Journal of Experimental Botany, Vol. 53, No. 371, pp. 1037-1045,
May 2002
© 2002 Oxford University Press
Original Papers |
Glasshouse behaviour of eight transgenic potato clones with a modified nitrate reductase expression under two fertilization regimes
1Station d'Amélioration de la Pomme de Terre et des Plantes à Bulbes, INRA, Domaine de Keraïber, 29260 Ploudaniel, France
2Unité de Nutrition Azotée des Plantes, INRA, Route de St Cyr, 78026 Versailles cedex, France
Received 13 June 2001; Accepted 26 December 2001
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Abstract |
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In this study, eight transformed Solanum tuberosum L. plants, affected in their nitrate assimilatory pathway by the introduction of a tobacco nitrate reductase gene (Nia2), were cultivated in glasshouse conditions at INRA Ploudaniel (West Brittany, France). Two irrigation regimes were compared and plants were sampled at four stages of vegetation. Yield, tuber dry matter content, total nitrogen content, nitrate content in the whole plant, and nitrate reductase activities were studied. High frequency irrigation with nutritive solutions negatively affects both yield and dry matter content in tubers. Moreover, the introduction of the tobacco Nia2 gene in the potato genome does not seem to affect the agronomical parameters of the initial genotype apart from the nitrate content of tubers. Five transgenic genotypes out of eight, in fact, showed a drastic decrease (of around 98%) in their tuber nitrate content. This nitrate decrease in the tubers was also correlated with the presence of the mRNA transgene, whereas the potato nitrate reductase transcript does not seem to be expressed in wild-type tubers. Regarding these genotypes, developmental stage and nutritive solution supply were found to have no effect on tuber nitrate content. In fact, tubers derived from these clones exhibited low nitrate content throughout the vegetation period, while nitrate accumulation in wild-type tubers is progressive and increases sharply with high nutritive solution supply.
Key words: Gene transcription, nitrate content, nitrate reductase, Solanum tuberosum L., transgenic plants.
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Introduction |
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Nitrogen is an essential nutrient which directly influences crop yield. The role of nitrogen in potato nutrition has already been the subject of several studies (Lorenz et al., 1974; Jenkins and Nelson, 1992; Cepl and Vokal, 1999; Ciecko et al., 1999; Mazurczyk and Wierzejska-Bujakowska, 1999; Davies, 2000; Hegney and McPharlin, 2000; Hlusek et al., 2000). Nitrogen is known to be one of the major factors determining potato yield. During the early stages of culture it favours canopy development and after that tuber initiation and swelling when supplied at the normal level (Van Kempen et al., 1996).
With culture intensification and the extensive use of fertilizers, nitrogen losses are potentially large in potato production. Because yield increases with nitrogen supply, fertilizer applications can reach very high values, up to 300 kg ha-1. Such excessive use of nitrogen can cause undesirable side-effects on the environment (Richards et al., 1990; Levallois et al., 1998), and can lead to poor quality tubers if their nitrate content is increased to levels considered to be over the acceptable limit for human consumption (Gravoueille et al., 1992; Marin et al., 1998).
