Plant Molecular Biology 48: 319–329, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
319
Three classes of proteinase inhibitor gene have distinct but overlapping
patterns of expression in Pisum sativum plants
Claire Domoney
∗
, Tracey Welham, Noel Ellis, Philippe Mozzanega and Lynda Turner
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK (
∗
author for correspondence; e-mail
claire.domoney@bbsrc.ac.uk)
Received 20 March 2001; accepted in revised form 11 September 2001
Key words: flower, gene expression, pea, proteinase inhibitor, root, trypsin inhibitor
Abstract
Genes representative of three gene classes encoding proteinase inhibitor proteins, with distinct spatial expression
patterns, were isolated and characterized from Pisum. Under standard plant growth conditions, one class is ex-
pressed exclusively in seeds, whereas the other two make minor contributions to seed inhibitor proteins but are
also expressed in other organs, predominantly in root endodermal and floral reproductive tissues. Two of the gene
classes contain few genes and are genetically linked at the Tri locus, whereas the third class displays complex
hybridization patterns to genomic DNA and maps to diverse genetic loci. Expression analysis of this last class
suggests that only a small number of these genes are expressed. The quantitative effect of the Tri locus on root
and floral inhibitor gene expression was examined in near-isogenic lines of pea. The proteins encoded by the three
classes are all members of the same family (Bowman-Birk) of enzyme inhibitors but are distinct in terms of overall
sequence, active site sequences and inhibitor function.
Introduction
Legume seeds contain proteins that are classified as
antinutritional based on their ability to inhibit animal
digestive enzymes. Such seed proteinase and amy-
lase inhibitors are therefore a target for improvements
in seed quality for animal feed. As a result, geno-
types with low inhibitor activity are selected as parents
in breeding programmes (Muel et al., 1998). How-
ever, both proteinase and amylase inhibitors have been
demonstrated to be effective in plant protection against
insect attack (Shewry and Lucas, 1997) and trans-
genic plants expressing heterologous inhibitor proteins
have shown the potential for exploitation of such plant
genes for crop improvement (Schroeder et al., 1995;
Duan et al., 1996; Xu et al., 1996; Graham et al.,
1997; Charity et al., 1999). Indeed in the case of
The nucleotide sequence data reported are in the EMBL, GenBank
and DDBJ Nucleotide Sequence Databases under the accession
numbers AJ276900 (TI1 gene), AJ296170 (TI6 gene), AJ291493
(TI9 gene), AJ296169 (TI6 gene class cDNA) and AJ296171 (TI6
gene class cDNA).
the complex family of amylase inhibitors and closely
related proteins in Phaseolus, it is only through trans-
genesis that the insect targets of the individual proteins
have been clearly defined (Altabella and Chrispeels,
1990) and small numbers of changes in amino acid
sequence have been implicated in gross changes in in-
hibitor: enzyme specificity (Mirkov et al., 1994, 1995;
Grossi de Sa et al., 1997). It is desirable, therefore, to
define the roles of individual inhibitors, as agents of
plant protection through inhibition of insect digestive
systems and as inhibitors of animal digestive enzymes
in feed, to permit optimal manipulation of the relevant
genes through plant breeding programmes or through
biotechnology.
In Pisum (pea), the major seed trypsin/chymotryp-
sin inhibitors have been characterized and genetically
mapped (Domoney et al., 1994, 1995) and have been
implicated in plant responses to water-deficit stress
(Welham and Domoney, 2000). The influence of the
genetic locus encoding these inhibitors on animal di-
gestion and performance is being investigated through
the production and testing of near-isogenic lines