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Keywords:

  • barley;
  • nitrate transport;
  • two-component transport

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

The analysis of genome databases for many different plants has identified a group of genes that are related to one part of a two-component nitrate transport system found in algae. Earlier work using mutants and heterologous expression has shown that a high-affinity nitrate transport system from the unicellular green algae, Chlamydomonas reinhardtii required two gene products for function. One gene encoded a typical carrier-type structure with 12 putative trans-membrane (TM) domains and the other gene, nar2 encoded a much smaller protein that had only one TM domain. As both gene families occur in plants we investigated whether this transport model has more general relevance among plants. The screening for nitrate transporter activity was greatly helped by a novel assay using 15N-enriched nitrate uptake into Xenopus oocytes expressing the proteins. This assay enables many oocytes to be rapidly screened for nitrate transport activity. The functional activity of a barley nitrate transporter, HvNRT2.1, in oocytes required co-injection of a second mRNA. Although three very closely related nar2-like genes were cloned from barley, only one of these was able to give functional nitrate transport when co-injected into oocytes. The nitrate transport performed by this two-gene system was inhibited at more acidic external pH and by acidification of the cytoplasm. This specific requirement for two-gene products to give nitrate transport function has important implications for attempts to genetically manipulate this fundamental process in plants.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

Nitrate is the primary nitrogen (N) source for many plants and so the cellular uptake process is of fundamental importance for the N cycle. Two families of carrier proteins that can provide an entry route for nitrate into cells have been identified and these are called Nitrate Transporters (NRT) 1 and 2 (Crawford and Glass, 1998; Forde, 2000). All the members of both families probably belong to the Major Facilitator Superfamily, and are typically proteins of around 450–600 amino acid residues and 12 putative trans-membrane (TM) domains (Pao et al., 1998). The assignment of genes to each of these families is based entirely on conserved elements within the sequences, but in the model plant Arabidopsis thaliana there are seven NRT2 family members (Orsel et al., 2002) and over 50 members of the NRT1 family (Williams and Miller, 2001).

One of the best ways to demonstrate the function of a gene is to express the protein in a heterologous expression system such as yeast or Xenopus oocytes. In Chlamydomonas a nitrate-regulated cluster of genes involved in nitrate transport and assimilation has been identified (Quesada et al., 1993) and mutant strains of the alga that are defective in some aspect of transport and assimilation have been used to assign functions to these genes (Galván et al., 1996). A combination of mutant analysis (Quesada et al., 1994), and oocyte expression (Zhou et al., 2000a) showed that for one of the NRT2 family members in Chlamydomonas two gene products were necessary to obtain a functional high-affinity nitrate transport system. In addition to the NRT2-type transporter, a second gene, CrNar2 encoding a smaller protein of about 200 amino acid residues, was identified as being essential for nitrate function (Quesada et al., 1994; Zhou et al., 2000a). However, not all members of the NRT2 family require a second gene product for function, a related gene from the fungus Aspergillus nidulans, showed high-affinity nitrate transport when expressed in oocytes (Zhou et al., 2000b).

Attempts to genetically manipulate the expression of NRT2 transporters in plants have given confusing results. The overexpression of a transporter did not lead to increased uptake or greater tissue accumulation of nitrate (Fraisier et al., 2000), but gene knockout plants have been used to show that two members of the NRT2 family in A. thaliana, are essential for high-affinity nitrate transport (Filleur et al., 2001). In this paper we show that the Chlamydomonas nitrate transport model applies to some higher plant NRT2-type transporters as they also have a highly specific two-gene requirement for function and this may explain some of the results obtained from genetically manipulated plants.