The underlying problem is that potato is a plant with high nitrogen requirements but with low nitrogen uptake, typically less than 60% of the supply (Loon et al., 1994). Once the plant has taken up nitrate, many authors consider the most important controlling step in the assimilation process to be the one catalysed by the enzyme nitrate reductase, NR (Beevers and Hageman, 1969; Davies and Ross, 1987; Maldonado et al., 1996). Indeed, expression of the NR gene is highly regulated in plants by both endogenous and environmental factors, such as nitrate, sugars, light or water availability (Meyer and Stitt, 2001). Furthermore, it has been shown that overexpression of this enzyme can reduce nitrate accumulation in Nicotiana plumbaginifolia leaves (Dorlhac de Borne et al., 1994; Quilleré et al., 1994). This could be due either to increased reduction and/or decreased root absorption of the nitrate ions (Gojon et al., 1998). In order to study nitrate metabolism in potato, a deleted NR gene from tobacco driven by the 35S promoter was introduced into different initial genotypes. This truncated NR coding sequence codes for a deleted NR protein (
NR) lacking 56 amino acids on its N-terminal domain. This protein has previously been expressed in N. plumbaginifolia and it has been shown that it is probably less affected by the post-translational regulation by light which normally operates on NR via phosphorylation of a conserved serine residue and binding of 14-3-3 proteins (Nussaume et al., 1995; Lillo et al., 1997). Although it did not seem to be inactivated in the dark like complete NR, the NR protein appears nevertheless to be phosphorylated and bound to endogenous 14-3-3 proteins in planta (Lillo et al., 1997; Provan et al., 2000). The 35S-driven expression of the NR protein also allowed a more consistent and significant decrease in nitrate content in leaves than the constitutive expression of the full-length NR coding sequence (Nussaume et al., 1995; Lejay et al., 1997).
A first study of 20 transgenic potato lines carrying the NR gene was performed under field conditions over a period of 2 years (Djennane et al., 2002). Nitrate content in tubers was found to have drastically decreased in some transgenic clones during the 2 years of culture. This decrease in tuber nitrate content was linked to the expression of the transgene in the leaves of transformed potato lines.
The aim of the present work is to study the impact of the expression of the NR transgene on both potato growth characteristics and nitrate accumulation under two nutrition regimes during the plant cycle in glasshouse conditions.
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Materials and methods |
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Material
Two genotypes, selected at the Institut National de la Recherche Agronomique (INRA) for their resistance to Phytophthora infestans: 92T.118.5 and 92T.110.29 (unpublished results) were used. These genotypes were transformed via Agrobacterium tumefaciens, using the technique described previously (Lecardonnel et al., 1999). The p
NR transformation plasmid used has been extensively described (Nussaume et al., 1995). Twenty-four primary transformants were regenerated (8 for the genotype 92T.110.29 and 16 for the genotype 92T.118.5). The presence of the tobacco Nia2 gene was checked through specific amplification of a 494 bp fragment with primers specific for the tobacco gene. All the transgenic material and the wild type (WT) were then observed in field experiments (data not shown). For each initial genotype, the transformed plants presenting an aberrant phenotype (dwarf, chlorotic, ...) were discarded and four transgenic plants presenting a wild-type aspect in field conditions in terms of morphology and growth were retained. In total, 10 genotypes (two control genotypes also called WT and eight primary transformants also called TL) are part of the present study (Table 1
).
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Growth conditions and sampling
Tubers of the transformed and wild-type potatoes (harvested during the 1999 field planting) were planted in April 2000 in a glasshouse at INRA Ploudaniel (West Brittany, France). The tubers were grown in individual pots (18 cm height and 20 cm diameter) containing the following substrate: 50% peat, 35% sand and 15% pine bark. The plants were irrigated with Soluveg Bleue® solution (Générale de Nutrition Végétale, La Rochelle, France), containing 12 mM N (19% N-
and 81% N-
). Thirty-two tubers were planted for each genotype. Half of the plants were irrigated with the nutritive solution only (NS1) while the other half was irrigated alternatively with nutritive solution and water (NS2). Watering was done by sub-irrigation: pots were half flooded for 2 h and then drained, as frequently as necessary (on average, every 5 d) according to plant development and glasshouse environmental conditions. Temperatures varied between 15–18 °C night and 25–35 °C day during the whole culture period.
Four plants were randomly sampled at different stages of development (Fig. 1): (S1) leaf development stage (25–26 DAP); (S2) inflorescence emergence/tuber initiation stage (43–50 DAP); (S3) full flowering stage (71–78 DAP), and (S4) senescence stage/complete maturity (130 DAP). The genotypes used in this study are late ripening genotypes.
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Analysed parameters
At each stage, four plants for each initial genotype and irrigation regime were harvested and different parameters were analysed.
