Journal of Experimental Botany, Vol. 51, No. 347, pp. 1017-1026,
June 2000
© 2000 Oxford University Press
Two cDNAs representing alleles of the nitrate reductase gene of potato (Solanum tuberosum L. cv. Desirée): sequence analysis, genomic organization and expression1
2 Plant Sciences Laboratory, Sir Harold Mitchell Building, Division of Environmental and Evolutionary Biology, School of Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK
3 Department of Cell and Environmental Physiology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
4 Max Planck Institut für Züchtungsforschung, Carl von Linne Weg 10, D-50829 Köln, Germany
Received 10 September 1999; Accepted 28 January 2000
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Abstract |
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Two, different, full-length cDNAs, StNR2 and StNR3, of 3049 and 3066 nucleotides, respectively, were isolated from a Solanum tuberosum L. cv. Desirée leaf cDNA library by an RT-PCR approach. Conceptual translation of the longest open reading frame of each cDNA showed that they could encode a protein of 911 amino acids in each case with an Mr of 102.6 and 102.5 kDa, respectively, and with 99.7% identity with each other. The cDNAs have a high degree of sequence similarity with all higher plant nitrate reductases (NRs) and possess structural characteristics expected of NADH-NR proteins, consistent with enzyme activity data. Southern analysis of genomic DNA suggested the presence of a single NR gene in the potato genome whilst studies using the mapping population F1840, and the full-length StNR2 cDNA as hybridization probe, identified a single NR locus within the potato genome that is located on chromosome XI. The two cDNAs identified here are probably derived from two transcribing alleles of this single gene. Distribution of total NR transcript and of NADH-NR activity, in different organs of compost-grown plants, depended on the level of nitrate supplied: at low nitrate level transcript and activity were detected only in leaf and stem tissue whilst at high nitrate level they could also be detected in root and stolon. An RT-PCR approach was used to differentiate between the transcripts derived from the two identified alleles and to show that the pattern of transcription of the two identified alleles of the potato nia gene, in the organs studied, is the same.
Key words: Gene expression, genomic organization, nitrate reductase, potato, Solanum tuberosum L. cv. Desirée.
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Introduction |
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After water supply, nitrogen, as nitrate, is the most important determinant of yield and maturity in the potato crop. Efficient use of nitrate fertilizer requires both that the crop incorporates sufficient N to attain its potential yield under prevailing conditions and also that excess uptake, leading to the storage of large quantities of nitrogen in stems and leaves, is avoided. Despite the potato crop being highly responsive to nitrogen fertilizer, its nitrogen use efficiency is low (Rourke, 1985
; Porter and Sisson, 1991).
Many physiological studies of nitrate assimilation in potato have been carried out and these have concentrated largely on measurements of the distribution of nitrate and of nitrate reductase (NR), the first enzyme of the nitrate assimilation pathway. These studies have shown that potato reduces nitrate primarily in the stem and the leaf, but not the root (Kapoor and Li, 1982; Davies and Ross, 1985) and that the leaf is the major site of nitrate reductase activity. High nitrogen inputs in the field situation induce higher activities of NR in young fully expanded leaves (Davies et al., 1987, 1988). In nitrogen-replete plants substantial amounts of nitrate accumulate in stems and often in greater amounts in the leaves. Stems can therefore represent a large storage pool for nitrate in the canopy. Early in the growing season the percentage of nitrate-N in certain leaf positions can rise to up to 30% of total N present, implying that the potential for nitrate assimilation is limiting at these developmental stages (Millard and Marshall, 1986). Approaches to the optimal use of nitrate-N at different growth stages include the development of mathematical modelling strategies to predict nitrogen requirements and to minimize leaching of surplus N (Greenwood et al., 1985), as well as the development of techniques for monitoring petiole sap nitrate levels (Williams and Maier, 1990; Waterer, 1997).