Identification of a widely occurring plant NAR2-family

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

The CrNAR2 sequence was used to identify related genes in A. thaliana (accession numbers AJ310933 and AJ311926) and using this information we were able to identify a family of higher plant NAR2-type genes (see Figure 1). Within this collection of predicted proteins between amino acids 140 and 180 there was a conserved region of sequence that can be used to define the higher plant family of NAR2-type genes. This conserved amino acid sequence run begins with a lysine residue and was found in all the higher plant NAR2s, K(2)K(2)LCY(2)S(3)RxWR(3)D(4)DK (Figure 1). The sequence similarity was not found in the algal homologues with only the Y(7)RxWR motif retained within these more distantly related NAR2s. This family sequence analysis had found nine NAR2-type genes within barley expressed sequence tag (EST) collections. Full-length cDNAs were obtained from three ESTs from young barley roots and sequence alignment of these three genes identified them as being very closely related (see Supplementary Material). Hydropathy plots of sequence alignments using the TMAP software (Persson and Argos, 1996) suggest that the predicted proteins have one TM domain, but the very end of the N-terminus is lipophilic and may be located in the membrane (see Supplementary Material).

image

Figure 1. The identification of a family of NAR2 genes. Alignment of 16 Nar2 family members showing the conserved regions of the predicted proteins. The lower two sequences shown are for two algae, Chlamydomonas reinhardtii and Dunaliella salina.

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Assaying nitrate transport activity

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

To facilitate the more rapid screening of various different co-expression combinations we required a test for a functional nitrate uptake system that can easily be used with large numbers of oocytes. Nitrate transporter activity has been demonstrated in oocytes by HPLC detection of accumulated nitrate and electrophysiological methods, either nitrate-elicited depolarizations of the membrane potential or by measuring the nitrate-elicited current using the two-electrode voltage clamp technique (Tsay et al., 1993). However, these techniques are technically difficult to perform and are not suitable for screening large numbers of oocytes. To provide an easier screen for a functional nitrate uptake system we developed an assay using 15N-enriched nitrate (see Experimental procedures). Figure 2 shows the results for oocytes injected with various different combinations of mRNA including both single types and mixtures of two types of mRNA. Measurements of the 15N-nitrate enrichment in oocytes showed that only one co-injection combination was able to provide significant uptake when compared with water-injected oocytes (Figure 2), HvNAR2.3 and HvNRT2.1. These results show that despite the close homology between the three HvNAR2 genes, only one was able to give functional nitrate uptake when oocytes were co-injected with mRNA encoding each of the three genes. Oocytes injected with one type of mRNA, encoding HvNRT2.1, did not show significant accumulations of 15N-nitrate when compared with water-injected controls (Figure 2).

image

Figure 2. Uptake of 15N nitrate into oocytes injected with water or mRNA mixtures as indicated. (a) Oocytes were incubated for 12 h. (b) Oocytes were incubated for 6 h (unshaded) or 12 h (shaded) as indicated. The delta-15N values are shown as the mean ± SD for six oocytes and uptake was measured from 500 μm nitrate modified Barth's saline.

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The two-gene interaction may be highly specific

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

Only oocytes co-injected with mRNA encoding HvNRT2.1 and HvNAR2.3 were able to show nitrate- and nitrite-elicited changes in the membrane potential (Figure 3). Oocytes injected with a single type of mRNA or other mixtures of mRNA failed to show any changes in the resting membrane potential when treated with nitrate or nitrite. Treatment of oocytes with 500 μm concentrations of nitrite elicited membrane potential changes from −17 mV to −4 mV, a depolarization of 13 mV (Figure 3). These nitrite-elicited changes in membrane potential were reversible; the membrane potential was restored when the nitrite was removed (Figure 3). Very similar results were obtained when oocytes were treated with nitrate (data not shown). Oocytes injected with water or mRNA encoding just HvNRT2.1, and co-injected with HvNAR2.1 and HvNAR2.2 showed no change in the membrane potential when treated with 500 μm nitrate or nitrite (data not shown). In addition we also screened oocytes co-injected with mRNA from a closely related wheat gene TaNRT2.3 (accession number AY053452) and the three barley NAR2 genes. These oocytes also failed to show any nitrate transport activity (data not shown).

image

Figure 3. Nitrite-elicited changes in membrane potential for oocytes injected with mRNA from HvNRT2.1. and HvNAR2.3. Control oocytes injected with water showed no response (data not shown) when treated with the same concentration of NaNO2 (500 μm). The oocytes had been injected with mRNA 4 days before this recording was obtained. The oocyte MBS was at pH 7.4, and the nitrite was prepared <4 h before the measurement. Similar electrical responses were also obtained with nitrate (data not shown). The top bar shows when additions (grey) and removals (white) of nitrite were made.