- (a) Biomass production and yield: daughter-tubers were weighed before and after desiccation at 80 °C for 72 h and the dry matter content was determined. Yield was determined only at final harvest.
- (b) Total nitrogen content in daughter-tubers: this parameter was determined only at final harvest (S4 stage). For all tubers of each plant, longitudinal cores were taken in the central part of each tuber from the stem-end to the rose-end. The cores were freeze-dried and ground to a fine powder. Aliquots of 7–9 mg of the powder were weighed in tin capsules and used for total nitrogen determination in a Fisons-Isochrom mass spectrometer.
- (c) Nitrate content: nitrate content was determined in petioles and daughter-tubers using Nitrachek/Merkoquant® method (Nitsch and Varis, 1991; Laurent and Lancelot, 1999; Djennane et al., 2002). Both petioles, taken from the three last-developed leaves, and the tubers of each plant were crushed. The juices were diluted, according to requirement, at 1/10 or 1/100 and nitrate was determined according to the procedure described by the manufacturer (Challenge Agriculture, France).
- (d) In vitro assay of total NR activity: at each stage, leaf (young leaves) and tuber material (500 mg) was ground in a mortar with liquid nitrogen and 4 ml of extraction buffer (50 mM HEPES pH 8.2, 0.1 M NaCl, 1 mM EDTA, 1 µM sodium molybdate) supplemented just before use with 5 µM leupeptin, 10 µM FAD and 2 mM DTT. For tuber NR extraction, 0.1 g of PVP was added. After 15–20 min at 4 °C, the extract is centrifuged at 19000 g for 20 min. In a reaction mixture containing 600 µl of 0.1 M phosphate buffer (pH 7.5), 100 µl of 0.1 M KNO3, and 100 µl of 1.5 mM NADH, 200 µl of the extract supernatant was added in a total volume of 1 ml and incubated for 15 min at 30 °C. The reaction was stopped by adding 1 ml of sulphanilamide 1% (w/v) in 3 N HCl and 1 ml of N-NED 0.02% (w/v). The nitrite formed is measured by colorimetric determination (A540nm). NR activity was expressed as nmol
min-1 g-1 FW. For each sample, the quantity of nitrite present before the NR activity (NRA) assay was measured and subtracted from the nitrite produced during the in vitro NR reaction.
- (e) Detection of the transgene and endogenous NR transcription in the tubers and the leaves by RT-PCR: total RNA was extracted from tubers (at harvest) or young leaves according to the protocol described previously (Fraisier, 2000). Deoxyribonuclease (DNase) digestion was performed with Ribonuclease-Free DNase (Pharmacia) in the presence of 40 units of ribonuclease inhibitor (Sigma). From 5 µg of total RNA, first strand cDNA was constructed using 10 µM anchor primer: 5'-GAG AAA TGA CCC TAA CGG CAT-3', 2 mM dNTPs, 10 mM DTT, and 200 units of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV, Sigma) in a final volume of 20 µl at 37 °C for 50 min. The resulting cDNA (1 µl) was used for amplification by PCR in a 25 µl reaction mixture containing 200 µM dNTPs, 1.5 mM MgCl2, 2 µM of two primers specific for tobacco NR: nia3: 5'-GAC TCT CCT GGC AAC TCC GTG-3' (sense) and nia4: 5'-GAG AAA TGA CCC TAA CGG CAT-3' (reverse) and 0.7 units of Hot Taq polymerase (Fisher). Reactions were performed in a MJ Research thermal cycler under the following regime: 95 °C for 5 min, 30 cycles of 95 °C for 1 min, 61 °C for 30 s, 72 °C for 1 min 15 s, and a final extension step of 72 °C for 10 min.
- (b) Total nitrogen content in daughter-tubers: this parameter was determined only at final harvest (S4 stage). For all tubers of each plant, longitudinal cores were taken in the central part of each tuber from the stem-end to the rose-end. The cores were freeze-dried and ground to a fine powder. Aliquots of 7–9 mg of the powder were weighed in tin capsules and used for total nitrogen determination in a Fisons-Isochrom mass spectrometer.
