotsaftis O, Plett D, Shirley N, Tester M, Hrmova M (2012) A two-staged model of Na+ exclusion in rice explained by 3D modeling
of HKT transporters and alternative splicing. PLoS ONE 7: e39865
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cram W, Pitman M (1972) The action of abscisic acid on ion uptake and water flow in plant roots. Australian Journal of Biological
Sciences 25: 1125-1132
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant
Physiology 133: 462-469
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Davenport RJ, Munoz-Mayor A, Jha D, Essah PA, Rus ANA, Tester M (2007) The Na+ transporter AtHKT1;1 controls retrieval of Na+
from the xylem in Arabidopsis. Plant, Cell & Environment 30: 497-507
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Evrard A (2013) Cell type-specific transcriptional responses of plants to salinity. PhD thesis, The University of Adelaide, Adelaide,
Australia
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Evrard A, Bargmann BR, Birnbaum K, Tester M, Baumann U, Johnson AT (2012) Fluorescence-activated cell sorting for analysis of
cell type-specific responses to salinity stress in Arabidopsis and rice. In S Shabala, TA Cuin, eds, Plant Salt Tolerance, Vol 913.
Humana Press, pp 265-276
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Felle HH (1994) The H+/Cl- symporter in root-hair cells of Sinapis alba (an electrophysiological study using ion-selective
microelectrodes). Plant Physiology 106: 1131-1136
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, Michaux-Ferriere N, Thibaud JB, Sentenac H (1998)
Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap. Cell 94: 647-655
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann C, Al-Rasheid KAS, Grill E, Romeis T, Hedrich R
(2010) Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities. Proceedings of the
National Academy of Sciences 107: 8023-8028
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese A, Al-Rasheid KAS, Romeis T, Hedrich R
(2009) Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proceedings
of the National Academy of Sciences 106: 21425-21430
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Geilfus C-M, Mithöfer A, Ludwig-Müller J, Zörb C, Muehling KH (2015) Chloride-inducible transient apoplastic alkalinizations
induce stomata closure by controlling abscisic acid distribution between leaf apoplast and guard cells in salt-stressed Vicia faba.
New Phytologist DOI: 10.1111/nph.13507
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Gille C, Birgit W, Gille A (2014) Sequence alignment visualization in HTML5 without Java. Bioinformatics 30: 121-122
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gilliham M, Tester M (2005) The regulation of anion loading to the maize root xylem. Plant Physiology 137: 819-828
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gong H, Blackmore D, Clingeleffer P, Sykes S, Jha D, Tester M, Walker R (2011) Contrast in chloride exclusion between two
grapevine genotypes and its variation in their hybrid progeny. Journal of Experimental Botany 62: 989-999
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Guan R, Qu Y, Guo Y, Yu L, Liu Y, Jiang J, Chen J, Ren Y, Liu G, Tian L, Jin L, Liu Z, Hong H, Chang R, Gilliham M, Qiu L (2014)
Salinity tolerance in soybean is modulated by natural variation in GmSALT3. The Plant Journal 80: 937-950
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Henderson SW, Baumann U, Blackmore DH, Walker AR, Walker RR, Gilliham M (2014) Shoot chloride exclusion and salt tolerance
in grapevine is associated with differential ion transporter expression in roots. BMC Plant Biology 14: 273
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Horie T, Hauser F, Schroeder JI (2009) HKT transporter-mediated salinity resistance mechanisms in Arabidopsis and monocot crop
plants. Trends in Plant Science 14: 660-668
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
James RA, Davenport RJ, Munns R (2006) Physiological characterization of two genes for Na+ exclusion in durum wheat, Nax1 and
Nax2. Plant Physiology 142: 1537-1547
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jeong J, Suh S, Guan C, Tsay Y-F, Moran N, Oh CJ, An CS, Demchenko KN, Pawlowski K, Lee Y (2004) A nodule-specific
dicarboxylate transporter from alder is a member of the peptide transporter family. Plant Physiology 134: 969-978
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jha D, Shirley N, Tester M, Roy SJ (2010) Variation in salinity tolerance and shoot sodium accumulation in Arabidopsis ecotypes
linked to differences in the natural expression levels of transporters involved in sodium transport. Plant, Cell & Environment 33:
793-804
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kamphake LJ, Hannah SA, Cohen JM (1967) Automated analysis for nitrate by hydrazine reduction. Water Research 1: 205-216