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Sensitivity of nitrate transport to pH changes

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

Figure 4 shows results for oocytes injected with mRNA encoding HvNRT2.1 and HvNAR2.3 during two-electrode voltage clamp analysis of the transport activity. These measurements showed that the nitrate-elicited currents could be fitted by Michaelis–Menten saturation kinetics (see Figure 4a) that then enabled the calculation of Km values for nitrate. The Km values for nitrate were 30 μm at −160 mV and these values were voltage-sensitive, increasing to 200 μm at −40 mV (Figure 4b). Figure 5(a) shows the pH sensitivity of the currents elicited by treatment with 200 μm nitrate. These nitrate-elicited currents became larger with increasing membrane voltage and as the pH changed from 8 to 6.5. At more acidic external pH values of <6.5 the current decreased, until at pH 5.5 no nitrate-elicited current could be measured even at −120 mV (see Figure 5a). The inhibition of the nitrate-elicited current measured at more acidic pH was entirely reversible when the oocytes were returned to more alkaline pH (data not shown). Acidification of cytosolic pH is known to occur when the external pH is changed from 7.6 to 6 (Miller et al., 1994), so oocytes co-injected with mRNA for HvNAR2.3 and HvNRT2.1 were also given other treatments that are known to acidify cytosolic pH to determine the effect on nitrate transport. The largest nitrate-elicited currents were obtained at pH 6.5 (Figure 5a), however when co-injected oocytes were treated with 3 mm butyrate at pH 6.5, both the 15N-nitrate uptake (see Figure 5b) and the nitrate-elicited current were abolished (not shown). Furthermore, the treatment of mRNA-injected oocytes with 10 mm ammonium at pH 6.5 also inhibited 15N-nitrate uptake (Figure 5b).

image

Figure 4. Current–voltage curve for nitrate concentrations for oocytes co-injected with mRNAs for HvNRT2.1 and HvNAR2.3. (a) Family of IV curves for the nitrate transport fitted with Michaelis–Menten kinetics with •: −160 mV, ∘: −140 mV, bsl00000: −120 mV, bsl00072: −100 mV, bsl00083: −80 mV and bsl00001: −40 mV. (b) Voltage-dependence of Km for nitrate (see Zhou et al., 1998 for calculation).

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image

Figure 5. Effects of changes in pH on nitrate transport activity in oocytes co-injected with mRNAs for HvNRT2.1 and HvNAR2.3. (a) Changes in the external pH on the nitrate-elicited (200 μm NaNO3) currents for oocytes co-injected with mRNAs. The data points are shown for the currents measured at a range of different membrane voltages (•: −120 mV, ∘: −110 mV, bsl00072: −100 mV, bsl00083: −90 mV, bsl00001: −80 mV, bsl00000: −70 mV, bsl00046: −50 mV). (b) 15N-nitrate uptake into oocytes was decreased at pH 5.5 and by treatment with 3 mm butyrate and 10 mm ammonium at pH 6.5.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

The finding that higher plants can have two-component transport systems has important implications for genetic studies and attempts to genetically manipulate membrane transport processes. The absolute requirement for two gene products for a functional transport system means that endeavours to engineer plants with increased nitrate uptake may need to identify and target for both components of the nitrate transport system. For example, this result can explain why the overexpression of NpNRT2.1 in tobacco plants failed to yield any increase in nitrate uptake (Fraisier et al., 2000). In these experiments two strong constitutive promoters drove expression of this gene and although the message was clearly present, there was no significant increase in nitrate uptake when compared with wild-type plant. The authors interpreted this result as being evidence for post-translational regulation of uptake, but it may also be that increased expression of a specific partner NAR2-type gene was necessary for function. Genetic manipulations of all of the component parts of the transport system are perhaps essential to achieve increased uptake. This result provides good evidence for the utility of the Chlamydomonas model for higher plants, as first the genetic studies and now the subsequent heterologous expression in oocytes have demonstrated that some of the NRT2-type nitrate transporters require two gene products for functional activity.