The first cDNA strand was also used for PCR assay using a pair of primers which amplify both potato and tobacco NR genes. The amplification was assessed using PCR in a 25 µl reaction mixture containing 200 µM dNTPs, 1.5 mM MgCl2, 2 µM of two primers: nia7: 5'-CCC TCA ATA CTC AAC CCG AGA-3' (sense) and nia9: 5'-GAG TCA GAC GTG TAA CCA GT-3' (reverse) and 0.7 units of hot Taq polymerase. Reactions were performed in a thermal cycler as described above with an annealing temperature of 60 °C. The resulting PCR products were separated on a 1.5% agarose gel.
Statistical analysis
The differences found between transformed and non-transformed plants and the two irrigation regimes were assessed by ANOVAR using the STAT-ITCF system.
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Results |
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Tuber yield
High nitrogen supply (NS1) seems to decrease potato yield expressed in g FW plant-1. Tuber yield decreased by about 16–27%, respectively, for genotypes 92T.110.29 and 92T.118.5 (Table 2
). By contrast, NS1 leads to an increased tuber number per plant (Table 2). No variation has been observed between WT and transgenic clones for the two genotypes.
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Daughter-tuber dry matter content
After tuber initiation, the tuber dry matter increases slightly until full flowering stage, then a sharp increase is recorded at final harvest. Dry matter content can thus reach 350 mg g-1 FW at S4 stage (Fig. 2).
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Low nitrate supply (NS2) markedly increases dry matter in tubers at harvest for both genotypes. An increase of 24% and 6% is observed, respectively, for the genotypes 92T.118.5 and 92T.110.29 (Fig. 2
). Again no effect of genetic transformation was observed.
Total nitrogen content in tubers
Total nitrogen was determined at final harvest (S4 stage). The genetic transformation did not significantly modify the total nitrogen in transgenic tubers compared with the WT for both studied varieties (Fig. 3); however, the NS regime did. Indeed, the NS1 regime increased total nitrogen content of about 57% and 61%, respectively, for 92T.118.5 and 92T.110.29.
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Nitrate content in petioles
Nitrate concentration in petioles is highly variable during growth (Table 3). However, a more or less pronounced decrease in nitrate level was usually observed at tuber initiation stage (S2). Then nitrate increased markedly in petioles at the full flowering stage (S3), especially when the nitrogen supply was high.
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Nitrate content in petioles is significantly affected by transgene introduction in some TLs under NS1. In the set of lines 92T.118.5, line 736.1 presents lower
in petioles at the S2 stage than WT. In the set of lines 92T.110.29, all TLs except 708.8 present a lower nitrate content at S2 stage (Table 3).
Under NS2, even though statistical differences were not revealed, content was reduced in the petioles of some TLs. For 736.1 decreases of about -91% and -16% were observed at the S2 and S3 stages, respectively, under the NS2 regime. For genotype 92T.110.29, all the TLs show a decreased nitrate content under NS2 of about -10% to -52% for the S2 and S3 stages (Table 3).
It is important to underline that large variations of nitrate content were observed between replicates (Table 3). These variations may have concealed a transgene effect.
Nitrate content in tubers
Nitrate content in very young tubers (S2) on control lines (WT) is less than 95 mg kg-1 FW, irrespective of nitrogen supply. During the following stages, nitrate concentrations usually increase and can reach very high values at harvest, reaching up to 1200 mg kg-1 FW under the NS1 fertilization conditions only (Table 4).