Other approaches would include the manipulation of nitrogen acquisition and assimilation using transgenic biology. However, by comparison with more traditional agronomic approaches, there has been little research on basic mechanisms of nitrogen capture and assimilation in potato using biochemical and molecular approaches. This contrasts sharply with the wealth of information available from a wide range of other crop plants (reviewed in Rouzé and Caboche, 1992). A particular target for study has been NR, which has been shown to be the subject of multifactorial control at the level of both gene expression and post-translational modification (reviewed in Kaiser and Huber, 1994; Kaiser et al., 1999) whilst its substrate, nitrate, or nitrate-derived signals, may also play a role in the regulation of carbon metabolism and the adjustment of root growth and architecture to the physiological status of the plant (Zhang and Forde, 1998; Zhang et al., 1999; Stitt, 1999). Other molecular and genetic studies have revealed information about the number and organization of the nia genes that encode NR (reviewed in Rouzé and Caboche, 1992). One such study on the identification of NR mutants in potato has been described (Chwilkowska et al., 1995).
In the present paper the lack of detailed molecular and biochemical information on nitrate assimilation in potato is addressed by describing the cloning and genomic organization of the potato nia gene(s) and the expression patterns in different potato tissues and under different nitrate levels.
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Materials and methods |
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Plant material
Plants of Solanum tuberosum cv. Desirée that had been grown in Levington's M2 compost (201 ppm total N: 124 ppm (2 mM)
and 77 ppm (4.5 mM) 
) in pots in a glasshouse for 4–6 weeks were used for the studies reported here. For the isolation of a partial NR cDNA, and the determination of reducing substrate specificity of the NR enzyme, plants were treated with modified half-strength Hoagland nutrient solution (Hoagland and Arnon, 1938) containing 25 mM nitrate as sole nitrogen source 18 h prior to tissue sampling. For Northern analysis, plants were treated with modified half-strength Hoagland nutrient solution either lacking a nitrogen source, or containing 1 mM, 5 mM or 25 mM nitrate as the sole nitrogen source, once a day for a period of 7 d prior to harvest. Cylinders (1 cm diameter) of tissue were removed longitudinally from the developing tubers and were cut into 1 mm thick discs. Potato tuber discs were incubated in modified half-strength Hoagland nutrient solution either lacking a nitrogen source or containing 25 mM or 50 mM nitrate as the sole nitrogen source for 18 h at room temperature with constant agitation before sampling.
DNA and RNA isolation
Total RNA, for use as a template for cDNA synthesis, was isolated from the leaf tissue of nitrate-treated plants as described previously (Grierson et al., 1985). Poly (A)+ RNA was isolated from this using an Oligotex dT kit (Qiagen, UK) following manufacturer's instructions. For Northern analysis the RNA miniprep extraction procedure of Verwoerd et al. (Verwoerd et al., 1989) was utilized. DNA was isolated from potato leaf tissue as described in Dellaporta et al. (Dellaporta et al., 1983).
Molecular cloning of potato nitrate reductase cDNAs
A potato partial NR cDNA was first amplified by RT-PCR using the method of Frohman et al. (Frohman et al., 1988). For reverse transcription an 18mer oligo-dT oligonucleotide was used as the 3' primer. cDNA was synthesized from 1 µg of poly (A) + RNA with RNase H minus M-MLV reverse transcriptase (Promega, UK) using the procedure detailed in the 1st-Strand cDNA synthesis System kit (Clontech, UK). For PCR amplification an oligonucleotide corresponding to the conserved sequence GGAAGAATGGTGAAATGG present at positions 1027–1044 of the coding sequences of the tobacco nia1 and nia2 genes (Vaucheret et al., 1989) and to positions 904–920 of the coding sequence of the tomato nia gene (Daniel-Vedele et al., 1989), was selected as a 5' gene-specific primer. An 18mer oligo-dT oligonucleotide was used as the 3' primer. Amplification, performed using an annealing temperature of 60 °C, yielded an expected 2 kb cDNA product (based on the positions of the oligonucleotide sequence in the tobacco and tomato NR gene sequences) which was purified using a Qia-Quick column (Qiagen, UK). This 2 kb partial NR cDNA was then used to isolate a full-length NR cDNA from a Solanum tuberosum cv. Desirée leaf cDNA library constructed in the vector 
ZapII (Kossmann et al., 1992).