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kanno Y, Hanada A, Chiba Y, Ichikawa T, Nakazawa M, Matsui M, Koshiba T, Kamiya Y, Seo M (2012) Identification of an abscisic
acid transporter by functional screening using the receptor complex as a sensor. Proceedings of the National Academy of
Sciences 109: 9653-9658
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kaplan B, Davydov O, Knight H, Galon Y, Knight MR, Fluhr R, Fromm H (2006) Rapid transcriptome changes induced by cytosolic
Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. Plant Cell 18: 2733-2748
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kohler B, Raschke K (2000) The delivery of salts to the xylem. Three types of anion conductance in the plasmalemma of the xylem
parenchyma of roots of barley. Plant Physiology 122: 243-254
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kohler B, Wegner LH, Osipov V, Raschke K (2002) Loading of nitrate into the xylem: apoplastic nitrate controls the voltage
dependence of X-QUAC, the main anion conductance in xylem-parenchyma cells of barley roots. The Plant Journal 30: 133-142
Pubmed: Author and Title
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kollist H, Jossier M, Laanemets K, Thomine S (2011) Anion channels in plant cells. FEBS Journal 278: 4277-4292
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kong X-Q, Gao X-H, Sun W, An J, Zhao Y-X, Zhang H (2011) Cloning and functional characterization of a cation-chloride
cotransporter gene OsCCC1. Plant Molecular Biology 75: 567-578
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K, Zazimalova E,
Benkova E, Nacry P, Gojon A (2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in
plants. Developmental Cell 18: 927-937
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lauchli A, James RA, Huang CX, McCully M, Munns R (2008) Cell-specific localization of Na+ in roots of durum wheat and possible
control points for salt exclusion. Plant, Cell & Environment 31: 1565-1574
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee SC, Lan W, Buchanan BB, Luan S (2009) A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA
signaling in plant guard cells. Proceedings of the National Academy of Sciences 106: 21419-21424
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Léran S, Varala K, Boyer J-C, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B, Gassmann W,
Geiger D, Gojon A, Gong J-M, Halkier BA, Harris JM, Hedrich R, Limami AM, Rentsch D, Seo M, Tsay Y-F, Zhang M, Coruzzi G,
Lacombe B (2014) A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants.
Trends in Plant Science 19: 5-9
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li J-Y, Fu Y-L, Pike SM, Bao J, Tian W, Zhang Y, Chen C-Z, Zhang Y, Li H-M, Huang J, Li L-G, Schroeder JI, Gassmann W, Gong J-M
(2010) The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium
tolerance. Plant Cell 22: 1633-1646
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lin SH, Kuo HF, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HL, Wang YY, Tsai CB, Gojon A, Tsay YF (2008) Mutation of
the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20: 2514 - 2528
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Luo GZ, Wang HW, Huang J, Tian AG, Wang YJ, Zhang JS, Chen SY (2005) A putative plasma membrane cation/proton antiporter
from soybean confers salt tolerance in Arabidopsis. Plant Molecular Biology 59: 809-820
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Marcotte WR, Russell SH, Quatrano RS (1989) Abscisic acid-responsive sequences from the em gene of wheat. Plant Cell 1: 969976
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Martin PK, Koebner RMD (1995) Sodium and chloride ions contribute synergistically to salt toxicity in wheat. Biologia Plantarum 37:
265-271
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mäser P, Hosoo Y, Goshima S, Horie T, Eckelman B, Yamada K, Yoshida K, Bakker EP, Shinmyo A, Oiki S, Schroeder JI, Uozumi N
(2002) Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT
transporters from plants. Proceedings of the National Academy of Sciences 99: 6428-6433
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Molendijk AJ, Ruperti B, Singh MK, Dovzhenko FA, Milia M, Westphal L, Rosahl S, Soellick TR, Uhrig J, Weingarten L, Huber M,
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Palme K (2008) A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase
interactors. Plant Journal 53: 909-923.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Møller IS, Gilliham M, Jha D, Mayo GM, Roy SJ, Coates JC, Haseloff J, Tester M (2009) Shoot Na+ exclusion and increased salinity
tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis. Plant Cell 21: 2163-2178
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Møller IS, Tester M (2007) Salinity tolerance of Arabidopsis: a good model for cereals? Trends in Plant Science 12: 534-540
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Moya JL, Gomez-Cadenas A, Primo-Millo E, Talon M (2003) Chloride absorption in salt-sensitive Carrizo citrange and salt-tolerant