A highly specific interaction between HvNRT2.1 and HvNAR2.3 is suggested by the fact that the closely related genes HvNAR2.1 and HvNAR2.2 are unable to functionally complement HvNRT2.1. Furthermore, none of the barley NAR2 mRNAs was able to complement function of TaNRT2.3, although there are probably a large family of NRT2-type genes in wheat. More work is necessary to determine how and where this interaction between the two gene products could be occurring; it might be between the RNAs, the protein and RNA, or the two proteins. A possible explanation for the lack of complementation by HvNAR2.1 and 2.2 mRNAs is that these two proteins, in contrast to HvNAR2.3, are incorrectly processed and targeted in oocytes. We cannot exclude this possibility and proof could be provided using a labelled antibody to show that all three of the HvNAR2 genes are processed and targeted in the same way. There is a precedent for this effect when a single site mutation was shown to alter membrane targeting of a glucose carrier protein in oocytes (Lostao et al., 1995).

Although there are examples of a second protein modifying the transport properties of another protein in mammals, for example phopholamban interaction with ATPases (Crambert et al., 2002) or syntaxin and channels (Bezprozvanny et al., 1995), the barley HvNRT2.2/NAR2.3 encoded carrier system is different because it has an absolute requirement for both gene products for function. No nitrate transport activity was measured in oocytes injected with a single species of mRNA. There are mammalian examples of membrane transport systems that require two proteins for function, specifically amino acid transporters (Palacin, 1994; Sato et al., 1999). However, these transporters do not require both gene products for their transport function, and in this respect they, like many channel proteins, can function in multimeric units that have different properties from monomeric units (Hanlon and Wallace, 2002). Accessory proteins can also be involved in linking to the cytoskeleton and for plants the auxin transporters are an example (Muday and Delong, 2001). The targeting and assembly of membrane protein complexes can require a second protein, for example some membrane receptors can require smaller accessory proteins for function. One such case is RIC-3; it also has two putative TM domains like NAR2 and is required for the production of functional acetylcholine receptors in Caenorhabditis elegans (Halevi et al., 2002). However, unlike NAR2, RIC-3 is not essential for the functional activity of the receptors as they can function without the much smaller protein.

The family of NRT2 transporters has be subdivided into different groups depending on the length of the C-terminus and central cytoplasmic loop using the predicted amino acid sequences and TM domains (Forde, 2000). In contrast to NRT2s from bacteria and fungi, the higher plant family members are characterized by having long C-termini. In fungi, the long C-terminus seems to be absent, but a longer central loop in the centre of the 12 putative TM-spanning domains in present. The oocyte expression of nrtA (formerly known as crnA; Unkles et al., 1991) has shown that this protein can function as a nitrate transporter by injection of a single type of mRNA (Zhou et al., 2000b). The long C-termini present in HvNRT2.1 and many of the higher plant family members may confer the requirement for a second, NAR2-type gene product. Like many of the higher plant NRT2 genes, the Chlamydomonas gene CrNRT2.1 also has a relatively long C-terminus.

The nitrate Km values for this nitrate transport system do change significantly with membrane voltage, with mean values of 30 μm at −160 mV and 70 μm at −60 mV. These values are remarkably close to measurements of the Km (30–80 μm) that were obtained for high-affinity range uptake of 13N-nitrate in nitrate-induced barley roots (Siddiqi et al.. 1990). Both HvNRT2.1 (Trueman et al., 1996; Vidmar et al., 2000) and HvNAR2.3 (G.-H. Yang, Y.P. Tong, Z.S. Li, The National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Beijing, China, unpublished data) are induced by nitrate supply and are strongly expressed in the roots. At the less physiologically relevant membrane potential of −40 mV, plant cells typically have membrane potentials of around −120 mV, the mean Km for nitrate increases to 200 μm.

