the Bowman – Birk class of inhibitors, based on sequence homology [7,8]. Two closely linked genes
encode the major pea seed inhibitors TI and map to a genetic locus on linkage group V [8,9]. A
proportion of the primary products of the two genes is post-translationally processed at the C-ter-
minus to produce isoforms which are more potent inhibitors of digestive enzymes [8]. Neither of the
two genes is expressed in vegetative organs of plants grown under normal environmental condi-
tions. However, one of the two was found to be expressed in roots of pea plants grown under
drought conditions [10], suggesting a possible physiological function for some of these proteins
in dehydrating tissue.
The high degree of homology between the two genes encoding the major seed TI suggests that a
single antisense gene could interfere with the ex- pression of both genes. However, an antisense TI
gene would need to be controlled by a promoter specifying its activity during the very late stages of
embryogenesis when expression of the two ho- mologous sense genes is maximal. Since such a
promoter had not been characterised previously, we have isolated the promoter from one of the TI
genes for construction of an antisense gene. We have used the promoter from the TI gene for
which the corresponding RNA had not been de- tected in any organ other than seeds. In this paper
we describe the behaviour of this promoter as a fusion with a GUS reporter gene during embryo
development in transgenic pea lines and in trans- genic plants subjected to drought stress. The ex-
ploitation of this promoter for an antisense TI gene and its introduction into transgenic peas is
also reported.
2. Materials and methods
2
.
1
. Plant material Seeds for transformation experiments were
Pisum sati6um cultivar cv. Puget wrinkled- seeded, obtained from Advanta Seeds UK, Lin-
coln, and the genotype BC117 round-seeded, a gift from C. Hedley, John Innes Centre. Both
batches of seeds were free from fungal contami- nants when used in tissue culture experiments.
All plants produced from transformation experi- ments were grown in John Innes No. 1 compost
with 30 extra grit. All primary transformants T
1
plants and the majority of T
2
and T
3
plants were grown in a controlled environment room with 16 h
light at 15°C, 8 h dark at 12°C and 70 relative humidity. A small number of T
2
plants were grown in a glasshouse with light supplementation
during winter. Immature embryos were harvested from plants
which were grown in the controlled environment rooms. Flowers were tagged on the day they were
fully open and embryos harvested at 33, 42 and 53 days after flowering.
Plants subjected to drought stress were sown in the controlled environment room and watered
daily for 26 days. After this period, some plants control were watered as before, whereas water
was withheld from a proportion of the plants. After a further 16 days, roots, leaves and stems
were harvested from control and drought-treated plants, frozen in liquid nitrogen and stored at
−
70°C.
2
.
2
. Vectors and bacterial strains
2
.
2
.
1
. TI promoter — glucuronidase gene construct
RSGIT Several genomic clones that hybridised to the
insert from a TI cDNA clone pTI 4 – 41, corre- sponding to one of two seed-expressed TI genes
[8,9], were isolated from a genomic library. The library, constructed using DNA from the pea cv.
Birte [11], was a gift from C. Forster and R. Casey. Clones that hybridised strongly to the
probe were analysed in detail; the transcribed re- gion of the TI gene in one clone TI
1
was identi- cal to the cDNA, pTI 12 – 36, representing the
second seed-expressed TI gene [8]. Most of the region immediately upstream of the transcribed
sequence was isolated from this genomic clone, following subcloning into a Bluescript vector, as a
1 kb KpnI-BglII promoter fragment. The se- quence between the BglII site in the promoter and
the transcription start in the TI gene was synthe- sised as a pair of complementary oligonucleotides
with the BglII site at one end and a HindIII site at the other, the latter site to facilitate ligation to
marker genes.
The TI
1
gene promoter was cloned into the plasmid vector, pJIT 166, containing a ß-glu-
curonidase GUS
coding sequence
with a
cauliflower mosaic virus CaMV terminator [12],
following removal of the double 35S CaMV pro- moter from this vector with KpnI and HindIII.
