Plant Science 159 2000 289 – 299
Temporal and spatial activity of a promoter from a pea enzyme inhibitor gene and its exploitation for seed quality improvement
T. Welham, C. Domoney
John Innes Centre, Norwich Research Park, Norwich NR
4 7
UH, UK Received 31 March 2000; received in revised form 28 July 2000; accepted 31 July 2000
Abstract
The promoter from one of the two seed-expressed genes encoding trypsinchymotrypsin inhibitors TI has been isolated and characterised in transgenic pea lines, following its re-introduction by Agrobacterium-mediated transformation, as a TI promoter-b-
glucuronidase GUS gene fusion. The promoter from this gene TI
1
directed expression of GUS enzyme at late stages of embryogenesis, comparable to those determined for activity of the homologous native TI genes. GUS expression was detected in
roots of plants subjected to drought stress conditions, indicating that the TI
1
gene, normally seed-specific in its expression, can be induced under these conditions. A second gene construct utilised the TI
1
gene promoter to direct expression of an antisense TI gene. Seed TI activities in some lines transformed with this construct were reduced significantly. A limitation of the pea
transformation methodology for antisense manipulations, in particular, is the observed frequency of non-transmission of transgenes from primary transformants up to 80. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Antisense gene; Drought response; Pea transformation; Pisum sati6um; Trypsin inhibitor www.elsevier.comlocateplantsci
1. Introduction
Legume seeds contain a range of components which are considered to be antinutritional. Among
these are the enzyme inhibitor proteins that can be potent inhibitors of animal digestive enzymes. As
a result, these proteins can limit the extent to which legume seeds are included in animal diets.
However, several plant enzyme inhibitors have been linked to protective functions in plants, par-
ticularly as protective agents against insect pests [1 – 3] and bacterial pathogens [4] and many in-
hibitors are wound-inducible [5]. Protease in- hibitors may also be involved in protecting seed
proteins, either by preventing premature hydroly- sis of storage proteins or by acting as enzyme-sta-
bilising agents [6]. Some classes of legume protease inhibitors are rich in sulphur-containing amino
acids and therefore are of nutritionally desirable composition.
Many legume seed products are processed by heating andor pelleting in order to denature and
inactivate inhibitors, processes that are not only expensive but can be damaging to other nutrients.
Genetic manipulation offers the potential to sup- press inhibitor gene activity through the introduc-
tion and expression of an appropriate antisense gene. However, suppression of inhibitor synthesis
in seeds would need to be achieved ideally without interfering with the expression of the same or
related genes in other parts of the plant as a protective response. It is important, therefore, to
establish the roles played by individual inhibitor genes in order to determine the scope for interfer-
ing with the expression of some without being detriment to the plant.
In pea seeds, the major enzyme inhibitor proteins are capable of inhibiting the digestive
enzymes, trypsin and chymotrypsin, and belong to
The TI
1
gene sequence is accession number AJ276900 in the EMBL database.
Corresponding author. Tel.: + 44-1603-450000; fax: + 44-1603- 450045.
E-mail address
:
claire.domoneybbsrc.ac.uk C. Domoney. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 5 8 - 7
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
