Proteinase inhibitors from seeds: prospects for their use in plant protection

Seeds constitute a rich source of proteins that can inhibit specific enzyme groups. Thus far, the best characterised are inhibitors of enzymes which play an integral part in the germination process. These include the proteinase inhibitors, and as such they may play a role in the prevention of precocious germination. However, these proteins are members of multi-gene families which also encode iso-inhibitors that can retard the activity of proteinases specifically from mammalian, insect or bacterial (but not plant) origin. A proposed role for this latter group of isoinhibitors is as seed defensive factors, and it is this group of proteins that is attracting interest from the biotechnology industry. A large number of proteinase inhibitors have now been identified and evidence for their proposed roles in seeds will be summarised. In addition, their potential as a source of genes that can be transformed into crop plants to enhance field resistance against insects pests will be evaluated. Additional key words: insect pest resistance, plant biotechnology, transgenic plants. Dermitions: Proteinases and Proteinase Inhibitors In modem nomenclature, proteinases are a subgroup of the peptidases (= proteases), which consists of the endopeptidases (the proteinases; EC 3.4.21-99) and the exopeptidases (EC 3.4.11-19). According to the mechanism of catalysis, proteinases can be grouped further into 'serine~, 'cysteine' (thiol), 'aspartic' (acid) and 'metallo' -proteinases which have been assigned the IUB designations of EC 3.4.21-24, respectively. The serine proteinases are the largest, most widespread and diverse group of these enzymes, and are found in both prokaryotes and eukaryotes. They consist of two superfamilies (the chymotrypsin superfamily and the subtilisin superfamily) and all enzymes in this group are characterised by the presence of two amino acids at the active site that are invariably serine and histidine. The cysteine proteinases are also widespread, and contain cysteine and histidine as the conserved amino acids at the catalytic site. The aspartic proteinases have thus far only been identified in eukaryotes. These enzymes are characterised by a ph optima usually in the range pH 3.55.5, and aspartic acid as the catalytically active amino acid. Metalloproteinases are found in both prokaryotes and eukaryotes, and are characterised by zinc (in most cases) being the catalytically active metal (Barrett, 1986; and references therein). Proteinase inhibitors are themselves proteins, and by definition, can repress the catalytic activity of proteinases (Laskowski and Kato, 1980). In accord with the proteinases, it is appropriate to group proteinase inhibitors into four major classes: serine, cysteine, aspartic and metalloproteinase inhibitors. Within each class, these inhibitors can be grouped further, usually into distinct families, with assignation to a particular family dependent upon amino acid sequence homology. For instance, at least 16 gene families of protein inhibitors of serine proteinases have been identified thus far (Garcia-Olmedo et al., 1987; Bode and Huber, 1992). In common with proteinases, the inhibitors of these enzymes also have specific amino acids at discrete sites (designated the reactive sites), where hydrolysis by the proteinase occurs. These amino acids occur as a pair, and it is these residues that determine the specificity of the enzyme:inhibitor interaction. However, and unlike natural peptide substrates, the consequence of cleavage at this site by the proteinase is comparatively trivial since Seed Symposium 1991. 107 Proteinase inhibitors from seeds for plant protection? the inhibitor binds essentially irreversibly into the active site of the enzyme (Laskowski et al., 1983; Ryan, 1989; Hubbard et al., 1991). Three facets commonly distinguish the enzyme I inhibitor association and together these account for an essentially stable and (in kinetic terms) an irreversible