Cleopatra mandarin citrus rootstocks is linked to water use. Journal of Experimental Botany 54: 825-833
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Munns R (2002) Comparative physiology of salt and water stress. Plant, Cell & Environment 25: 239-250
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Munns R, Gilliham M (2015) Salinity tolerance of crops - what is the cost? New Phytologist: DOI: 10.1111/nph.13519
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Munns R, James RA, Xu B, Athman A, Conn SJ, Jordans C, Byrt CS, Hare RA, Tyerman SD, Tester M, Plett D, Gilliham M (2012)
Wheat grain yield on saline soils is improved by an ancestral Na+ transporter gene. Nature Biotechnology 30: 360-364
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59: 651-681
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nakashima K, Fujita Y, Katsura K, Maruyama K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Transcriptional
regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of
Arabidopsis. Plant Molecular Biology 60: 51-68
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Marcotte WR, Russell SH, Quatrano RS (1989) Abscisic acid-responsive sequences from the em gene of wheat. Plant Cell 1: 969976
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Narusaka Y, Nakashima K, Shinwari ZK, Sakuma Y, Furihata T, Abe H, Narusaka M, Shinozaki K, Yamaguchi-Shinozaki K (2003)
Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis RD29A gene in
response to dehydration and high-salinity stresses. The Plant Journal 34: 137-148
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M, Uchimiya H, Hashimoto M, Iba K (2008) CO2 regulator
SLAC1 and its homologues are essential for anion homeostasis in plant cells. Nature 452: 483-486
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Newstead S, Drew D, Cameron AD, Postis VLG, Xia X, Fowler PW, Ingram JC, Carpenter EP, Sansom MSP, McPherson MJ, Baldwin
SA, Iwata S (2011) Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and
PepT2. The EMBO Journal 30: 417-426
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jorgensen ME, Olsen CE, Dreyer I, Hedrich R, Geiger D, Halkier BA (2012)
NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488: 531-534
Pubmed: Author and Title
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Parker MB, Gascho GJ, Gaines TP (1983) Chloride toxicity of soybeans grown on atlantic coast flatwoods soils. Agronomy Journal
75: 439-443
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Peña L (2004) Transgenic Plants: Methods and Protocols Humana Press, Totowa, New Jersey
Pitman M, Luttge U, Lauchli A, Ball E (1974) Effect of previous water stress on ion uptake and transport in barley seedlings.
Functional Plant Biology 1: 377-385
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pitman MG (1977) Ion transport into the xylem. Annual Review of Plant Physiology 28: 71-88
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pitman MG (1982) Transport across plant roots. Quarterly Reviews of Biophysics 15: 481-554
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Plett D, Safwat G, Gilliham M, Skrumsager Møller I, Roy S, Shirley N, Jacobs A, Johnson A, Tester M (2010) Improved salinity
tolerance of rice through cell type-specific expression of AtHKT1;1. PLoS ONE 5: e12571
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Plett DC, Møller IS (2010) Na+ transport in glycophytic plants: what we know and would like to know. Plant, Cell & Environment 33:
612-626
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Qiu L, Wu D, Ali S, Cai S, Dai F, Jin X, Wu F, Zhang G (2011) Evaluation of salinity tolerance and analysis of allelic function of
HvHKT1 and HvHKT2 in Tibetan wild barley. TAG Theoretical and Applied Genetics 122: 695-703
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Qiu Q-S, Guo Y, Dietrich MA, Schumaker KS, Zhu J-K (2002) Regulation of SOS1, a plasma membrane Na+/H+ exchanger in
Arabidopsis thaliana, by SOS2 and SOS3. Proceedings of the National Academy of Sciences 99: 8436-8441
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ren Z-H, Gao J-P, Li L-G, Cai X-L, Huang W, Chao D-Y, Zhu M-Z, Wang Z-Y, Luan S, Lin H-X (2005) A rice quantitative trait locus for
salt tolerance encodes a sodium transporter. Nature Genetics 37: 1141-1146
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rengasamy P (2002) Transient salinity and subsoil constraints to dryland farming in Australian sodic soils: an overview. Australian
Journal of Experimental Agriculture 42: 351-361
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rengasamy P (2006) World salinization with emphasis on Australia. Journal of Experimental Botany 57: 1017-1023
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roberts SK (1998) Regulation of K+ channels in maize roots by water stress and abscisic acid. Plant Physiology 116: 145-153
