JXB Advance Access originally published online on July 12, 2005
Journal of Experimental Botany 2005 56(419):2379-2388; doi:10.1093/jxb/eri230
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Systematic silencing of a tobacco nitrate reductase transgene in lettuce (Lactuca sativa L.)
1INRA, Unité de Génétique et Amélioration des Fruits et Légumes, UR 1052, domaine St Maurice BP 94, F-84143 Montfavet cedex, France
2INRA, Laboratoire de Nutrition Azotée des Plantes, UR 511, RD10 route de Saint-Cyr, F-78026 Versailles cedex, France
To whom correspondence should be addressed. Fax: +33 4 32 72 27 02. E-mail: Marianne.Mazier{at}avignon.inra.fr
Received 4 January 2005; Accepted 19 May 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A population of 50 independent transgenic lettuces transformed with a nitrate reductase coding sequence under the control of the 35S promoter was studied. None of them showed significantly lower nitrate levels when compared with the untransformed plants, despite the presence of nitrate reductase (NR) activity that derives from the transgene in at least four of the transformants. No repercussion on total NR activity (endogenous+transgenic) was detected in these plants. Nevertheless, 28% of the transformants showed phenotypes characteristic of a general silencing of the NR genes as already described in tobacco and potato, i.e. bleaching of the leaves leading to the death of the plant. By northern blots, it was shown that the transgene was silenced in these chlorotic plants and also in the plants that did not show symptoms of chlorosis. Thus a silencing process specifically directed against the NR mRNA derived from the transgene occurred very early in the development of all the plants studied, whatever homologous endogenous NR mRNA is present in the plant. In some cases this transgene-specific silencing was shown subsequently to extend to the homologous endogenous NR mRNA. These results suggest that, in lettuce, the level of nitrate reductase mRNA is under tight expression control and this is able specifically to target transgenic transcripts by a post-transcriptional gene silencing (PTGS) mechanism during the first stage of development of the plantlet.
Key words: Lettuce, mRNA, nitrate, nitrate reductase, PTGS, transgenic plants
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nitrate
is the major source of nitrogen for higher plants. The first enzymatic step of the nitrate assimilation pathway is catalysed by nitrate reductase (NR). The reduction of nitrate into nitrite by NR is often considered to be one of the major rate-limiting reactions of this pathway. NR is highly regulated, not only at the transcriptional level but also at the post-transcriptional level and is influenced by several endogenous and environmental factors. These regulations affect both the amount and the activity of the NR protein in the cytosol (for reviews see Campbell, 1999
; Kaiser and Huber, 2001). Lettuce (Lactuca sativa L.) is an economically important leafy vegetable. Under winter conditions, mainly because of poor light, lettuce accumulates high levels of nitrate in leaves compared with other vegetables. In these growth conditions, the maximal nitrate contents in leaves authorized by the EC are often exceeded. As a consequence, nitrate content remains an essential criterion for marketing this crop in Europe.
In the past 15 years, Nicotiana plumbaginifolia and N. tabacum transgenic plants constitutively over-expressing a gene coding for a tobacco NR (tobacco Nia2 cDNA driven by the 35S promoter from the CaMV) have been obtained (Vincentz and Caboche, 1991; Dorlhac de Borne, 1993). These plants showed elevated NR activity (NRA) and reduced leaf nitrate content compared with wild types (WT) (Quilleré et al., 1994). Recently, the same strategy was applied to Solanum tuberosum (Djennane et al., 2002a, b, 2004) and L. sativa (Curtis et al., 1999). Some transgenic potatoes with highly reduced nitrate levels in tubers were obtained. By contrast, at maturity, none of the transgenic lettuces showed differences in their 
content compared with WT. Lettuces with leaf nitrate contents slightly lower than WT were observed only at the mid-culture stage. The authors of this study (Curtis et al., 1999) proposed that this difference between mid-culture stage and mature lettuces might be due to reduced activity of the 35S promoter in older lettuce plants. Another study has shown that, in lettuce, the 35S promoter was less stable than the PetE promoter (McCabe et al., 1999b). However, genes other than Nia2, driven by the p35S have already been used in lettuce and did not show any failure of expression: examples include genes coding for a ß-1,3-glucanase (Dede, 1998), a ß-glucuronidase (McCabe et al., 1999a), or a soybean ferritin (Goto et al., 2000). For this reason, alternative explanations concerning the results obtained by Curtis et al. (1999) with the 35S::Nia2 construct should be considered.
In order to determine the reasons for the inability of the 35S::Nia2 transgene to reduce nitrate contents in lettuce, a new population of transgenic lettuce containing a 35S::Nia2 construct was produced. The systematic and efficient silencing of the tobacco NR transgene in lettuce is described here. It is shown that a mechanism of gene silencing was responsible for the lack of expression of the transgene and its lack of effects on nitrate contents in lettuce leaves. In some of the transgenic genotypes obtained, this mechanism was identified as a post-transcriptional gene silencing (PTGS) mechanism.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth conditions
Lactuca sativa cultivar Jessy (Syngenta previously Caillard; Domaine du Moulin, 84260 Sarrians, France), a butterhead type of lettuce usually grown in glasshouses, was used for transformation.