The pH dependence of the HvNRT2.1 and HvNAR2.3 nitrate transport system is different from other nitrate transporters that have been functionally characterized (e.g. Tsay et al., 1993; Zhou et al., 2000b). This system is inhibited at more acidic external pH values (Figure 5) when the electrochemical gradient of protons, the driving force for a H+-cotransport mechanism, has actually increased. However, one important consequence of treating oocytes with more acidic solutions is that cytosolic acidification occurs (Bröer et al., 1998; Miller et al., 1994), a process that decreases the driving force for uptake. Consistent with this idea, the uptake of nitrate was also inhibited by treatment with butyrate and ammonium at pH 6.5 (Figure 5b), treatments that acidify the cytosolic pH of an oocyte (e.g. Bröer et al., 1998; Burckhardt and Frömter, 1992). Microelectrode measurements of pH have reported an acidification of the cytosol from 7.4 to 7.25 when the external pH was changed from 7.6 to 6 (Miller et al., 1994) and in oocytes co-injected with HvNRT2 and HvNAR2.3 mRNA and given these treatments (data not shown). However, at pH 6 and 5.5 (Figure 5a) the inhibition of the nitrate-elicited current was independent of membrane voltage; a result that cannot be explained by changes in the driving force for nitrate because an increase in membrane voltage was not able to compensate for the decreased pH gradient across the plasma membrane. Another explanation might be the protein–protein interaction that is necessary for nitrate transport being sensitive to pH, with the putative NAR2:NRT2 complex dissociating at higher H+ concentrations and so no current was detected at pH 5.5. These nitrate uptake measurements showing inhibition by treatment with butyrate and ammonium at pH 6.5 (Figure 5b) support the idea that there might be an interaction between the two proteins that is dependent on cytosolic pH. This result may have ecological and physiological significance, for example in acidic and anaerobic soils – the main form of N available to plant roots changes from nitrate to ammonium (Kronzucker et al., 1997). Under these conditions if cytoplasmic pH acidifies then this process will inhibit nitrate uptake by this type of two-component nitrate transport system.

The pH dependence of nitrate uptake by plant roots does not show inhibition at pH 6 like that found for the NAR2:NRT2 system in oocytes. High-affinity nitrate uptake by barley seedlings was maximal between external pH 6 and 7 over the range pH 3–9 (Aslam et al., 1995). However, in these experiments net uptake was actually assayed by depletion from the nutrient solution and these measurements sum changes in both influx and efflux although short duration (1 min) uptake intervals were used. Measurements of high-affinity 13N-nitrate influx in nitrate-induced barley roots showed maximal influx at pH 6 that was 20% lower at pH 4.5, but influx was only measured at three different external pH values (Glass et al., 1990). In other plant types, electrophysiological measurements of nitrate-elicited changes in membrane potential have shown the largest responses at acidic external pH (McClure et al., 1990; Ullrich and Novacky, 1981). These electrical responses are likely to be a direct measure of nitrate influx (reviewed by Miller et al., 2001). However, all in planta assays of nitrate transport are complicated by the fact that they are likely to be the combined transport activity of several different gene products.

In the NCBI dbEST database, there are in total 42 ESTs of HvNAR2 genes from the root and leaf. Of these, 10 ESTs are for HvNAR2.1, three for HvNAR2.2 and 26 for HvNAR2.3. These data indicate that at the mRNA level, HvNAR2.3 is likely to be the most abundant of these three NAR2 genes in barley plants. Of these 39 ESTs for these three NAR2s, 17 are from a nitrogen-starved root cDNA library; suggesting that HvNAR2s are upregulated by nitrogen starvation. Northern analysis of HvNAR2.3 indicated this gene most abundantly expressed in roots and was upregulated by nitrate and N-starvation, but down-regulated by ammonia and glutamine (G.-H. Yang, et al., unpublished data). Searching bacterial and fungal sequence databases failed to identify any related genes, we therefore believe that all green plants can have this second gene requirement for functional nitrate transport as it is found within green algae and mosses (e.g. Physcomitrella, data not shown). This evolutionary step coincides with the development of photosynthesis and may provide a link between nitrate reduction in the cytoplasm and nitrite reduction in the chloroplast. During evolution as plants developed chloroplasts it became important to match the uptake of nitrate to the supply of available reductant. The lack of a close link between nitrate reduction in the cytoplasm and nitrite reduction in the chloroplast may lead to toxic accumulations of nitrite. Nitrite is the substrate for nitrate reductase generation of nitric oxide (Desikan et al., 2002), a signal molecule that is being increasingly shown to be important in plants. Tight regulation of the activity of these two enzymes and the link between nitrate entry to the cytoplasm and nitrite transfer across the chloroplast envelope may require a two-component nitrate/nitrite transport system that is rapidly inactivated by changes in cytosolic pH.