Approximately 200 000 phage were transferred to Hybond N+ membrane filters (Amersham, UK) according to manufacturer's instructions. Hybridization was carried out at 65 °C overnight, in 2xSSPE, 5xDenhardt's solution, 0.5% (w/v) SDS, and 500 µl of denatured salmon sperm DNA (10 mg ml-1), with the 2 kb RT-PCR product that had been radiolabelled using a Megaprime kit (Amersham UK). The final wash was at 0.1xSSPE/0.1% SDS and filters were exposed to Kodak XAR-5 film at -70 °C. Seven positive plaques were isolated after a second round of screening. After in vivo excision (Short et al., 1988) and sizing of the cDNA inserts, plasmid pStNR2, that was estimated to contain a full length NR cDNA, StNR2, was studied further.
In order to isolate other full-length NR cDNA species the library was rescreened, as described above, using a 400 bp Bam HI fragment of StNR2 encoding the 5' end of the cDNA. This resulted in the isolation of 15 further cDNAs of c. 3 kb. Partial sequencing showed that two of these, StNR3 and StNR4, differed from StNR2. Although both represented the same cDNA species StNR4 lacked 150 bp at the 5' end and StNR3, which was shown to be full length, was studied further.
Southern and Northern analysis
For Southern analysis, genomic DNA (10 µg) was digested according to the manufacturer's instructions and separated on a 1xTBE, 0.8% (w/v) agarose gel and transferred to Hybond N+ membrane (Amersham, UK) as described by Southern (Southern, 1975). The filter was then hybridized overnight, using the conditions described above for library screening, against a radiolabelled probe prepared from 25 ng of StNR2 cDNA using a Megaprime kit (Amersham, UK).
Total RNA (10 µg) from each sample was separated on a 1xMOPS/1% agarose/6% formaldehyde denaturing gel and transferred to Hybond Nmembrane (Amersham, UK) according to the manufacturer's instructions. A radiolabelled probe was made as described for Southern analysis and hybridization was carried out overnight at 42 °C in 2xSSPE, 5xDenhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide, and 500 µl of denatured salmon sperm DNA (1 mg ml-1). Filters were then finally washed in 0.1xSSPE/0.1% SDS and exposed to Kodak XAR-5 film at -70 °C.
Mapping the location of the potato nitrate reductase gene
Mapping studies were carried out using the full-length StNR2 cDNA as a hybridization probe on population F1840 as described in Gebhardt et al. (Gebhardt et al., 1991).
RT-PCR analysis of StNR2 and StNR3 expression
5 µg of DNAase-treated RNA was reverse transcribed as described in Simpson et al. (Simpson et al., 1992) using a common forward primer for both cDNAs (CATAGCCTCTCAACGTG). PCR amplification was then carried out using either an StNR2-specific reverse primer (TTATTCCGGGATTCATC) or an StNR3-specific reverse primer (TTATTCCGGGATTCAT
A) (sequence difference underlined) in conjunction with the common forward primer, using 30 cycles of 94 °C for 1 min; 62 °C for 2 min; and 72 °C for 1 min. The reaction was then extended for another 10 min at 72 °C. Taq polymerase was added after the initial denaturation step on the first cycle (‘hot start’) to increase primer specificity. Control amplifications, using either pStNR2 or pStNR3, demonstrated the specificity of each reverse primer.
DNA sequencing
Plasmid DNA was isolated using Qiagen Midiprep columns (Qiagen, UK). Double-stranded sequencing was carried out by a combination of Exonuclease III deletions (Henikoff, 1984) and synthetic oligonucleotide primers by the method of Sanger et al. (Sanger et al., 1977) using Sequenase V2.0 (USB, USA).
