Journal of Experimental Botany, Vol. 56, No. 419, pp.

23792388, September 2005

doi:10.1093/jxb/eri230 Advance Access publication 12 July, 2005

RESEARCH PAPER

Systematic silencing of a tobacco nitrate reductase

transgene in lettuce (Lactuca sativa L.)
Vincent Dubois1,*, Emmanuel Botton1, Christian Meyer2, Aline Rieu1, Magali Bedu2, Brigitte Maisonneuve1
and Marianne Mazier1,
1

Received 4 January 2005; Accepted 19 May 2005

Abstract
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 signicantly 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
(endogenous1transgenic) 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 specically 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-specic 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 specically to target transgenic
transcripts by a post-transcriptional gene silencing
(PTGS) mechanism during the rst stage of development of the plantlet.

Key words: Lettuce, mRNA, nitrate, nitrate reductase, PTGS,

transgenic plants.

Introduction
Nitrate (NO
3 ) 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)

* Present address: Instituto de Biologa Molecular y Celular de Plantas, Universidad Politecnica de Valencia, Avenida de Los Naranjos s/n, 46022 Valencia,
Espana.
y
To whom correspondence should be addressed. Fax: +33 4 32 72 27 02. E-mail: Marianne.Mazier@avignon.inra.fr
Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.

Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013

INRA, Unite de Genetique et Amelioration des Fruits et Legumes, UR 1052, domaine St Maurice BP 94,
F-84143 Montfavet cedex, France
2
INRA, Laboratoire de Nutrition Azotee des Plantes, UR 511, RD10 route de Saint-Cyr,
F-78026 Versailles cedex, France

2380 Dubois et al.

Materials and methods

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 8C. 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 8C, 8 h darkness at 12 8C). 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 lmol m2 s1), 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 lAbbe, 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 l1 kanamycin-containing regeneration
medium and transplanted individually onto rooting medium [macro
MS, micro Heller, vitamins B, sucrose 30 g l1, agar Biomar 6 g l1
(Quarrechim, 9 rue Paul Langevin BP 361 34504 Beziers Cedex,
France), pH 5.8] with 50 mg l1 kanamycin. Vigorous plants were
transplanted to pots containing peat in a growth chamber (22/16 8C,
16 h photoperiod), with manual irrigation with water, before transfer
to a glasshouse where they were watered each day with Coc-Lesaint
nutritive solution. Primary transformants (R0 generation) were selfpollinated 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).

Fig. 1. Structure of pSCK-NR T-DNA. The chimeric Nia2 gene was composed of the CaMV 35S promoter (p35S) derived from the expression cassette
of pDH51 (Pietrzak et al., 1986); the leader sequence (L), the coding sequence (Nia2 cDNA) and the 39 end (39) of the Nia2 gene from Nicotiana
tabacum (Vincentz and Caboche, 1991). The chimeric nptII gene driven by the nos promoter was used for the selection of the transformed plants. LB,
T-DNA left border; RB, T-DNA right border.

Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013

and reduced leaf nitrate content compared with wild types

(WT) (Quillere 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 NO
3 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 b-1,3-glucanase
(Dede, 1998), a b-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.

Nitrate reductase silencing in lettuce

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 Rene 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 23
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, 53 Denhart, 2.5 mM
EDTA, 5% dextran sulphate, 2.5 mg DNA salmon sperm) for 16
24 h at 65 8C. The filters were then washed once in 23 SSC and 0.1%
SDS for 20 min at 65 8C followed by one wash in 13 SSC and 0.05%
SDS for 20 min at 65 8C, and a final wash in 0.53 SSC and
0.05% SDS for 1020 min at 65 8C. Filters were exposed and
analysed by autoradiography.
RNA analysis
Total RNA from lettuce was prepared with the TRI REAGENT
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 Rene Razel, Saclay,
F-91898 Orsay cedex, France) in 103 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 1624 h at 65 8C. The filters were then washed four
times in 23 SSC and 0.1% SDS for 5 min at room temperature followed
by one wash in 0.23 SSC and 0.1% SDS for 510 min at 65 8C.

