The proteinase inhibitors of plants and micro-organisms

To understand the present widespread interest in protease inhibitors (PI), one must look back to the vast amount of research that has been conducted for over 50 years. Intensive investigations, like wave motions on the water, followed a highly cyclical pathway depending upon the interest and support for research in various disciplines. Nutritionists were concerned with the potential adverse effects of the inhibitors of trypsin and chymotrypsin in the intestinal tract of man and animals (Liener and Kakade, 1980; Liener, 1981; Rackis and Gumbmann, 1981); biochemists recognized that the peculiar properties of PI’s may yield fundamental information concerning protein-protein interactions especially the mechanism of proteolytic hydrolysis (Richardson, 1981; Ryan, 1979); medical scientists envisioned an important role for PI’s in the treatment of various metabolic disorders (Katunuma et al., 1983; Vogel et al., 1968; Weyer, 1968), and biologists foresaw an important physiological role of PI’s to regulate plant growth, protein synthesis and resistance to microbial and insect pests (Richardson, 1977, 1981; Ryan, 1973, 1979).

Protease inhibitors in plant foods: content and inactivation.

Genomic blotting of restriction fragments of Russet Burbank DNA indicated that at least 6 copies of Inhibitor I are present in the tetraploid potato genome. A library of potato genes in bacteriophage λ was screened for the presence of Inhibitor I genes using a wound-inducible tomato Inhibitor I cDNA as a hybridization probe. One phage with an insert of 13.1 kb was isolated that hybridized most strongly with the probe. A 4.2 kb Eco RI fragment containing the gene was isolated from the clone and 2.2 kb region was sequenced that included about 800 bp of both the 5′ and 3′ regions. The gene contained two introns of 479 and 417 bp respectively, and the splice junctions were typical of other eukaryotic genes. Putative TATAA and CAAT boxes were identified. The nucleotide sequence, when compared with a wound-inducible tomato Inhibitor I cDNA, exhibited over 90% identity. The gene codes for a prepro-Inhibitor I protein of 96 amino acids. The putative pre-sequence of 19 amino acids, differs in only one residue from that of tomato Inhibitor I. The potato pro-sequence, however, is lacking a tetrapeptide that is found in the tomato pro-sequence in the region of pro-peptide processing. This deletion, together with a substitution of a Gln for a Leu (4 residues toward the N terminus) provides an explanation for the differences at the N-termini between tomato and potato Inhibitor I natural proteins by providing different processing sites in the two pro-inhibitors. Thus, amino acid sequence differences between the N termini of tomato and potato Inhibitor I are easily explained by the mutational events. The different proposed pro-processing sites of the tomato and potato inhibitors suggest that a processing protease may be present in the vacuole with a specificity for Asn-X and Gln-X bonds.

Molecular characterization of a wound-inducible inhibitor I gene from potato and the processing of its mRNA and protein.

Seven small (approximately 6,000 D) wound-inducible proteinase inhibitor proteins were isolated from leaves of pepper (Capsicum annuum) plants that are members of the potato inhibitor II family. N-terminal sequences obtained indicated that the pepper leaf proteinase inhibitors (PLPIs) exhibit homology to two GenBank accessions that code for preproteins containing three isoinhibitors domains each that, when post-translationally processed, can account for the mixture of isoinhibitors that are reported herein from pepper leaves. A constitutive level of PLPI proteins was found in pepper leaves, and these levels increased up to 2.6-fold upon wounding of the lower leaves. Exposing intact plants to methyl jasmonate vapors induced the accumulation of PLPIs. Supplying excised young pepper plants with water through the cut stems induced PLPI proteins to levels higher than those found in intact plants, but with high variability. Supplying the excised plants with systemin did not result in an increase of PLPI levels that were statistically higher than levels found in excised plants. Gel-blot analyses of PLPI induction revealed the presence of two mRNA bands, having slightly different mobilities in agarose gels. Only the low M(r) mRNA is present in untreated control plants, and it appears to be responsible for the constitutive levels of PLPI found in leaves. Both mRNA species are wound- and methyl jasmonate-inducible. Only the low- M(r) species is weakly induced by systemin, indicating a differential expression of the two PLPI species.

Wound-inducible proteinase inhibitors in pepper. Differential regulation upon wounding, systemin, and methyl jasmonate

CHAPTER 2 – Insect-Induced Chemical Signals Regulating Natural Plant Protection Responses

Qualitative and quantitative estimation of a proteinaceous chymotrypsin inhibitor in tissues of potato plants has revealed a transitory existence in all tissues except xylem, flowers, and seeds, where the inhibitor was not found. The distribution pattern in both aerial tissues and in tubers during development and senescence suggests that the concentrations of this protein in all tissues are influenced by meristematic regions of the plants. The transitory existence coinciding with breaking of apical dominance suggests that in the potato plant the protein may have some role in the process of establishing and maintaining meristematic tissue. A search for the protein in other Solanaceae species and in plants from non-related species has proven unsuccessful.

R. W. Van Denburgh

Papers

Transitory Aspects of a Single Protein in Tissues of Solanum tuberosum and Its Coincidence With the Establishment of New Growth.