The resulting plasmid, pJP04, therefore, contained a TI gene promoter that included an oligonucle-
otide linker to the GUS gene, but no TI gene transcribed sequence. The TI promoter-GUS gene
was excised from pJP04 using XhoI and cloned into the binary vector RS66; the vector RS66 is
one of a series of vectors that includes E6 nos bar; see below and contains a 35S CaMV pro-
moter-bar gene-CaMV terminator cassette Fig. 1A. The bar gene encodes a modifying enzyme,
phosphinothricin acetyltransferase, that facilitates selection of transformed plants [13]. The TI-GUS
TIG gene fusion was inserted in both orienta- tions giving the plasmids RSGIT and RSTIG;
only results obtained with RSGIT are reported here. Both RSTIG and RSGIT plasmids were
shown to be active in a transient expression assay based on GUS enzyme detection in pea axes fol-
lowing particle bombardment [14].
2
.
2
.
2
. TI promoter-antisense TI construct E
6
ASTI A near-full-length TI cDNA in pUC 19 pTI
38-38 was isolated from a library as previously described [9]. The sequence of this cDNA is identi-
cal to that of pTI 5-72 [8], except that it is two bases shorter at the 5 end. A SphI site that exists
midway in the mature protein sequence was used to clone the TI
1
gene promoter as a HindIII – SphI fragment such that the cDNA was in antisense
orientation relative to the promoter. For this cloning, the TI
1
promoter was isolated as a HindIII – BglII
fragment and
complementary oligonucleotides used as above to provide the re-
gion between the BglII site and the transcription start in the TI
1
gene; in this case, a BglII – SphI fragment was provided by the annealed oligonu-
cleotides. The TI
1
-antisense TI gene was cloned as a HindIII – BamHI fragment into the binary vec-
tor, E6 nos bar one of a series described by Schneider et al.[15]; the vector employed here
contained a bar gene with a CaMV terminator under the control of a nopaline synthase nos
promoter,
giving the
plasmid E6ASTI.
The BamHI site at the 5 end of the cDNA was used
to clone a nos terminator as a BamHI – BglII fragment from the plasmid, pJIT 2 a gift from P.
Mullineaux, giving the plasmid E6ASTIJIT Fig. 1B; only results obtained with E6ASTIJIT are
reported here.
The plasmids containing the above constructs were transformed into Agrobacterium tumefaciens
EHA 105 [16] by electroporation.
2
.
3
. Pea transformation Peas were transformed as described in [17].
Briefly, cotyledonary meristems of germinating seeds were inoculated by making cuts with a
scalpel blade which had been dipped in a culture of A. tumefaciens carrying either RSGIT or
E6ASTI plasmids. Inoculated explants cotyledon pieces with attached axes were cultured on selec-
tion medium according to [17]. Shoots produced by explants were excised and placed directly onto
Fig. 1. Diagrammatic representations not to scale of the two constructs A and B and the TI
1
promoter C used in transformation experiments. A The cloning of a TI
1
pro- moter-GUS gene with a CaMV terminator into the polylinker
PL of the binary vector RS66. The resulting plasmid is RSGIT. A 2.8 kb fragment can be excised from this construct
using EcoRI see Fig. 2. B The cloning of a TI
1
promoter- antisense TI gene with a nopaline synthase nos terminator
into the polylinker PL of the binary vector E6 nos bar. The resulting plasmid is E6ASTIJIT. Long arrows show the
direction of transcription from the transgenes. The promoter for the bar gene differs in the two vectors. C The positions
of features within the promoter, described in the text, relative to the transcription start in the TI
1
gene.
selective media
containing 3.75
mgl phos-
phinothricin PPT. Shoots surviving selection were grafted onto root stocks provided by non-
transgenic seedlings.
2
.
4
. Analysis of transgenic plants and seeds Primary transformants T
1
were analysed by painting 2 – 4 leaflets with the herbicide, Herbiace,
at 3 mgml [17].
2
.
4
.