Cultures were started by soaking lettuce seeds on filter paper with water, leaving them in the dark for 48 h at 4 °C. Before cultivation in a tunnel, germinated seeds were first grown on compost in 7 cm pots (Compost H1 from Tref, Tref Substrates Coevorden B.V., Marconiweg 6, 7741 KM Coevorden, Nederland) in a glasshouse for 3 weeks.
Before culture in a hydroponic system, germinated seeds were placed on vermiculite impregnated with distilled water for 10 d in a growth chamber of 1.60 m2 with fluorescent light (28 tubes of 36 W) (16 h daylight at 16 °C, 8 h darkness at 12 °C). Hydroponic cultures were set up in a growth chamber under different culture conditions: three nitrate concentrations of the nutrient solution (0.5, 5, and 12 mM), two light intensities (80 and 140 µmol m–2 s–1), and a long (16 h) or a short photoperiod (8 h). Aeration and stirring of the nutrient solution was performed using a compressed air pump and a magnetic stirrer, respectively.
Plasmid and bacterial strain
The binary vector pSCKDH51 is a derivative of pSCK (Biocem, Groupe Limagrain, Av. de Bois l'Abbé, 49070 Beaucouze, France), where all the restriction sites from the polylinker were deleted, except the EcoRI site in which the plant expression cassette p35S::t35S (obtained as an EcoRI fragment from plasmid pDH51 (Pietrzak et al., 1986) was introduced. The vector pSCK-NR (Fig. 1) was constructed by cloning a SalI-SacI fragment containing the complete Nia2 cDNA of pBCSL16 (Vincentz and Caboche, 1991) in the SmaI site of pSCKDH51, between the promoter and terminator sequences of the CaMV. The resulting binary vector, pSCK-NR, was introduced by electroporation into the disarmed Agrobacterium tumefaciens strain C58C1 (pGV2260) (Deblaere et al., 1985).
|
Production of transgenic plants
Leaves excised from 10-d-old seedlings (cultured aseptically) were co-cultured with A. tumefaciens strain C58 pGV2260 carrying pSCK-NR as described in Dinant et al. (1997)
. Transformed buds were selected on 200 mg l–1 kanamycin-containing regeneration medium and transplanted individually onto rooting medium [macro MS, micro Heller, vitamins B, sucrose 30 g l–1, agar Biomar 6 g l–1 (Quarrechim, 9 rue Paul Langevin BP 361 34504 Beziers Cedex, France), pH 5.8] with 50 mg l–1 kanamycin. Vigorous plants were transplanted to pots containing peat in a growth chamber (22/16 °C, 16 h photoperiod), with manual irrigation with water, before transfer to a glasshouse where they were watered each day with Coïc-Lesaint nutritive solution. Primary transformants (R0 generation) were self-pollinated and their kanamycin-resistant R1 progenies were again self-pollinated to obtain R2 seeds. Homozygous R1 plants and their R2 progenies were identified by analysing the segregation of resistant versus susceptible R2 seedlings on a kanamycin-containing rooting medium (Chupeau et al., 1989).
In vitro selection procedures
Analysis of kanamycin resistance of the transformants was performed by sowing sterilized seeds (30 min dipping in a solution of Teepol 0.05% (Kiwi-France, 93 594 Le Blanc Mesnil cedex, France) and 0.15% (w/v) Inov'chlore (Top Hygiene previously Inov'chem; ZAC des Peyrardes, 42 273 St Just St Rambert, France) in Petri dishes on semi-solid basal rooting medium containing 75 mg l–1 kanamycin. After 48 h exposure in darkness at 4 °C, Petri dishes were transferred to a growth room (20 °C, 16 h photoperiod). Ten to 15 d after sowing, kanamycin resistance was analysed.
The counter-selection system described by Nussaume et al. (1991) was exploited to select the Nia2 cDNA-expressing genotypes. In order to improve the accuracy of the test, 8 mM of glutamine was added to the chlorate-containing nitrate-free medium. When cultured in this medium, seedlings expressing the transgene 35S::Nia2 failed to survive on the selective medium due to constitutive NR expression, whereas non-transformed seedlings were unaffected because of the transcriptional negative regulation of the endogenous Nia gene by glutamine. Sterile seeds were sown in Petri dishes on A medium (KH2PO4 0.45 mM, MgSO4 0.45 mM, K2SO4 1.18 mM, CaCl2 1.5 mM, CaSO4 0.2 mM, glutamine 8 mM, KClO3 0.5 mM, MES 3.5 mM, EDDHA-Fe 0.01 g l–1, Heller's micro salts 1x, and agar Biomar 6 g l–1, pH 5.8). After 48 h exposure in darkness at 4 °C, Petri dishes were transferred in a growth chamber (20 °C, 16 h photoperiod).