Sequence analysis
Sequence analyses were carried out using the University of Wisconsin Genetics Computer Group (UWGCG) package (Devereux et al., 1984). Multiple protein sequence alignments were carried out using the Clustal V program (Higgins and Sharp, 1988) and then analysed by protein parsimony using the PHYLIP package (Felsenstein, 1993).
Tissue extraction and nitrate reductase assay
Leaf tissue was extracted with a buffer (10 ml g-1 fresh weight) containing 100 mM TRIS (pH 8.5), 1 mM EDTA, 5 mM DTT, 0.1 mM FAD, 0.1 mg ml-1 PVP, and 10 µg ml-1 leupeptin. The crude homogenate was centrifuged at 20 000 g for 20 min and supernatant used as the source of enzyme. NR assays were performed essentially as described by Wray and Fido (1990). The reaction mix contained 0.5 ml 100 mM phosphate buffer (pH 7.5), 0.1 ml 100 mM potassium nitrate, 0.1 ml 5 mM NADH or NADPH, and 0.1 ml distilled water and the reaction was started by the addition of 0.2 ml of enzyme extract. After incubation at 25 °C for 10 min the reaction was stopped by the addition of 0.2 ml of a 1 : 1 (v/v) mixture of 1 M zinc acetate and 0.3 mM phenazine methosulphate (Scholl et al., 1974). After 10 min 1 ml of 1% (w/v) sulphanilamide (in 3 M HCl) and 1 ml of 0.02% (w/v) NED were added. The optical density at 540 nm was read after 15 min. Controls lacked enzyme extract. When NADPH was used as reducing substrate, NR activity was also measured in the presence of 50 µg lactate dehydrogenase and 5 mM pyruvic acid to competitively eliminate NADH produced by the conversion of NADPH to NADH by phosphatase enzyme that might be present in the tissue extract (Dailey et al., 1982). Protein was determined by the method of Bradford (Bradford, 1976) using bovine serum albumen as the standard protein.
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Results and discussion |
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Cloning and sequence analysis of two cDNA clones, StNR2 and StNR3, encoding potato nitrate reductase
Sequencing of the two cDNA clones, StNR2 and StNR3, on both strands revealed them to be 3049 and 3066 nucleotides in length, respectively. In common with other NR cDNAs both StNR2 and StNR3 possess an untranslated leader sequence, with the first translation initiation codon occurring 87 bp and 104 bp downstream of the 5' end, respectively. This gives an open reading frame of 911 amino acids in both cases, starting with the sequence MAASV that is conserved throughout all higher plant nitrate reductases (Miyazaki et al., 1991
). In addition, both cDNAs possess a 3' untranslated region of 227 nucleotides with a putative polyadenylylation signal motif AATAA (Joshi, 1987) present 26 bp downstream of the translation stop codon.
Analysis of the deduced amino acid sequence of StNR2 and StNR3
Conceptual translation showed that the longest ORFs of StNR2 and StNR3 could encode proteins, StNR2 and StNR3, of 102.6 kDa and 102.5 kDa, respectively, that show a high degree of similarity with each other (99.7%) and with all forms of higher plant NR so far described. The greatest similarity was exhibited with the NR amino acid sequences from tomato (95% identity, 97% homology), petunia (87% identity, 94% homology), tobacco (89% identity, 93% homology) and spinach (68% identity, 79% homology) (Fig. 1). An alignment of all available plant/algal, and some fungal, full length NR protein sequences, and calculation of phylogenetic distances by protein parsimony using PHYLIP, demonstrated the existence of several families (data not shown) with the potato NR sequences, StNR2 and StNR3, being placed closest to the tomato NR (Daniel-Vedele et al., 1989) in a group also containing the other three available sequences from the Solanaceae, the NIA1 and NIA2 proteins from tobacco (Vaucheret et al., 1989) and the NIA protein from petunia (Salanoubat and Ha, 1993).