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Nitrate content in the daughter-tubers of five transgenic clones (736.1, 756.1, 756.2, 708.1, 708.8) is dramatically reduced. Indeed, at harvest, a decrease in tuber nitrate concentration of about 98% compared with the untransformed control lines, is observed for all 92T.110.29 transformants with the NS1 high nitrogen supply. Under the NS2 irrigation regime, these transformants show a nitrate decrease of about 90% (Table 4
). Tuber nitrate concentration appears to be less affected by plant development stage and is only slightly affected by the nutritive solution supply in these transgenic lines (Table 4). Other transgenic clones (708.2, 712.2, 729.1) were not affected by the introduction of the Nia2 gene and accumulated as much or more nitrate in their tubers than their corresponding control lines.
Nitrate reductase activity
Nitrate reductase activities (NRA) were measured at each sampling stage using young leaves. A significant variation in NRA between different samples from the same line was observed.
For genotype 92T.118.5 under the NS1 regime, clone 736.1 seems to have more NRA at the first stage, but no statistical differences were detected. At the third stage, 3 TLs (729.1, 736.1 and 708.2) exhibit high NRA with an increase of, respectively, 144%, 44%, and 15% compared with the WT. For the set of lines 92T.118.5, NRA decreases at the second sampling stage and increases later (Table 5) for both NS regimes, following nitrate content fluctuations in petioles.
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min-1g-1 FW) of wild types (WT) and transgenic lines of the genotypes 92T.118.5 and 92T.110.29 at different stages of vegetation and with different nutritive solutions (each value represents the mean±SD of four replicates)Genotype 92T.110.29 does not show the same trend as genotype 92T.118.5. In fact, in this genotype NRA increases largely at the second stage under the NS2 regime and continues to increase in WT only, while it decreases significantly in TLs.
Nitrate reductase activity in tubers was measured with the same protocol as in leaves, but no activity was detected.
Transcriptional expression of the transgene in tubers
Transgene expression in tubers was studied at the RNA level using RT-PCR. Total RNA was extracted from tuber cores of each transformant at final harvest (S4).
For each RNA extraction, two samples were examined using RT-PCR, either treated with M-MLV reverse transcriptase or not, in order to detect possible DNA contamination in the RNA extract. A pair of primers (nia3 and nia4) specific for the tobacco NR coding sequence were designed in order to amplify the RNA derived from the tobacco Nia2 transgene only, and not from the endogenous NR gene. Using these two specific primers, one major band of 404 bp from some transgenic plants was amplified, but not from the control untransformed potato plants (Fig. 4). A band of the same size was amplified using the same primers and genomic DNA from one of the transformants (Fig. 4, C+). This band is present in all transgenic plants for the genotype 92T.110.29 (Fig. 4B) and only in 736.1 derived from 92T.118.5 (Fig. 4A). An unspecific band of approximately 350 bp appeared after M-MLV treatment, in extracts taken from all WTs and TLs (Fig. 4). Identical profiles were obtained in both regimes of nutritive solution supply (NS1 and NS2).
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Using another pair of primers (nia7 and nia9) which can amplify both tobacco and potato NR sequences, the expected band of 494 bp in tubers was obtained but only in some transgenics (Fig. 5
), i.e. those expressing the tobacco NR gene (Fig. 5A). This indicates that expression of the transgene occurs in tubers while the endogene seems to be silent. In leaves, the band is amplified from both WT and transgenic samples (Fig. 5B), which shows that these primers can indeed detect the potato NR gene expression.
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Discussion |
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Yield and biomass production
In this study, the introduction of the Nia2 tobacco gene in the potato genome does not seem to affect any of the agronomic characters of the transgenic lines. TLs and respective WTs are similar for most of the observed characters: yield and tuber number which are studied here, and other characters such as plant height and phenotype (data not shown).
Under these experimental conditions, NS1 produced more vigorous and larger plants than NS2 (data not shown). Tuber number per plant was also increased. By contrast, tuber yield is decreased, indicating a delay in tuber induction and/or competition between tubers for growth. It is known that excessive nitrogen supply favours the development of aerial parts at the expense of tuber growth (Ellissèche, 1996; Van Kempen et al., 1996). On the contrary, it was observed that, under field conditions, tuber yield was increased as long as high nitrogen was applied (160 and 240 kg N ha-1), but progeny tuber number was not affected (Jenkins and Nelson, 1992). So far, transgenic clones have not exhibited any modification of these parameters when compared to their corresponding controls.