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roberts SK, Snowman BN (2000) The effects of ABA on channel-mediated K+ transport across higher plant roots. Journal of
Experimental Botany 51: 1585-1594
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roy SJ, Gilliham M, Berger B, Essah PA, Cheffings C, Miller AJ, Davenport RJ, Liu LH, Skynner MJ, Davies JM, Richardson P,
Leigh RA, Tester M (2008) Investigating
glutamate
gene
co-expression
in Arabidopsis thaliana. Plant, Cell &
Downloaded
fromreceptor-like
on June 14, 2017
- Published
by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Environment 31: 861-871
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roy, SJ, Huang W, Wang XJ, Evrard E, Schmöckel SM, Zafar ZU, Tester M (2013) A novel protien kinase involved in Na+ exclusion
revealed from positional cloning. Plant, Cell & Environment 36: 553-568
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Roy SJ, Negrão S, Tester M (2014) Salt resistant crop plants. Current Opinion in Biotechnology 26: 115-124
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sali A, Blundell T (1994) Comparative protein modelling by satisfaction of spatial restraints. Protein Structure by Distance Analysis
64: C86
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sanders D (1980) The mechanism of Cl- transport at the plasma membrane of Chara corallina I. Cotransport with H+. The Journal of
Membrane Biology 53: 129-141
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schachtman DP, Tyerman SD, Terry BR (1991) The K+/Na+ selectivity of a cation channel in the plasma membrane of root cells
does not differ in salt-tolerant and salt-sensitive wheat species. Plant Physiology 97: 598-605
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in
Arabidopsis. Plant Cell 18: 1121-1133
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Segonzac C, Boyer JC, Ipotesi E, Szponarski W, Tillard P, Touraine B, Sommerer N, Rossignol M, Gibrat R (2007) Nitrate efflux at
the root plasma membrane: Identification of an Arabidopsis excretion transporter. Plant Cell 19: 3760-3777
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shelden MC, Howitt SM, Kaiser BN, Tyerman SD (2009) Identification and functional characterisation of aquaporins in the
grapevine, Vitis vinifera. Functional Plant Biology 36: 1065-1078
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shen M-y, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Science 15: 2507-2524
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shi H, Quintero FJ, Pardo JM, Zhu J-K (2002) The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+
transport in plants. Plant Cell 14: 465-477
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shi HZ, Ishitani M, Kim CS, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+
antiporter. Proceedings of the National Academy of Sciences 97: 6896-6901
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Two different novel cis-acting
elements of ERD1, a CLPA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced
senescence. The Plant Journal 33: 259-270
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Skerrett M, Tyerman SD (1994) A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots.
Planta 192: 295-305
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded
Google Scholar: Author Only Title Only Author
and Title from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Stacey G, Koh S, Granger C, Becker JM (2002) Peptide transport in plants. Trends in Plant Science 7: 257-263
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Storey R, Walker RR (1999) Citrus and salinity. Scientia Horticulturae 78: 39-81
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Subramanian C, Woo J, Cai X, Xu X, Servick S, Johnson CH, Nebenführ A, Von Arnim AG (2006) A suite of tools and application
notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET). The Plant Journal 48: 138152
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sun J, Bankston JR, Payandeh J, Hinds TR, Zagotta WN, Zheng N (2014) Crystal structure of the plant dual-affinity nitrate
transporter NRT1.1. Nature 507: 73-77
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sunarpi, Horie T, Motoda J, Kubo M, Yang H, Yoda K, Horie R, Chan W-Y, Leung H-Y, Hattori K, Konomi M, Osumi M, Yamagami M,
Schroeder JI, Uozumi N (2005) Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na+ unloading from xylem
vessels to xylem parenchyma cells. The Plant Journal 44: 928-938
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Takano J, Noguchi K, Yasumori M, Kobayashi M, Gajdos Z, Miwa K, Hayashi H, Yoneyama T, Fujiwara T (2002) Arabidopsis boron
transporter for xylem loading. Nature 420: 337-340
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tamura K, Peterson D, Peterson N, Stecher G, Nei M and Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Taochy C, Gaillard I, Ipotesi E, Oomen R, Leonhardt N, Zimmermann S, Peltier JB, Szponarski W, Simonneau T, Sentenac H (2015)
