Plant Molecular Biology 41: 105–114, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Expression and sequence requirements for nitrite reductase
Laboratoire de Biologie Cellulaire, INRA, 78026 Versailles Cedex, France (
author for correspondence);
address: Friedrich Miescher Institute, P.O. Box 2543, 4002 Basel, Switzerland
Received 28 September 1998; accepted in revised form 19 July 1999
Key words: co-suppression, nitrite reductase, tobacco, transgene
We have previously reported that the introduction of a full-length tobacco nitrite reductase Nii1 cDNA under the
control of the 35S promoter triggers co-suppression of endogenous Nii genes in 25% of tobacco transformants.
Here we show that introduction of chimeric Nii1-uidA, uidA-Nii1 and Nii1-uidA-Nii1 transgenes carrying 186 bp
of the 5
end and/or 241 bp of the 3
end of the Nii1 cDNA do not trigger co-suppression of endogenousNii genes. In
addition, we show that when introduced by crossing or transformation into co-suppressed transgenic tobacco lines
carrying full-length Nii1 transgenes, these chimeric transgenes are not silenced. These results therefore suggest that
ends of the Nii1 cDNA are not sufﬁcient to trigger co-suppression and are not targets for homology-
dependent RNA degradation. Surprisingly, co-suppression was released in a double transformant obtained by
introduction of one of these constructs into the co-suppressed transgenic tobacco line 461-2.1 homozygous for
a full-length Nii1 transgene, and in one plant regenerated from untransformed leaf discs (plant 461-2.1
reappearance of co-suppression at very low frequency (less than 10
progeny of plant 461-2.1
apparent absence of structural modiﬁcation of the transgene locus suggest a metastable epigenetic modiﬁcation.
The steady-state level of Nii mRNAs in the plant 461-2-.1
was higher than in wild-type plants but lower than in
hemizygous plants 461-2.1 which never trigger silencing. These results therefore conﬁrm that transcription of the
transgene above a particular threshold is required to trigger co-suppression.
In recent years, it has been demonstrated that introduc-
tion into plants of a transgene containing sequences
homologous to an endogenous gene can result in si-
lencing (also called co-suppression) of the transgene
and of all homologous host gene copies (see Meyer,
1995; Stam et al., 1997; Depicker and Van Montagu,
1997 for recent review). As opposed to transcrip-
tional gene silencing, which is characterized by (1) a
block of transcription activity, (2) a heavy methylation
in promoter sequences, and (3) meiotic heritability
of silencing and methylation (Meyer et al., 1993;
Vaucheret, 1993; Neuhuber et al., 1994; Park et al.,
1996), post-transcriptionalgene silencing (PTGS) (co-
suppression) is characterized by (1) normal transcrip-
tion rates in the nucleus whereas the corresponding
mRNAs do not accumulate into the cytosol, (2) an oc-
casional methylation in the transcribed region, and (3)
meiotic reversibility of silencing and progressive in-
activation during development at each generation (De
Carvalho et al., 1992; Hobbs et al., 1993; Vaucheret
et al., 1995; English et al., 1996).
Various hypotheses have been formulated to ex-
plain PTGS of transgenes and homologous host genes.
One is a biochemical switch model in which the over-
expression of a gene product up to a threshold level
could increase the speciﬁc degradation of this product
(Meins and Kunz, 1994). Another model was pro-
posed where transgenes produce unintended antisense
RNAs (Grierson et al., 1991). However, there has
been considerable debate on whether these antisense
RNAs are involved in post-transcriptional silencing.
Jorgensen et al. (1996) have concluded that partic-