Tuber dry-matter content is an important component of tuber quality, especially in potatoes required for processing. Again, there is no significant effect of the introduction of the tobacco NR gene in the potato genome on this character. This is conform to what was observed on transgenic tobacco and Arabidopsis leaves (Quilleré et al., 1994; Nejidat et al., 1997).
At harvest, lower tuber DM content was observed on both genotypes studied and their corresponding transgenic clones under NS1 supply. In fact, MacKerron and Young found that DM content in potato tubers was slightly higher with lower N-application in field conditions (MacKerron and Young, 1999). Jenkins and Nelson found the same results after year one of their experiment but not during the second year (Jenkins and Nelson, 1992). Van Kempen et al. also demonstrated that high nitrogen supply could affect DM content in progeny tubers by decreasing it (Kempen et al., 1996).
In this experiment, DM reached a very high value (about 300–350 mg g-1 FW) while in field conditions, DM content in tubers is about 18–25% (Jenkins and Nelson, 1992; Gravoueille, 1996); this can be attributed to the glasshouse experimental conditions.
Nitrogen status of the plants
Measurement of total nitrogen in mature tubers does not reveal clear evidence of a transgene effect, which is quite surprising since one could expect that activation of the NR enzyme would have led to an increased accumulation of total nitrogen in the various organs. However, Gojon et al. demonstrated in tobacco that over-expression of NR leads to a global reduction in nitrate uptake by the plant (Gojon et al., 1998). Moreover, it is worth underlining that the total nitrogen fraction in potato tubers is normally very low: about 1.15–2% of DM (Gravoueille, 1996). A more global study of total nitrogen status of the entire plants would be needed to be able to reach a conclusion on this point.
The main consequence of tobacco Nia2 gene expression in transformed potato plants is a significant decrease in the tuber content, which is consistent with previous field observations (Djennane et al., 2002). Five transgenic clones were largely affected in their tuber nitrate content. It is worth underlining that the decrease is mainly observed in tubers, but rarely in aerial parts. However, nitrate content in aerial parts was determined on petiole juice extracts of three freshly developed leaves. With this measurement method, only nitrate-sap content was estimated. There are no data regarding nitrate content in leaves, where the major part of nitrate reduction and/or accumulation in vacuoles takes place.
Nitrate content in tubers of 736.1, 756.1, 756.2, 708.1, and 708.8 was not or only slightly affected by the NS frequency and stage of development, and therefore always exhibited low nitrate content from their initiation to their complete maturity compared with the WT. This result is possibly due to the presence of an active NR in tubers of these transgenic clones.
At final harvest, nitrate contents in WT and some TL tubers reached very high levels under the NS1 regime, far from the recommended content value for human consumption (100–200 mg kg-1 FW) (Gravoueille et al., 1992). These high values could be explained by the incomplete maturity of tubers due to their delayed initiation. In fact, some authors (Reust and Quinche, 1988; Ciecko et al., 1999) detected a final decrease of tuber nitrate content at maturity. It is also known that nitrate levels in tubers increase with nitrate supply (Gravoueille et al., 1992).
Nitrate reductase activity
Under the present sampling conditions, transgenic clones did not clearly exhibit greater NRA in leaves compared with controls, excepted for clone 756.2 at the first stage and 729.1 at the third stage under the NS1 regime. By contrast, Nicotiana plumbaginifolia plants, transformed with the complete Nia2 cDNA sequence under the control of the 35S CaMV promoter, exhibited 25–150% higher NR activity than the wild type (Quilleré et al., 1994). The number of NRA measured may be too small under these experimental conditions to provide a good idea of the real NRA potential of TLs. It would be worth comparing the NRAs of WT and TLs more frequently under the same experimental conditions or under more controlled conditions (for instance, in vitro plantlets) because NRA is highly regulated by endogenous and exogenous factors (nitrate availability, light, sugars, hormones, ... Faure et al., 1997; Meyer and Stitt, 2001). Especially under the present culture conditions, sub-irrigating every 5 d with water and/or nutritive solution may have been a great source of variation for nitrate availability for plants and thus for NRA. Furthermore, there is a possible compensation process between endogenous and transgenic NRA in TLs which could explain the lack of a clear difference between WT and TLs.