Genomic DNA analysis
Genomic DNA was extracted from leaf tissues using a modified CTAB method (Bernatzky and Tanksley, 1986). Twelve micrograms of DNA were digested with the appropriate restriction enzyme as recommended by the manufacturer (Life Technologies, Invitrogen Sarl, BP 96, 95613 Cergy-Pontoise cedex, France). After separation on a 0.8% (w/v) agarose gel and denaturation and neutralization using a standard procedure (Sambrook et al., 1989), the DNA was transferred onto a Hybond N+ membrane (Amersham Biosciences, Europe GmbH, Parc Technologique, rue René Razel, Saclay, F-91898 Orsay cedex, France). The probes, an 805 bp HindIII fragment from pABDI (Paszkowski and Whitham, 2001) containing the nptII gene, or a 2x 500 bp HindIII/EcoRI fragment from the pUCEn4 plasmid (Leprince, 1996) containing the p35S sequence, were labelled by random priming. Hybridizations with 32P-labelled probes were performed in the hybridization buffer (750 mM NaCl, 125 mM sodium citrate, 0.6% SDS, 50 mM Na2HPO4/NaH2PO4 pH 7.5, 5x Denhart, 2.5 mM EDTA, 5% dextran sulphate, 2.5 mg DNA salmon sperm) for 16–24 h at 65 °C. The filters were then washed once in 2x SSC and 0.1% SDS for 20 min at 65 °C followed by one wash in 1x SSC and 0.05% SDS for 20 min at 65 °C, and a final wash in 0.5x SSC and 0.05% SDS for 10–20 min at 65 °C. Filters were exposed and analysed by autoradiography.
RNA analysis
Total RNA from lettuce was prepared with the TRI REAGENTTM according to the protocol described by the manufacturer (Euromedex, 24 rue des Tuileries, BP 74 684 Souffelweyersheim, 67 458 Mundolsheim cedex, France).
Thirty micrograms of total RNA were separated on a 1% (w/v) agarose gel containing 8% (v/v) formaldehyde. The RNA were capillary-blotted to Hybond N+ membrane (Amersham Biosciences, Europe GmbH, Parc Technologique, rue René Razel, Saclay, F-91898 Orsay cedex, France) in 10x SSC.
Hybridizations with 32P-labelled probes (in 7% SDS; 300 mM Na2HPO4/NaH2PO4 pH 7.2; EDTA 1 mM pH 8.0 buffer) were performed for 16–24 h at 65 °C. The filters were then washed four times in 2x SSC and 0.1% SDS for 5 min at room temperature followed by one wash in 0.2x SSC and 0.1% SDS for 5–10 min at 65 °C.
The probes (a Nia2 cDNA 119 pb fragment and a Ls.Nia1 120 pb fragment) were labelled by polymerase chain reaction. Primers 1 (5'-GGATTCTGCTGCATCATCACCAAATA-3') and 2 (5'-TTCACGAGGGACTAAGGCTACGTTTCTTG-3') were used for amplification of the ls.Nia1 probe and primers 3 (5'-TGACTCTCCTGGCAACTCCGTGCACGGAT-3') and 4 (5'-CTCTCTTGGAATTAGGGCCACACTCCTCT-3') for the Nia2 probe. Forty-five cycles of amplifications were performed for both probes [94 °C (1 min), 55 °C (1 min), and 72 °C (30 s)].
Determination of nitrate reductase activity
Nitrate reductase activity was measured in the presence of MgCl2 or of EDTA using the same protocol for both activities, except that 10 mM MgCl2 was added to the buffers instead of 15 mM EDTA. One gram of leaf material was ground in a mortar with liquid nitrogen and 8 ml of extraction buffer (50 mM HEPES–KOH, 10 mM MgCl2 (or 15 mM EDTA), 7.5 mM cysteine, 10 µM FAD, 5 µM leupeptin, 0.2 g PVP per 8 ml buffer, pH 7.6). The extract was centrifuged for 15 min at 5500 g at 2 °C and the soluble fraction assayed for NR activity. The reaction mixture contained 800 µl of activity buffer (50 mM HEPES–KOH, 10 mM MgCl2, 10 mM KNO3, 180 µM NADH, pH 7.6) and 100 µl of the extract. The reaction was carried out for 25 min at 30 °C and was terminated by the addition of 100 µl of 1 M sodium acetate. After 10 min of centrifugation (15 000 g), the quantity of 
formed during the enzymatic reaction was determined at 540 nm after mixing 100 µl of reaction mixture in the presence of 0.5 ml sulphanilamide (10 g l–1 HCl 3N) and 0.5 ml N-(1-naphthyl)ethylenediamine dihydrochloride (0.2 g l–1 H2O).