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The deduced proteins, StNR2 and StNR3, possess the three catalytic domains defined for NRs (Crawford et al., 1988
; Rouzé and Caboche, 1992) (Fig. 1
). The MoCo, haem and FAD domains are located at amino acid positions 84–479, 532–610 and 649–911 and are composed of 395, 78 and 262 amino acids, respectively. In common with other higher plant forms StNR2 and StNR3 possess an 83 amino acid N-terminal domain that may be involved in post-transcriptional regulation of NR by phosphorylation (Nussaume et al., 1995).
The MoCo domain of StNR2 and StNR3 contains the single cysteine (Cys-188) conserved in all eukaryotic NRs (Barber and Neame, 1990; Hoff et al., 1992; Rouzé and Caboche, 1992). This is found within the highly conserved region TLXCAGNRR (K/S)E(Q/M) which is represented as 185TLVCAGNRRKEQ196 in the deduced amino acid sequence of StNR2 and of StNR3 (Fig. 1). Both the StNR2 and StNR3 MoCo domain amino acid sequences differ from all other NR sequences (Rouzé and Caboche, 1992) by possessing an isoleucine instead of valine at position 241.
In the hinge region between the MoCo and haem domains, StNR2 and StNR3 possess a conserved serine residue (Ser 528) that has been shown to be present also in spinach (Ser 543) (Douglas et al., 1995) and Arabidopsis (Ser 534) (Su et al., 1996) NRs. As with other characterized NRs this residue is contained within the motif LK(K/R)(T/S)XS(T/S)PFM (Huber et al., 1996) which is represented as 523LKKSISTPFM532 in the case of the amino acid sequences encoded by StNR2 and StNR3 (Fig. 1). Post-translational control of NR activity is effected by phosphorylation at this conserved serine residue. Residues marked in bold are conserved between higher plant NRs and are believed to be involved in recognition of the phosphorylation site by kinases (Bachmann et al., 1996; Huber et al., 1996). Subsequent interaction of the phosphorylated protein with a member of the 13–2-2 protein family brings about inactivation of NR (Douglas et al., 1995).
In the haem domain region of the amino acid sequences of StNR2 and StNR3, the 11 residues conserved throughout members of the cytochrome b5 superfamily are present including the two histidine residues (His-571 and His-594) demonstrated to bind the haem Fe (Beck von Bodman et al., 1986; Meyer et al., 1991) (Fig. 1).
The FAD domain contains conserved residues that have been implicated in pyridine nucleotide binding and catalysis. Three-dimensional comparisons of the FAD-containing fragment of maize NR with other flavoprotein reductases (Lu et al., 1994), and site-directed mutagenesis (Shiraishi et al., 1998), suggest that NADPH-dependent NRs have a positively charged residue adjacent to the 2' phosphate binding site whilst NADH-specific forms possess a negatively charged residue at this position (Campbell, 1999). Amino acids adjacent to the conserved CG dipeptide motif (positions 883 and 884 in StNR2 and StNR3) (Fig. 1) are also thought to be involved in some manner in the determination of pyridine nucleotide preference. In NADH NRs, three proline residues next to the CG dipeptide are conserved as CGPPP (for example in tomato, petunia and tobacco NRs). In NAD(P)H forms such as those from barley (Miyazaki et al., 1991) and birch (Schöndorf and Hachtel, 1995) only two proline residues are present (CGPPA). Schöndorf and Hachtel have demonstrated that substitution of a proline for alanine in the birch NAD(P)H NR greatly increased preference for NADH, perhaps by affecting the structural topology of the dinucleotide binding pocket and making it less accessible to the 2' phosphate of NADPH (Schöndorf and Hachtel, 1995). In the case of potato NR the sequence data show that both StNR2 and StNR3 possess the motif 883CGPPP887 characteristic of NADH-specific NRs. A thiol that plays an important role in catalysis has been localized to the cysteine residue of the conserved CG dipeptide (Barber and Solomonson, 1986; Dwivedi et al., 1994). StNR2 and StNR3 also possess the conserved lysine residue corresponding to Lys-725 (Hackett et al., 1988), a conserved serine residue (Ser-742) (Yubishi et al., 1991) and a conserved histidine residue (His-870), all believed to be involved in NADH binding, and two conserved arginine residues (Arg-706 and Arg-868) that may also be involved in the catalytic activity of the FAD domain (Baijal and Sane, 1988).