1
. GUS acti6ity measurements of plants transformed with RSGIT
Mature T
2
seeds were assayed for GUS activity. A small meal sample was taken from individual
seeds by drilling a small hole in the cotyledons away from the axis. Meal was extracted with 100
mM Tris, 150 mM NaCl pH 8 250 mg meal per ml buffer for 1 h at 4°C with continuous stirring.
The meal residue was removed by centrifugation and GUS activity in the supernatant was esti-
mated using a GUS-light™ kit Tropix. Between 1 and 5 ml of the extracts were assayed using 200
ml diluted glucuron substrate, according to the manufacturer’s instructions. Emitted light was
measured as relative light units RLU using a luminometer Lumat 9501. The total protein con-
centration of supernatants was determined using a dye-binding kit Bio-Rad and bovine serum albu-
min as a standard.
Immature embryos from T
2
plants transformed with the RSGIT construct were harvested at three
different stages of development measured by days after flowering. Three embryos from every stage
were ground using a pestle and mortar and ex- tracted with 100 mM Tris, 150 mM NaCl pH 8
200 mg fresh weight per ml buffer. GUS activity was measured using supernatants as before.
Vegetative organs from drought-treated and control watered plants were freeze-dried and
ground to a fine powder using a pestle and mortar. The samples were extracted with buffer and GUS
activity measured as described for mature seeds.
2
.
4
.
2
. Southern blot analysis DNA was prepared from 1 to 2 leaves using a
small scale DNA preparation method [18]. DNA was digested with EcoRI and run on 0.8 agarose
gels. DNA was blotted onto nitrocellulose and hybridised [19] with a [
32
P]-labelled bar gene probe and, where appropriate, a GUS gene probe. Bar
and GUS DNA for labelling was isolated from the plasmids pJIT 84 and pJIT 166, respectively, using
appropriate restriction enzymes and labelled using an oligolabelling kit Pharmacia. Blots were
washed in 0.1 × SSC, 0.1 SDS at 50°C and exposed to pre-flashed X-ray film at − 70°C.
2
.
4
.
3
. Northern blot analysis Total RNA was extracted from organs of plants
subjected to drought or control conditions and analysed by Northern hybridisation as previously
described [20]. Blots were hybridised as outlined for Southern blots using a GUS probe or a trypsin
inhibitor cDNA probe; the latter was derived from the plasmid pTI 4 – 41 [8,9]. Blots were washed in
0.1 × SSC, 0.1 SDS at 50°C and exposed to pre-flashed X-ray film at − 70°C. Autoradio-
graphs
were scanned
using a
Joyce-Loebl Chromoscan.
2
.
4
.
4
. Western blot analysis Organs from drought-treated and control plants
were extracted directly in sample buffer 50 mg freeze-dried and ground material per ml buffer
and boiled, or were extracted with 100 mM Tris 150 mM NaCl pH 8 50 mg freeze-dried and
ground material per ml buffer and the protein concentration
of supernatants
determined as
above; supernatants were diluted 1:1 with 2 × sample buffer and boiled. Sample aliquots, based
on equivalent dry weights or equivalent protein amounts, were analysed on sodium dodecyl sul-
phate-polyacrylamide gels SDS-PAGE. Condi- tions for SDS-PAGE were as described in [21],
with the exception that mercaptoethanol in the sample buffer was replaced by dithiothreitol
DTT at 17mM. Separated polypeptides were electroblotted onto nitrocellulose and blots pro-
cessed [21], using anti-ß-glucuronidase-rabbit IgG Molecular Probes as a primary antibody for
detection of GUS protein on blots.
2
.
4
.
5
. Trypsin inhibitor TI
measurements Meal samples from individual T
3
seeds from plants transformed using E6ASTI were extracted
in 0.05 M HCl 25 mg meal per ml. TI activity measurements were performed as previously de-
scribed [10,22], with the modification that 0.05 M Tris pH 7.5 was used in the assay instead of 0.01
M NaOH.
2
.
5
. Statistical methods Segregation ratios were analysed using a X
2
analysis. A two-sample t-test Minitab was applied in
the analysis of TI activity measurements and com- parison of populations of T
3
seeds.
3. Results