Determination of nitrate and protein content in leaves
Nitrate and protein concentrations were determined using the same leaf extracts as for NR activity. Nitrate content was determined by colorimetry using an Aquatec 5400 Analyser (Foss France, 35 rue des peupliers, 92000 Nanterre, France), and soluble protein by the method of Bradford (1976).
Inoculation of plants and detection of green fluorescence
The recombinant virus LMV0-GFP (German-Retana et al., 2001) was used in this study. This recombinant isolate was constructed by inserting the GFP reporter gene (coding for a modified Aequorea victoria green fluorescent protein) between the P1 and HC-Pro genes of a full-length infectious cDNA copy of the viral genome. An artificial cleavage site specific to the NIa viral proteinase was introduced between the GFP and the helper component (HC-Pro) sequences. This virus thus expressed the GFP reporter as a free protein. The recombinant virus was propagated under containment glasshouse conditions on lettuce plants of cv. Trocadero. Mechanical inoculation of lettuce plants was performed as described by Dinant et al. (1997) for pot-grown plants or as described by Mazier et al. (2004) for in vitro-grown plants. GFP fluorescence was detected visually in whole plants with a 100 W, hand-held, long-wave UV spot-light (Model B-100, UV Products, Upland, CA).
Statistical analyses
The data were subjected to the Student-Newman-Keuls multiple range test (GLM procedure of SAS package).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Production and characterization of the transgenic plants
Fifty independent transgenic plants of Lactuca sativa cv. Jessy (primary transformants: R0) were obtained from transformation experiments performed on leaf explants with A. tumefaciens strain C58 pGV2260 containing the binary vector pSCK-NR (Fig. 1). After transfer to a greenhouse for self-pollination, 14 of these R0 transformants developed leaf chlorosis. This phenotype appeared at different developmental stages depending on the transgenic line considered (Fig. 2). Three of the chlorotic transgenic lettuce genotypes died before seed production.
|
One-hundred R1 seeds from each of the 36 remaining R0 transformants were sown on rooting medium containing 75 mg l–1 kanamycin to analyse the segregation of resistance for this antibiotic (Table 1). Twenty-six genotypes showed a ratio 3:1 of kanamycin-resistant to susceptible plants consistent with the presence of only one functional nptII locus. Twelve R1 resistant plants of each of the 26 transformants were grown in the greenhouse for self-pollination and selection for homozygous plants. Five other transgenic genotypes (JSNR 8, 18, 23, 28, and 54) were sensitive to kanamycin. The five remaining transformants (JSNR 15, 36, 38, 48, and 59) showed in their progeny a ratio of kanamycin-resistant to kanamycin-sensitive plants different from that expected for 1 or 2 independent transgene insertions, respectively 3:1 and 15:1.
|
The 26 selected R2 homozygous genotypes were further submitted to a chlorate sensitivity test (Nussaume et al., 1991
) to detect transgenic lines showing NR activity (NRA) due to expression of the transgene. Only 4 (JSNR 5-5, 21-8, 45-12, and 58-7) of the 25 R2 genotypes studied were significantly more susceptible to chlorate than WT (Table 2).
|
Analysis of T-DNA integration was performed by Southern blot on DNA extracted from the R0 transformants. Probing of DNA with the nptII and the p35S cDNA probes revealed that the right border was partially missing in 15 transformants. Eight genotypes (JSNR 5, 9, 21, 26, 32, 45, 46, and 58) presented a unique insertion of the transgene p35S::Nia2 and JSNR 35 a double T-DNA insertion arranged as an inverted repeat (IR). A multiple and complex T-DNA insertion was detected for JSNR 14. Table 3 summarizes the results obtained by Southern blot analysis.
|
Physiological analysis of the transformants
The 11 homozygous transgenic lettuce presenting a single transgene locus were cultivated in the glasshouse in order to study their nitrate content compared with WT. Each genotype was present in three plots (each plot comprised three plants of the WT and three plants of the corresponding transgenic genotype) randomly distributed in the greenhouse. Plants were irrigated with a nutritive solution containing 12 mM of nitrate. Plants were collected at maturity 3 months after sowing. The fresh weight, dry weight, and nitrate content of these plants were then analysed (Fig. 3). Differences in phenotype (leaf colour, leaf texture) and in heading earliness were observed during culture. In these culture conditions, no significant differences in the measured parameters were found between the transformants and the WT (data not shown).
|
Chlorate susceptible genotypes (JSNR 5-5, 21-8, 45-12, and 58-7) were also cultivated hydroponically with different nitrate concentrations (0.5, 5, and 12 mM) and different light intensities (80 and 140 µmol m–2 s–1). Plants were harvested 45 d after sowing and nitrate concentrations and total NR activity in leaves were analysed. Again no significant differences were detected between the WT and the transgenic genotypes (data not shown).
During culture, some plants of genotype JSNR 21-8 showed a chlorotic phenotype at mid-stage development. This phenotype was not observed on JSNR 5-5, 45-12, and 58-7.