The probes (a Nia2 cDNA 119 pb fragment and a Ls.Nia1

120 pb fragment) were labelled by polymerase chain reaction.
Primers 1 (59-GGATTCTGCTGCATCATCACCAAATA-39) and 2
(59-TTCACGAGGGACTAAGGCTACGTTTCTTG-39) were used
for amplification of the ls.Nia1 probe and primers 3 (59-TGACTCTCCTGGCAACTCCGTGCACGGAT-39) and 4 (59-CTCTCTTGGAATTAGGGCCACACTCCTCT-39) for the Nia2 probe. Forty-five
cycles of amplifications were performed for both probes [94 8C
(1 min), 55 8C (1 min), and 72 8C (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 HEPESKOH, 10 mM MgCl2
(or 15 mM EDTA), 7.5 mM cysteine, 10 lM FAD, 5 lM leupeptin,
0.2 g PVP per 8 ml buffer, pH 7.6). The extract was centrifuged for
15 min at 5500 g at 2 8C and the soluble fraction assayed for NR
activity. The reaction mixture contained 800 ll of activity buffer
(50 mM HEPESKOH, 10 mM MgCl2, 10 mM KNO3, 180 lM
NADH, pH 7.6) and 100 ll of the extract. The reaction was carried
out for 25 min at 30 8C and was terminated by the addition of 100 ll
of 1 M sodium acetate. After 10 min of centrifugation (15 000 g),
the quantity of NO
2 formed during the enzymatic reaction was
determined at 540 nm after mixing 100 ll of reaction mixture in the
presence of 0.5 ml sulphanilamide (10 g l1 HCl 3N) and 0.5 ml
N-(1-naphthyl)ethylenediamine dihydrochloride (0.2 g l1 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 uorescence
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
Production and characterization of the
transgenic plants

Fifty independent transgenic plants of Lactuca sativa cv.

Jessy (primary transformants: R0) were obtained from

Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013

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) Inovchlore (Top Hygiene previously Inovchem; ZAC
des Peyrardes, 42 273 St Just St Rambert, France) in Petri dishes on
semi-solid basal rooting medium containing 75 mg l1 kanamycin.
After 48 h exposure in darkness at 4 8C, Petri dishes were transferred
to a growth room (20 8C, 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 l1, Hellers micro salts 13, and agar Biomar
6 g l1, pH 5.8). After 48 h exposure in darkness at 4 8C, Petri dishes
were transferred in a growth chamber (20 8C, 16 h photoperiod).

2381

2382 Dubois et al.

Fig. 2. Typical chlorosis symptoms observed in some transgenic plant

lines. Fully chlorotic plants at different stages of development. When cosuppression was initiated at an early stage of development, chlorosis
propagated quickly to the whole plant within 45 d. The affected plants
showed a slowing down of development and finally died quickly. WT,
wild-type genotype.

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

Table 1. Segregation analysis of kanamycin resistance in R1

progeny
R0 geno
R1 (selfed progeny from R0) nptII segregationa
type name
Number of
Number of
kanamycin-resistant
kanamycin-susceptible
plants
plants
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR

2
3
4
5
6
7
8
9
10
14
15
16
17
18
21
23
26
27
28
30
31
32
33
35
36
38
39
45
46
47
48
50
54
57
58
59

72
72
71
72
71
76
0
70
65
71
44
71
74
0
71
0
67
69
0
73
76
71
67
72
54
59
67
76
76
64
59
61
0
69
71
4

26
25
23
25
22
22
98
26
29
24
50
28
23
97
23
95
26
25
97
20
21
27
25
27
45
36
28
23
20
32
37
23
93
25
26
95

,b

23:1

0.12
0.03
0.01
0.03
0.09
0.34

0.22
1.72
0.03
39.84
0.57
0.09

0.01

0.67
0.13

0.61
0.58
0.34
0.23
0.27
22.09
8.42
1.07
0.16
0.89
3.55
9.39
0.25

0.13
0.17
273.65

a
Seeds obtained by selfing of the primary transformants (R1) were
germinated on a selective medium containing 75 mg l1 of kanamycin.
b 2

3:1 > 3:84: significantly different from the hypothesis of a single
insertion (P <0.05).

Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013

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 l1 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

Nitrate reductase silencing in lettuce

2383

Table 2. Chlorate sensitivity of R2 seedlings

Seeds obtained by selfing of the R1 transformants (R2) were sown on
medium containing 0.5 mM of potassium chlorate.
Genotypes

Root development (cm)a

Leaf developmentb

WT
JSNR
WT
JSNR
WT
JSNR
WT
JSNR

33.164.2
19.263.8***c
35.665.1
13.262.1***
40.764.0
15.365.1***
32.265.8
15.763.6***

3.660.4
1.660.5***
3.260.7
0.560.2***
3.460.6
1.360.3***
3.260.7
0.760.4***

5-5
21-8
45-12
58-7

Root development was determined by measuring the root length.

Leaf development was determined by classifying each plant in one of
the five following groups: 0, only cotyledons developed; 1, one fully
developed leaf; 2, two fully developed leaves; 3, three fully developed
leaves; 4, four fully developed leaves.
c
***Mean is less than the mean of the control seedlings at P <0.001%.
b

Total DNA (12 lg) of each plant was digested by EcoRV or HindIII and
hybridized to a nptII or a p35S probe (see Fig. 1 for Southern
hybridization results).
R0 genotype name

JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR
JSNR

2
3
4
5
6
7
9
10
14
16
17
21
26
27
30
31
32
33
35
39
45
46
47
50
57
58

nptII probea

p35S probeb

EcoRV

HindIII

EcoRV

HindIII

1
1
1
2
1
1
1
1
4
1
1
1
1
1
1
1
1
1
1
1
1
2
1
3
1
1

1
1
1
2
1
2
1
1
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
1
1

0
0
0
1
0
0
1
0
4
0
0
1
1
0
0
0
1
0
2
0
1
1
0
2
0
1

0
0
0
1
0
0
1
0
3
0
0
1
1
0
0
0
1
0
2
0
1
1
0
2
0
1

Number of bands which hybridized to an internal 0.8 kb fragment

from the nptII coding sequence (nptII probe).
b
Number of bands which hybridized to an internal 0.5 kb fragment
from the sequence of the 35S promoter from CaMV (p35S probe).