Genomic organization of the nitrate reductase gene in potato
Southern analysis of genomic DNA from the tetraploid Solanum tuberosum cv. Desirée, a dihaploid line (PDH 1151) derived from Solanum tuberosum cv. Pentland Crown, a wild, non-tuberizing diploid Solanum brevidens and a diploid tomato (Lycopersicon esculentum cv. Money Maker) was carried out in order to estimate the copy number of NR genes in potato. DNA samples were digested with either EcoRI (data not shown) or XbaI and hybridized with either the full-length StNR2 cDNA, or a BamHI fragment encoding the 5' 400 bp of this cDNA. The hybridization patterns found suggest the presence of a single NR gene in the potato genome (Fig. 2).
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Mapping studies performed using the full-length StNR2 cDNA as a hybridization probe of mapping population F1840 (Gebhardt et al., 1991
) identified a single NR locus within the potato genome that is located on chromosome XI (Fig. 3). This suggests that the two distinct NR cDNAs are alleles of a single gene.
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A comparison between the two potato NR cDNA clones for the rate of synonymous and non-synonymous nucleotide substitution was determined using the NewDiverge programme. At the whole protein level the ratio for synonymous and non-synonymous nucleotide substitution was very high, Ks/Ka=26.31. The ratio for individual conserved domains such as MoCo (59.85) and FAD (7.66) were also high. The high synonymous and non-synonymous nucleotide substitution ratios suggest that the domains have been under stringent selection and that the two cDNA clones are probably derived from functional alleles of the single nitrate reductase gene.
Reducing substrate specificity of potato nitrate reductase
NR exists in three forms that are either NADH specific (EC 1.6.6.1), NAD(P)H bispecific (EC 1.6.6.3) or NADPH specific (EC 1.6.6.3). NR activity was measured in cell-free extracts of leaf tissue from potato plants to determine the reducing substrate specificity of the potato enzyme. An approximately 30-fold greater specific activity was observed with NADH as the reducing substrate compared to NADPH (Table 1). When lactate dehydrogenase and pyruvate were included in the assay to eliminate NADH that might be formed by phosphatase-mediated conversion of NADPH to NADH (Dailey et al., 1982), the level of NADPH activity was even lower. This suggests that potato possesses an NADH-specific NR, in common with most higher plants, rather than the NAD(P)H bispecific form found in a small minority. When NADH and NADPH are present together in the reaction there is a 10% reduction in activity suggesting some form of inhibition, as noted previously (Dailey et al., 1982). The presence of an NADH-specific form of NR in cell-free extracts is consistent with the deduced amino acid sequence data described above.
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Distribution of nitrate reductase transcript and nitrate reductase activity
A combination of Northern analysis and NR activity measurements using the optimized extraction and assay procedures described in the Materials and methods was used to study the distribution of NR transcript and activity in organs of the potato under different levels of nitrate (Fig. 4). For Northern analysis total RNA from leaf, root, stem, stolon, and tuber tissue of plants that had been grown in compost and watered with half-strength Hoagland nutrient solution, either lacking nitrate (0 mM) or containing either 1 mM, 5 mM or 25 mM nitrate, daily for a period of 7 d prior to sampling was blotted onto nitrocellulose after electrophoretic separation and probed with the full length potato NR cDNA, StNR2. In all cases, even in the absence of an exogenous nitrate treatment in the supplied solution, NR transcript was present in leaf and stem and transcript abundance increased with increasing nitrate level. In the case of root and stolon NR transcript was not detectable at 0 and 1 mM nitrate, but became apparent at 5 mM nitrate and increased further after treatment with 25 mM nitrate. NR transcript could not be detected in tuber tissue at any nitrate level.