Nia2 mRNA presence in the transformants
In order to understand why plants, that in the chlorate test showed NR activity derived from the transgene, did not present any changes in their nitrate contents, the presence of the Nia mRNA was checked by northern blots in different environmental conditions and at various developmental stages.
Firstly the genotypes JSNR 5-5, 45-12, and 58-7 were studied 45 d after sowing in hydroponic culture with 5 mM of nitrate. In this experiment, the genotype JSNR 21-8 was not retained because of its tendency to develop chlorosis at this stage of development. The mRNA of the transgene was not detected by northern blot in these three genotypes (Fig. 4). However, LsNia mRNA was present in all of them (Fig. 4).
|
By contrast, the tobacco Nia2 mRNA was detected in cotyledons of plantlets cultivated in the chlorate test conditions with glutamine as the nitrogen source in the genotypes JSNR 5-5, 21-8, and 58-7 (Fig. 5). In leaves of the same plants, the transgene tobacco Nia2 mRNA was not detected by northern blot analysis (Fig. 6). In plantlets cultivated with nitrate as the nitrogen source instead of glutamine, the tobacco Nia2 mRNA was not detected either in the cotyledons or in the leaves.
|
|
Infection of chlorotic plants by LMV-0-GFP
Infection of Jessy by the recombinant virus LMV-0-GFP (German-Retana et al., 2001
) revealed that this recombinant virus was able to infect L. sativa cv. Jessy (Fig. 7) without producing any serious symptoms.
|
Chlorotic R1 JSNR 60 plants infected by LMV-0-GFP turned green again (Fig. 8a) around 20 d post-inoculation, whereas control non-infected plants still produced chlorotic leaves. In the non-inoculated chlorotic plants of JSNR 60, northern blot analysis showed a very low level of LsNia and tobacco Nia2 mRNA compared with WT (Fig. 8b). By contrast, in the inoculated JSNR 60 plants leaves, northern blot analysis revealed the accumulation of LsNia and tobacco Nia2 mRNA (Fig. 8b).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Earlier studies have shown that the introduction of the p35S::Nia2 transgene into the genome of tobacco and potato led to a marked decrease in nitrate content in leaves and tubers, respectively (Vincentz and Caboche, 1991
; Dorlhac de Borne, 1993; Djennane et al., 2002a). The same effect was not obtained in two lettuce cultivars (Curtis et al., 1999) even though, at an early developmental stage (22-d-old plants), some of these transgenic lettuces showed a decrease in their leaf nitrate content. In the work presented here, six other lettuce cultivars were used (but only the results obtained with Jessy are presented) to verify if the results obtained by Curtis et al. (1999) were cultivar-dependent. Although 50 independent transgenic lines were studied, there was no success in reducing the leaf nitrate content in mature transgenic lettuce (corresponding to the size of the lettuce at harvest for market), using the p35S::Nia2 construction. In contrast to Curtis et al. (1999), a significant decrease in leaf nitrate content in younger plants (mid-stage culture) was not observed. It has been shown that these results were due to a decrease in NR mRNA derived from the transgene expression occurring early in plant development (first two leaves), to levels undetectable by northern blots upon ageing of the plants.
In transgenic lines showing symptoms of chlorosis, in addition to the transgene mRNA decay, a certain decrease in the quantity of the lettuce LsNia1 mRNA was also observed. Similar symptoms of chlorosis have been described in previous studies in tobacco (Dorlhac de Borne et al., 1994; Palauqui and Vaucheret, 1995; Palauqui et al., 1996), Arabidopsis (C Meyer, unpublished results), and potato (Djennane et al., 2002a) transformed with the same p35S::Nia2 transgene. In tobacco, it was demonstrated that this chlorosis was the result of PTGS directed specifically against the mRNA of the transgene and the mRNA of the endogenous NR gene (Dorlhac de Borne et al., 1994).
To confirm that a similar mechanism was occurring in the transgenic lettuce showing symptoms of chlorosis, an attempt was made to use the properties of PTGS suppressor shown by the HC-Pro component of some potyviruses (Marathe et al., 2000; Li and Ding, 2001; Mallory et al., 2001; Mette et al., 2001). Lettuce mosaic virus (LMV) is the most widespread virus in cultivated lettuce in the world (Dinant and Lot, 1992). Recently, infectious cDNA copies of some LMV isolates have been obtained, tagged with a green fluorescent protein (GFP) gene (German-Retana et al., 2001). Twenty days after the infection of chlorotic plants by LMV0-GFP, new green leaves developed on the infected plants whereas the non-infected control plants continued to develop yellowish leaves. Northern blot analysis of these plants showed the mRNA of the transgene and the endogenous NR gene only in the infected plants. These results demonstrate, firstly, that, like other potyviruses, the LMV0 is able to suppress the PTGS already installed in a plant and, secondly, that, as suspected, the symptoms of chlorosis observed in some of the lettuce lines are indeed due to a mechanism of PTGS directed specifically against the NR transgene and endogenous mRNAs. As demonstrated in this study, silencing properties of LMV can provide a useful tool to identify the occurrence of PTGS in lettuce, greatly improved by the visual non-destructive detection of the virus allowed by the GFP tag.