The decrease in NR activity at the second stage for variety 92T.118.5 may be due to tuber initiation (Murti and Balasimha, 1983). In the case studied here, maximum activity was observed at full flowering stage (71–78 DAP) on 70% of clones. Others have observed that NR activity was maximum in leaves taken on 45 and 30-d-old plants, respectively, and declined toward maturity (Marwaha, 1998; Fonseka et al., 1997). These differences in the results may be explained by genotypic effects. In this study's experiments, late ripening cultivars were in fact studied with a 130 d culture cycle.
The five transformants presenting low tuber nitrate contents in the present study were previously studied under field conditions and also exhibited a large decrease in their content with 95% mean of decrease (Djennane et al., 2002). They also exhibited high chlorate sensitivity in vitro when plantlets were cultured on Murashige and Skoog medium supplemented with 5 mM 
suggesting an increase or a differential expression of the NR enzyme.
No nitrate reductase activity was detectable in tubers of both WT and TLs while a large decrease in nitrate content in tubers was observed. Kapoor and Li also reported low activity of nitrate reductase in untransformed roots and tuber tissues (Kapoor and Li, 1982) and Marwaha demonstrated that roots showed very low activity at all stages and that mean activities in the roots were approximately 10–13 times lower as compared to leaves (Marwaha, 1998). One possible explanation of this study's results, is that the protocol used was not adapted for tuber NR enzyme extraction. In fact, Palmer revealed NR activity in potato tubers by giving them special ageing treatment before measuring activity (Palmer, 1979). Trials are currently underway to try to evaluate NR activity in transgenic tubers.
Although NR activity could not be measured in transgenic tubers, the presence of the tobacco Nia2 gene transcripts in tubers was demonstrated using RT-PCR. This can be explained by the use of 35S CaMV promoter which usually drives high constitutive expression level of transgenes (Graham et al., 1997), even in the tubers (Artsaenko et al., 1998). Further studies are still needed to verify that transgene mRNA is effectively translated into an active NR.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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Introduction and expression of a truncated tobacco NR gene in potato led to a drastic decrease of tuber nitrate content in five transgenic lines out of eight. This decrease was observed in all culture conditions tested and was effective over all the plant cycle period. By RT-PCR experiments it was possible to demonstrate that the transgene was transcribed in tubers, while no endogenous NR transcripts were found in wild-type tubers. Further studies are needed to verify if there is an active NR in transgenic tubers. In this case, is it the only reason for nitrate level reduction in tubers or are there more complicated regulation mechanisms of absorption, assimilation and accumulation of nitrate induced by the transgene expression in the whole plant?
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Acknowledgements |
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We thank Dr Yves Chupeau and Daniel Ellissèche for helpful discussions and critical reading of the manuscript. We thank Catherine Souchet and Alain Label-Richardson for technical assistance, Jean-Paul Dantec for care of the plants in the glasshouse.
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Footnotes |
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3 To whom correspondence should be addressed. Fax: +33 2 2962 6330. E-mail: jchauvin{at}rennes.inra.fr
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Abbreviations |
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DAP, days after plantation; DM, dry matter; dNTPs, deoxynucleotide triphosphates; DTT, dithiothreitol; FW, fresh weight; N-NED, N-naphthyl-1-ethylenediamine dichlorohydrate; NR, nitrate reductase; NRA, nitrate reductase activity; PVP, polyvinylpyrrolidone; RT-PCR, reverse transcriptase-polymerase chain reaction; TL, transgenic line; WT, wild type..
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References |
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