Two-component signal transduction systems are employed by eubacteria, archaebacteria and cell-wall-containing eukaryotes including fungi and plants (Lohrmann and Harter, 2002). These systems involve reversible phosphorylation between a sensor kinase and a response regulator, to mediate the adaptation to changing environmental conditions. These sensors are usually integral membrane proteins that respond to a particular chemical or physical signal by altering the phosphorylation state of a partner regulatory protein. The regulatory protein may be a transcription factor that has its conformation or affinity for promoter sequences changed by phosphorylation (Stock et al., 1995). There is no evidence for this type of interaction in the barley NRT2/NAR2 system, although putative phosphorylation sites have been identified on HvNRT2.1 (Forde, 2000). The expression of NAR2-type genes had been previously reported as being induced by wounding (Titarenko et al., 1997) and pathogens (Marois et al., 2002). These are conditions that are not obviously linked to changes in nitrogen nutrition and so this family of genes may have other functions that have yet to be identified.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

Cloning and sequencing of HvNAR2 cDNAs

The protein sequence of CrNAR2, the component of high-affinity nitrate transport systems of Chlamydomonas reinhardtii (Quesada et al., 1994), was used to search for homologues in higher plants. Nine barley EST clones of roots of barley (Hordeum vulgare cv. Optic) were identified in GenBank dbEST database. Three EST clones, EBroO1_SQ003-M04, EBroO2_SQ001-E09 and EBroO8_SQ002-J14, were obtained from the Scottish Crop Research Institute (SCRI), UK and sequenced by ABI PRISM® BigDyeTM terminators 2.0 DNA sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequences analysis showed that each of these three clones contain a full-length cDNA, and each was named with the information submitted to GenBank under the accession numbers AY253448 (HvNAR2.1), AY253449 (HvNAR2.2) and AY253450 (HvNAR2.3).

Synthesis of mRNA from full-length cDNAs of HvNRT2.1 and HvNAR2

The full-length HvNRT2.1 cDNA (formerly named BCH1, accession no. U34198) was cloned in pBluescript phagemids (Trueman et al., 1996). HvNRT2.1 cDNA was excised with NotI and blunted with Taq DNA polymerase (Invitrogen, Paisley, UK). The HvNRT2.1 cDNA was then subcloned into EcoRV site of the oocyte expression vector pT7TS containing the 5′-UTR and 3-UTR of the Xenopusβ-globin gene (Cleaver et al., 1996) using a T/A cloning strategy. Briefly, NotI excised HvNRT2.1 insert was blunted and tailed at 72°C for 30 min in a PCR mixture supplement with 0.2 mmol of dGTP, dCTP and dATP. The pT7TS vector was digested with EcoRV and treated with CIAP, and tailed at 72°C for 30 min in a PCR mixture containing only 1 mmol of dTTP. Finally the HvNRT2.1 cDNA and the pT7TS vector were ligated together. The plasmid with HvNRT2.1 in the sense orientation was named pT7TSHvNRT2.1 and used for mRNA synthesis.

The full-length HvNAR2.1, HvNAR2.2 and HvNAR2.3 cDNAs were subcloned into pGEM® T Easy vector (Promega, Southampton, UK), and then digested with EcoRI followed by subcloning in EcoRI site of oocyte expression vector pXE1, a modified expression vector of pBSTA that also includes the 5′-UTR and 3′-UTR of the Xenopus β-globin gene (Shih et al., 1998). The plasmids with the HvNAR2 cDNAs in the sense orientation were named pXE1HvNAR2.1, pXE1HvNAR2.2 and pXE1HvNAR2.3, and these were used for mRNA synthesis. For in vitro synthesis of mRNA, pT7TSHvNRT2.1 was linearized by digestion with PstI, pXE1HvNAR2.1 by NotI, pXE1HvNAR2.2 and pXE1HvNAR2.3 by SacII. Capped full-length mRNAs were synthesized using a T7 RNA transcription kit (mMESSAGE mMACHINE, Ambion, Austin, TX, USA).