Of the four genotypes sensitive to chlorate (JSNR 5-5, 21-8, 45-12, and 58-7), only JSNR 21-8 showed symptoms of chlorosis during development. Compared with the other transformants that developed chlorosis, JSNR 21-8 was well characterized and mRNA decay specific to the transgene was observed initially and later in plant development (mid-culture stage) a silencing extending to the endogenous NR gene (symptoms of chlorosis). These results suggest that the PTGS mechanism observed in JSNR plants is firstly directed specifically against the Nia2 mRNA and subsequently extends to the endogenous NR mRNA. It is hypothesized that the delay in the appearance of chlorosis observed between the different transformants could be explained by differences in the level of expression of the transgene in these lines. This hypothesis suggests that the stronger the expression of the transgene, the sooner the PTGS would be extended to the endogenous NR mRNAs. The strong transgene expression observed in JSNR 60 after silencing of the PTGS by LMV0-GFP confirmed this hypothesis as JSNR 60 developed chlorosis in the T1 generation early in development.
For the three other transgenic genotypes sensitive to chlorate (JSNR 5-5, 45-12, and 58-7), mechanisms other than PTGS could be involved in the absence of accumulation of the transgenic mRNA as chlorosis symptoms were not observed in these plants. In the case of JSNR 5-5, Southern blot analysis has already shown that this genotype presents a double T-DNA insertion arranged as an inverted repeat (IR). Transgenes of T-DNAs that are organized as IRs often show low or no expression indicating that the genes are totally silenced or silenced to some degree by transcriptional gene silencing (TGS: Stam et al., 1997, 1998).
The Southern hybridizations revealed that 14 out of the 25 transgenic genotypes tested did not show a complete integration of the right border of the T-DNA. It is generally accepted that the right border is well preserved during the integration of a T-DNA. This could be explained by covalent binding of the VirD2 protein to the T-DNA right border during integration (Gelvin, 2000) which could protect it from exonuclease activities. This suggests that, as shown by the chlorotic phenotypes, strong expression of the transgene NR could be detrimental to the cells expressing it and hence counter-selected in vitro. This would favour plants showing an aberrant integration of the T-DNA and/or a low expression of the NR transgene.
It is well known that the PTGS mechanism is initiated earlier in R2 plants than in R1 plants of a given genotype (Fagard and Vaucheret, 2000; Pang et al., 1996). The study by Curtis et al. (1999) was conducted on R1 plants, which could explain the differences observed in nitrate content between the results of these authors and the results presented here since R2 plants were used in this study (homozygous for the transgene). A decrease in p35S promoter activity upon ageing of the lettuce plants was proposed by Curtis et al. (1999) to explain the differences they obtained between 22-d-old and 84-d-old plants. More recently, the p35S promoter has been used in a number of studies in lettuce, none of which mentioned any problems in transgene expression or stability (Dede, 1998; Kapusta et al., 1999, 2001; Mohapatra et al., 1999; McCabe et al., 1999a; Goto et al., 2000; Niki et al., 2001).
Taken together, these results suggest that the problems found with expressing a tobacco NR transgene in lettuce may be due to the NR sequence itself. Nitrate reduction could also be strongly regulated in lettuce due to the important role of nitrate as an osmoticum regulator in winter for this plant (Behr and Wiebe, 1992; Blom-Zandstra and Lampe, 1985; Blom-Zandstra et al., 1988). As in lettuce, insertion of the p35S::Nia2 transgene in Arabidopsis did not allow reduction in nitrate levels (C Meyer, unpublished results). All Arabidopsis transgenic plants developed a PTGS mechanism directed specifically against the transgene mRNA and the endogenous NR mRNA. Transgene efficiency was only obtained in a Solanaceae context: in tobacco (Dorlhac de Borne, 1993; Vincentz and Caboche, 1991) and potato (Djennane et al., 2002a). Thus, the transgene origin could be a determining factor in PTGS initiation. Use of a Ls.Nia transgene in lettuce could allow verification of this hypothesis.
This study strongly suggests that it would be very difficult to obtain lettuce with low leaf nitrate levels using the transgene p35S::Nia2, and also suggests that a PTGS mechanism directed against the transgene mRNA and initiated early in plant development is one of the explanations for the absence of accumulation of NR mRNA in lettuce.
|
Acknowledgements |
|---|
This work was partially supported by a grant from the French MENRT (French Ministry of Research). We thank Dr Yves Chupeau, Dr Hervé Vaucheret, Dr Françoise Rousselle-Bourgeois, Dr Jose Luis Garcia Martinez, Daphne Goodfellow, Rebecca Stevens, and Isabelle Quilleré for helpful discussions and S German-Retana for providing LMV-O-GFP recombinant virus. The authors are grateful to Jean Pierre Meunier for care of the plants in the Versailles glasshouse, Fabrice Flamain, Eric Martin, and Vérane Sarnette for technical assistance in plant culture, Doriane Bancel for help in the nitrate content measure, and Marie Therese Ledecker for help in NR activity determination.