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The distribution of NADH-NR enzyme activity was very similar to that of NR transcript, both with respect to tissue and nitrate treatment (Fig. 4a
). Activity was present in the leaf and stem even in the absence of an exogenous nitrate treatment and increased 8- and 2-fold, respectively, as nitrate concentration was increased. However, NR activity was detected in root and stolon tissue only at the 10 mM and 25 mM nitrate levels. NR activity could not be detected in tuber tissue at any nitrate level. These activity measurements are consistent with previous data from potato (Kapoor and Li, 1982; von Meltzer, 1987) which showed that enzyme activity was highest in leaves and young stems but very low in root and tuber. This pattern was seen at low exogenous nitrate levels, but when nitrate is abundant there is clearly a shift in the distribution of both transcript and activity to the root and stolon, increasing the range of organs potentially capable of performing nitrate reduction. Indeed, at the highest nitrate dosage the NR specific activity in root tissue was twice that observed for leaf and stem. Presumably at high nitrate levels the capacity for nitrate transport to leaf tissue is not high enough to prevent accumulation of nitrate in other organs.
Although no NR transcript or NR activity could be detected in whole tubers, irrespective of the level of nitrate presented to the plant, previous studies utilizing excised tuber slices (Palmer, 1981, 1982) have demonstrated that nitrate reductase activity can be induced in this tissue. In order to confirm these observations and also demonstrate that, like the whole plant situation, transcript abundance correlates with NR activity, NR transcript abundance and NR activity level were measured in tuber tissue discs incubated in half-strength Hoagland nutrient solution containing either 25 mM or 50 mM nitrate. The data (Fig. 4b) suggest that tubers have the capacity both to transcribe the NR gene and produce NR activity. Tuber tissue discs incubated in the absence of a nitrate source possess no detectable NR transcript or activity. Thus, in compost-grown whole plants, the tuber nitrate concentration may not be of a sufficiently high level to induce NR expression due perhaps to tissue-specific control mechanisms or threshold limits relating to transport, or else constraints are placed on the transcription of the NR gene in planta that are not exerted in tuber tissue discs.
Use of a full-length NR cDNA probe as described above, coupled with the great overall sequence similarity, did not allow this study to distinguish between StNR2 and StNR3 transcripts by Northern hybridization. The authors, therefore, resorted to a RT-PCR approach to determine whether the expression patterns of the two NR alleles represented by the cloned cDNAs were similar or not. Total RNA from leaf, root, stem, and stolon tissue from potato plants grown in the presence of 25 mM nitrate, and that were shown by Northern analysis to contain NR transcript (Fig. 4), was reverse transcribed. The cDNA product was used as a template for PCR using primers, derived from a knowledge of the nucleotide sequences, that are specific for either StNR2 or StNR3. Transcripts corresponding to both StNR2 and StNR3 are present in all tissues of whole plants (Fig. 5a) as well as in tuber slices incubated in the presence of nitrate (Fig. 5b). Control reactions using PCR amplification from plasmid DNA (Fig. 5c) show that these two cDNAs correspond to different unique transcripts since no cross-amplification with the gene-specific sets of primers used was apparent.
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These data are consistent with the idea that the two cDNA clones StNR2 and StNR3 are derived from two transcribing alleles of a single NR gene, nia.
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Acknowledgments |
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This work was funded wholly by the European Communities' BIOTECH Programme as part of the Project of Technological Priority 1993–1996. We thank Professor L Willmitzer
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Notes |
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1 The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers U76701 (S. tuberosum StNR2 cDNA) and U95317 (S. tuberosum StNR3 cDNA).
5 To whom correspondence should be addressed. Fax: +44 1334 463366. E-mail:jlw{at}st\|[hyphen]\|andrews.ac.uk
6 Present address: Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex TW11 0LY, UK.
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Abbreviations |
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NR, nitrate reductase..
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References |
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