Plant Molecular Biology 37: 561–569, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Expressing foreign genes in the pistil: a comparison of S-RNase constructs in
different Nicotiana backgrounds
Jane Murfett and Bruce A. McClure
Department of Biochemistry, 117 Schweitzer Hall, Columbia, MO 65211, USA (
author for correspondence)
Received 19 September 1997; accepted in revised form 22 January 1998
Key words: Nicotiana, pollination, promoter, S-RNase, transgenic plants
Transgenic plant experiments have great potential for extending our understanding of the role of speciﬁc genes
in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on
pollination. We have examined the efﬁciency of six different S-RNase constructs in Nicotiana species and hybrids.
gene. Among the six expression constructs, two utilized the cauliﬂower mosaic virus (CaMV) 35S promoter with
duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included
promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S
-RNase gene, and a promoter
from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia,
SI N. alata S
hybrids, N. langsdorfﬁi,andN. langsdorfﬁi
SC N. alata hybrids.
Stylar speciﬁc RNase activities and S
-RNase transcript levels were determined in transformed plants. Constructs
including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of
-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was
observed in post-anthesis styles. Expression levels of the S
-RNase transgenes was dependent on the genetic
backgroundof the host. Higher levels of S
-RNase expressionwere observedin N. plumbaginifolia
SC N. alata
hybrids than in N. plumbaginifolia.
S-RNases are stylar glycoproteinsthat areknownfrom
genetic and molecularstudies to control the speciﬁcity
of pollen rejection in self-incompatible (SI) species in
the Solanaceae, Rosaceae, and Scrophulariaceae, and
to contribute to controlling some types of interspecif-
ic pollination [4, 27, 28, 29, 33]. We are interested
in manipulating S-RNase expression to model pollen
rejection systems in transgenic plants. Therefore, we
have evaluated the efﬁciency of several systems for
expressing foreign genes in the pistil.
The most important considerations in designing
expression constructs are to choose an appropriate
promoter and genetic background for transformation.
Several promoters from genes expressed primarily or
exclusivelyin the pistil have been described and tested
for activity in transgenic plants. The tomato 9612 and
9617 cDNA were among the ﬁrst sequences described
that were expressed predominantly in the pistil .
The corresponding promoters were isolated and used
to drive expression of the GUS reporter gene in trans-
genic plants. In transgenic tomato, a 1.2 kb promoter
fragment from the 9612 gene was used to drive GUS
expression in pistils and anthers, but the same con-
struct was not expressed in N. tabacum . The gene
corresponding to the 9617 cDNA has been referred to
as ChiP [1, 23, 24, 27, 34] but current nomenclature
refers to this gene as Chi2;1 . A 1.4 kb promoter
fragment from the Chi2;1 gene has been shown to
direct GUS expression in pistils with lower levels of
expression detected in stamens, sepals and petals .
The promoter from the tobacco sp41a gene, encod-
-1,3-glucanase that is highly expressed in the
transmitting tract, has also been shownto give efﬁcient
reporter gene expression .