Oocyte preparation, injection, 15NOinline image uptake and electrophysiology

Oocytes were prepared, stored in nitrate-free modified Barth's saline (MBS) and injected as described previously (Zhou et al., 1998). Healthy oocytes at stage V and VI were used for injection with 50 nl di-ethyl pyrocarbonate-treated water, or 50 nl HvNRT2.1 mRNA (1 ng nl−1), or a mixture of 25 nl HvNRT2.1 mRNA (2 ng nl−1) and 25 nl (2 ng nl−1) HvNAR2 mRNA. The oocytes were used in 15NOinline image uptake and electrophysiological analysis 3–7 days after injection with mRNA or water. The uptake of 15NOinline image by oocytes was measured in MBS at pH 7.4. For experiments at more acidic external pH, the buffer 2-[N-morpholino]ethanesulphonic acid was added at the same concentration in place of N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]. For measuring 15N enrichment, eight to 12 oocytes were incubated in saline containing 0.2–0.5 mm95 atom%15NOinline image for 3–12 h at 18°C. The oocytes were then thoroughly washed three times with ice-cooled nitrate-free saline and dried at 60°C. The 15N/14N ratio of single dried oocytes was measured using an Isotope Ratio Mass Spectrometer (model Integra CN; PDZ Europa, Crewe, UK). The delta-15N was calculated as the ratio of the sample in excess of air divided by the standard atmospheric 15N/14N ratio atom % (Mariotti, 1984). Samples with positive delta (δ)15N values are enriched in 15N content with respect to the atmospheric standard.

The nitrate-elicited currents were assayed in oocytes using the two-electrode voltage-clamp method and using the pCLAMP software (v8; Axon Instruments, Foster City, CA, USA). All electrophysiological measurements were made in MBS at pH 7.4 unless stated otherwise. Experiments were performed in a continuously perfusing system with saline at a rate of 1 ml min−1 throughout the recording. Only oocytes that had resting potentials more negative than −30 mV were used for the voltage-clamp experiments.

Steady-state current was measured as described previously (Zhou et al., 2000b) with some modification. The oocyte membrane potential was clamped at −50 mV, from this value the membrane was pulsed to a range of different test potentials for 120 msec from +40 to −160 mV with either 10 or 20 mV incremental steps, followed by a 1-sec pulse interval at the holding potential. The membrane currents reached steady-state within 50 msec of clamping, and the mean values were calculated from the last 20 msec at each clamping voltage. The anion-elicited currents were used to obtain current–voltage difference curves (IV) and calculate the kinetics of transport as described previously (Zhou et al., 1998). In all experiments, the oocytes were allowed to adjust for at least 5 min after changing the external pH before any treatments were applied.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

The authors thank the Royal Society of the UK for a Study Visit Award that enabled Dr Yiping Tong to visit Rothamsted and for support from the China State Major Basic Research Project (2004CB117202 and G1998010205), and the Natural Science Foundation of China (30390083) and the European Union Research Training Network (HPRN-CT-2002-00247). Rothamsted Research is grant-aided by the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. The authors also wish to thank Brian Forde (University of Lancaster) for providing the HvNRT2.1 cDNA, Emilio Fernandez (University of Cordoba) for the sequence of CrNAR2, David Caldwell and Robbie Waugh (SCRI) for the barley ESTs and Maureen Birdsey (Rothamsted) for help with the 15N analysis.

The nucleotide sequences reported in this paper have been submitted to the GenBankTM Data Bank with accession numbers AY253448 (HvNAR2.1), AY253449 (HvNAR2.2), AY253450 (HvNAR2.3) and AY053452 (TaNRT2.3).

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Identification of a widely occurring plant NAR2-family
  6. Assaying nitrate transport activity
  7. The two-gene interaction may be highly specific
  8. Sensitivity of nitrate transport to pH changes
  9. Discussion
  10. Experimental procedures
  11. Acknowledgements
  12. Supplementary Material
  13. References
  14. Supporting Information

Fig. S1. Comparison of deduced amino acid sequences of HvNAR2.1, HvNAR2.2 and HvNAR2.3 using ClustalW (http://www.ch.embnet.org/software/ClustalW.html). The TM domains predicted by TMAP are underlined and the N-terminus is predicted to be on the cytoplasmic side of the membrane.

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TPJ_2310_sm_FigS1.doc21KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.