|
Footnotes |
|---|
* Present address: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Avenida de Los Naranjos s/n, 46022 Valencia, España.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Behr U, Wiebe HJ. 1992. Relation between photosynthesis and nitrate content of lettuce cultivars. Scientia Horticulturae 49, 175–179.[CrossRef]
Bernatzky R, Tanksley SD. 1986. Genetics of actin-related sequences in tomato. Theoretical and Applied Genetics 72, 314–315.[CrossRef][Web of Science]
Blom-Zandstra G, Lampe JEM. 1985. The role of nitrate in the osmoregulation of lettuce (Lactuca sativa L.) grown at different light intensities. Journal of Experimental Botany 36, 1043–1052.
Blom-Zandstra G, Lampe JEM, Ammerlaan HM. 1988. C and N utilization of two lettuce genotypes during growth under non-varying light conditions and after changing the light intensity. Physiologia Plantarum 74, 147–153.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[CrossRef][Web of Science][Medline]
Campbell WH. 1999. Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology. Annual Review of Plant Physiology and Plant Molecular Biology 50, 277–303.[CrossRef][Web of Science][Medline]
Chupeau MC, Bellini C, Guerche P, Maisonneuve B, Vastra G, Chupeau Y. 1989. Transgenic plants of lettuce (Lactuca sativa) obtained through electroporation of protoplasts. Biotechnology 7, 503–508.[CrossRef]
Curtis IS, Power JB, de Laat AMM, Caboche M, Davey MR. 1999. Expression of a chimeric nitrate reductase gene in transgenic lettuce reduces nitrate in leaves. Plant Cell Reports 18, 889–896.[CrossRef]
Deblaere R, Butebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J. 1985. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer. Nucleic Acids Research 13, 4777–4788.
Dede Y. 1998. Development of the downy mildew pathogen Bremia lactucae on transgenic lettuce expressing a bacterial ß-1,3-glucanase. Turkish Journal of Agriculture and Forestry 22, 313–321.
Dinant S, Lot H. 1992. Lettuce mosaic virus. Plant Pathology 41, 528–542.
Dinant S, Maisonneuve B, Albouy J, et al. 1997. Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains. Molecular Breeding 3, 75–86.
Djennane S, Chauvin JE, Meyer C. 2002a. Glasshouse behaviour of eight transgenic potato clones with a modified nitrate reductase expression under two fertilization regimes. Journal of Experimental Botany 53, 1037–1045.
Djennane S, Chauvin JE, Quilleré I, Meyer C, Chupeau Y. 2002b. Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers. Transgenic Research 11, 175–184.[CrossRef][Web of Science][Medline]
Djennane S, Quillere I, Leydecker MT, Meyer C, Chauvin JE. 2004. Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs. Planta 219, 884–893.[Web of Science][Medline]
Dorlhac de Borne F. 1993. Obtention de tabacs industriels transgéniques a teneur réduites en nitrate. PhD thesis, Paris Sud University, France.
Dorlhac de Borne F, Vincentz M, Chupeau Y, Vaucheret H. 1994. Co-suppression of nitrate reductase host genes and transgenes in transgenic tobacco plants. Molecular and General Genetics 243, 613–621.
Fagard M, Vaucheret H. 2000. (Trans)gene silencing in plants: how many mecanisms? Annual Review of Plant Physiology and Plant Molecular Biology 51, 167–194.[CrossRef][Web of Science][Medline]
Gelvin SB. 2000. Agrobacterium and plant genes involved in t-DNA transfer and integration. Annual Review of Plant Physiology and Plant Molecular Biology 51, 223–256.[CrossRef][Web of Science][Medline]
German-Retana S, Redondo E, Le Gall O, Candresse T. 2001. The use of green fluorescent protein tagging to follow the lettuce mosaic virus invasion in succeptible and tolerant lettuce. 10th international MPMI meeting, Madisson.
Goto F, Yoshihara T, Saiki H. 2000. Iron accumulation and enhanced growth in transgenic lettuce plants expressing the iron- binding protein ferritin. Theoretical and Applied Genetics 100, 658–664.
Kaiser WM, Huber SC. 2001. Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. Journal of Experimental Botany 52, 1981–1989.
Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M, Lisowa O, Yusibov V, Koprowski H, Plucienniczak A, Legocki AB. 1999. A plant-derived edible vaccine against hepatitis B virus. The FASEB Journal 13, 1796–1799.
Kapusta J, Modelska A, Pniewski T, Figlerowicz M, Jankowski K, Lisowa O, Plucienniczak A, Koprowski H, Legocki AB. 2001. Oral immunization of human with transgenic lettuce expressing hepatitis B surface antigen. Advances in Experimental Medecine and Biology 495, 299–303.