The Arabidopsis root stele transporter NPF2. 3 contributes to nitrate translocation to shoots under salt stress. The Plant Journal
DOI:10.1111/tpj.12901
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tavakkoli E, Fatehi F, Coventry S, Rengasamy P, McDonald GK (2011) Additive effects of Na+ and Cl- ions on barley growth under
salinity stress. Journal of Experimental Botany 62: 2189-2203
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Teakle NL, Flowers TJ, Real D, Colmer TD (2007) Lotus tenuis tolerates the interactive effects of salinity and waterlogging by
'excluding' Na+ and Cl- from the xylem. Journal of Experimental Botany 58: 2169-2180
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Teakle NL, Tyerman SD (2010) Mechanisms of Cl- transport contributing to salt tolerance. Plant, Cell & Environment 33: 566-589
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91: 503-527
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tilman D, Balzer C, Hill J, Befort BL (2011) Global food demand and the sustainable intensification of agriculture. Proceedings of
the National Academy of Sciences 108: 20260-20264
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tregeagle JM, Tisdall JM, Blackmore DH, Walker RR (2006) A diminished capacity for chloride exclusion by grapevine rootstocks
following long-term saline irrigation in an inland versus a coastal region of Australia. Australian Journal of Grape and Wine
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Research 12: 178-191
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tsay YF, Chiu CC, Tsai CB, Ho CH, Hsu PK (2007) Nitrate transporters and peptide transporters. FEBS Letters 581: 2290 - 2300
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tyerman SD (1992) Anion channels in plants. Annual Review of Plant Physiology and Plant Molecular Biology 43: 351-373
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Uozumi N, Kim EJ, Rubio F, Yamaguchi T, Muto S, Tsuboi A, Bakker EP, Nakamura T, Schroeder JI (2000) The Arabidopsis HKT1
gene homolog mediates inward Na+ currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant
Physiology 122: 1249-1260
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its
gene product binds to the conserved MYB recognition sequence. Plant Cell 5: 1529-1539
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vahisalu T, Kollist H, Wang YF, Nishimura N, Chan WY, Valerio G, Lamminmaki A, Brosche M, Moldau H, Desikan R, Schroeder JI,
Kangasjarvi J (2008) SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452: 487491
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vahisalu T, Puzõrjova I, Brosché M, Valk E, Lepiku M, Moldau H, Pechter P, Wang Y-S, Lindgren O, Salojärvi J, Loog M,
Kangasjärvi J, Kollist H (2010) Ozone-triggered rapid stomatal response involves the production of reactive oxygen species, and
is controlled by SLAC1 and OST1. The Plant Journal 62: 442-453
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Waters S, Gilliham M, Hrmova M (2013) Plant high-affinity potassium (HKT) transporters involved in salinity tolerance: structural
insights to probe differences in ion selectivity. International Journal of Molecular Sciences 14: 7660-7680
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
White PJ, Broadley MR (2001) Chloride in soils and its uptake and movement within the plant: A review. Annals of Botany 88: 967988
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wu S, Zhang Y (2007) LOMETS: A local meta-threading-server for protein structure prediction. Nucleic Acids Research 35: 33753382
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wu SJ, Ding L, Zhu JK (1996) SOS1, a genetic locus essential for salt tolerance and potassium acquisition. Plant Cell 8: 617-627
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu G, Magen H, Tarchitzky J, Kafkafi U (1999) Advances in chloride nutrition of plants. In LS Donald, ed, Advances in Agronomy,
Vol Volume 68. Academic Press, pp 97-150
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yamashita T, Shimada S, Guo W, Sato K, Kohmura E, Hayakawa T, Takagi T, Tohyama M (1997) Cloning and functional expression
of a brain peptide/histidine transporter. Journal of Biological Chemistry 272: 10205-10211
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded
Google Scholar: Author Only Title Only Author
and Title from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression
analysis. Nature Protocols 2: 1565-1572
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhou J-J, Theodoulou FL, Muldin I, Ingemarsson B, Miller AJ (1998) Cloning and functional characterization of a Brassica napus
transporter that is able to transport nitrate and histidine. Journal of Biological Chemistry 273: 12017-12023
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.