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 differ-

Fig. 3. Nitrate content of transgenic JSNR R2 genotypes. Plants were

cultured in the greenhouse and harvested at maturity (90 d after sowing).
Each genotype was present on three parcels (each one composed of three
plants of the WT and three plants of the corresponding transgenic
genotype) randomly distributed in the greenhouse. Fresh weight was
measured at maturity (a). Dry weight was determined after drying the
sample for 3 d at 70 8C (b). Nitrate content was determined by colorimetry
using a Aquatec 5400 Analyser (Tecator) (c). Each plot represents the
average of the three plants of a parcel. Results were statistically analysed
and no significant differences were obtained between WT and transgenic
genotypes for the three characters analysed.

ences 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 lmol m2 s1). 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).

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Table 3. T-DNA integration in primary transformants (R0):

overall results obtained by Southern blot on 26 R0 genotypes

2384 Dubois et al.

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

Fig. 5. Detection of LsNia and NtNia2 mRNAs in cotyledons of four

selected R2 genotypes sensitive to chlorate. Total RNA was isolated from
cotyledons (30 lg lane1) and analysed by northern blot with a probe
which covers an internal 120 bp fragment from the L. sativa Nia gene
(LsNia) coding sequence or with a probe which covers an internal 119 bp
fragment from the N. tabacum Nia2 gene (NtNia2) coding sequence.
(a, c) Plantlets grown in vitro on medium B without 0.5 mM potassium
chlorate (containing glutamine as the only nitrogen source). (b, d,)
Plantlets grown in vitro on basal medium A (containing nitrate as
nitrogen source). WT, wild-type lettuce; N. tabacum, N. tabacum
XHFD8.

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.

Fig. 4. Presence of LsNia and NtNia2 mRNAs in 45-d-old plants. Plants

were cultivated in a hydroponic system. Total RNA were isolated from
the leaves (30 lg lane1) and analysed by northern blot with a probe
which covers an internal 120 pb of the Lactuca sativa Nia gene (LsNia)
coding sequence or a probe which covers an internal 119 pb of the
Nicotiana tabacum Nia2 gene (NtNia2) coding sequence. WT: wild-type
lettuce; N. tabacum: Nicotiana tabacum XHFD8.

Fig. 6. Presence of LsNia and NtNia2 mRNAs in the two first leaves of
some R2 transgenic lines sensitive to chlorate. Total RNA was isolated
from the leaves (30 lg lane1) and analysed by northern blot with a probe
which covers an internal 120 bp fragment from the L. sativa Nia gene
(LsNia) coding sequence or a probe which covers an internal 119 bp
fragment from the N. tabacum Nia2 gene (Ntnia2) coding sequence. (a, c)
Plantlets grown in vitro on medium B without 0.5 mM potassium chlorate
(containing glutamine as the only nitrogen source). (b, d) Plantlets grown
in vitro on basal medium A (containing nitrate as nitrogen source). WT,
wild-type lettuce; N. tabacum, Nicotiana tabacum XHFD8.

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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.

Nitrate reductase silencing in lettuce

2385

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
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 cultivardependent. 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.

Fig. 8. Reversion of nitrate reductase silencing 20 d post-inoculation in

a JSNR chlorotic genotype with LMV-0-GFP. (a) JSNR 60 R1 plants
were grown on soil in pots and showed the typical chlorotic symptoms
due to the silencing of nitrite reductase (plant 1). Infection with LMV-0GFP reverted the nitrate reductase silencing and the plants turned green
(plants 2 and 3) (b) Northern blot analysis showing the levels of LsNia or
NtNia2 RNA in JSNR 60 plants infected or not infected by LMV-0-GFP.

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

Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013

Fig. 7. Visualization under UV light of green fluorescent protein (GFP)

in LMV0-GFP-infected lettuce (Lactuca sativa cv. Jessy) 20 d postinoculation. 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).

2386 Dubois et al.

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; BlomZandstra 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,

Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013

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

Nitrate reductase silencing in lettuce

Dr Herve Vaucheret, Dr Francxoise Rousselle-Bourgeois, Dr Jose
Luis Garcia Martinez, Daphne Goodfellow, Rebecca Stevens, and
Isabelle Quillere 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 Verane 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.

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