Leprince AS. 1996. Isolement de lignées cellulaires de Nicotiana tabacum anergiées pour les besoins en cytokinines. PhD thesis, Paris-VI University, France.
Li WX, Ding SW. 2001. Viral suppressors of RNA silencing. Current Opinion in Biotechnology 12, 150–154.[CrossRef][Web of Science][Medline]
Mallory AC, Ely L, Smith TH, Marathe R, Anandalakshmi R, Fagard M, Vaucheret H, Pruss G, Bowman L, Vance VB. 2001. HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. The Plant Cell 13, 571–583.
Marathe R, Smith TH, Anadalakshmi R, Bowman LH, Fagard M, Mourrain P, Vaucheret H, Vance VB. 2000. Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing. The Plant Journal 22, 51–59.[CrossRef][Web of Science][Medline]
Mazier M, German-Retena S, Flamain F, Dubois V, Botton E, Sarnette V, LeGall O, Candresse T, Maisonneuve B. 2004. A simple and efficient method for testing lettuce mosaic virus resistance in in vitro cultivated lettuce. Journal of Virological Methods 116, 123–131.[CrossRef][Web of Science][Medline]
McCabe MS, Mohapatra UB, Debnath SC, Power JB, Davey MR. 1999a. Integration, expression and inheritance of two linked T-DNA marker genes in transgenic lettuce. Molecular Breeding 5, 329–344.
McCabe MS, Schepers F, van der Arend A, Mohapatra U, de Laat AMM, Power JB, Davey MR. 1999b. Increased stable inheritance of herbicide resistance in transgenic lettuce carrying a petE promoter-bar gene compared with a CaMV 35S-bar gene. Theoretical and Applied Genetics 99, 587–592.
Mette MF, Matzke AJM, Matzke MA. 2001. Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing. Current Biology 11, 1119–1123.[CrossRef][Web of Science][Medline]
Mohapatra U, McCabe MS, Power JB, Shepers F, van der Arend A, Davey MR. 1999. Expression of the bar gene confers herbicide resistance in transgenic lettuce. Transgenic Research 8, 33–44.
Niki T, Nishijima T, Nakayama M, Hisamatsu T, Oyama-Okubo N, Yamazaki H, Hedden P, Lange T, Mander LN, Koshioka M. 2001. Production of dwarf lettuce by overexpressing a pumpkin gibberellin 20-oxidase gene. Plant Physiology 126, 965–972.
Nussaume L, Vincentz M, Caboche M. 1991. Constitutive nitrate reductase: a dominant conditional marker for plant genetics. The Plant Journal 1, 267–274.
Palauqui JC, Elmayan T, Dorlhac de Borne F, Crété P, Charles C, Vaucheret H. 1996. Frequencies, timing, and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants. Plant Physiology 112, 1447–1456.[Abstract]
Palauqui JC, Vaucheret H. 1995. Field trial analysis of nitrate reductase co-suppression: a comparative study of 38 combinations of transgene loci. Plant Molecular Biology 29, 149–159.[CrossRef][Web of Science][Medline]
Pang SZ, Jan FJ, Carney K, Stout J, Tricoli DM, Quemada HD, Gonsalves D. 1996. Post-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene dosage and plant development. The Plant Journal 9, 899–909.[CrossRef]
Paszkowski J, Whitham SA. 2001. Gene silencing and DNA methylation processes. Current Opinion in Plant Biology 4, 123–129.[CrossRef][Web of Science][Medline]
Pietrzak M, Shillito RD, Hohn T, Potrykus I. 1986. Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Research 14, 5857–5868.
Quilleré I, Dufossé C, Roux Y, Foyer CH, Caboche M, Morot-Gaudry JF. 1994. The effects of deregulation of NR gene expression on growth and nitrogen metabolism of Nicotiana plumbaginifolia plants. Journal of Experimental Botany 45, 1205–1211.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd edn. New York: Cold Spring Habor Laboratory Press.
Stam M, Mol JNM, Kooter JM. 1997. The silence of genes in transgenic plants. Annals of Botany 79, 3–12.
Stam M, Viterbo A, Mol JNM, Kooter JM. 1998. Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for post-transcriptional silencing of homologous host genes in plants. Molecular and Cellular Biology 18, 6165–6177.
Vincentz M, Caboche M. 1991. Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants. The EMBO Journal 10, 1027–1035.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Mazier, E. Botton, F. Flamain, J.-P. Bouchet, B. Courtial, M.-C. Chupeau, Y. Chupeau, B. Maisonneuve, and H. Lucas Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco Plant Physiology, May 1, 2007; 144(1): 18 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kawachi, Y. Sunaga, M. Ebato, T. Hatanaka, and H. Harada Repression of Nitrate Uptake by Replacement of Asp105 by Asparagine in AtNRT3.1 in Arabidopsis thaliana L. Plant Cell Physiol., October 1, 2006; 47(10): 1437 - 1441. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||