Six small molecular mass, wound-inducible trypsin and chymotrypsin inhibitor proteins from tobacco (Nicotiana tabacum) leaves were isolated to homogeneity. The isoinhibitors, cumulatively called tobacco trypsin inhibitor (TTI), have molecular masses of approximately 5500 to 5800 D, calculated from gel filtration analysis and amino acid content. The amino acid sequence of the entire 53 residues of one isoinhibitor, TTI-1, and the sequence of 36 amino acid residues from the N terminus of a second isoinhibitor, TTI-5, were determined. The two isoinhibitors differ only at residue 11, which is threonine in TTI-1 and lysine in TTI-5. The isoinhibitors are members of the potato inhibitor II family and show considerable identity with the small molecular mass members of this family, which include the eggplant inhibitor, two small molecular mass trypsin and chymotrypsin inhibitors from potatoes, and an inhibitor from pistils of the ornamental plant Nicotiana alata. Antibodies produced against the isoinhibitors in rabbits were used in radial immunoassays to quantify both the systemic wound inducibility of TTI in tobacco leaves and its constitutive levels in flowers.

The primary structure-activity relationships of systemin, an 18-amino acid polypeptide from tomato leaves that regulates the expression of two wound-inducible proteinase inhibitor genes in tomato and potato plants, were investigated. Analogs of systemin, the only example of a polypeptide signal from plants, were synthesized with progressive deletions of amino acids from both the NH2 terminus and COOH terminus and assayed in young excised tomato plants. All of the analogs exhibited severely decreased proteinase inhibitor-inducing activities, indicating that the entire 18-amino acid sequence is necessary for maximal activity. Deletion of the COOH-terminal Asp abolished inducing activity. Progressive replacement of each amino acid of the entire polypeptide with Ala revealed two regions, near residues Pro13, where Ala substitution reduced activity to less than 0.2%, and Thr17, which totally inactivated the analog. Other replacements with Ala had little or only moderate effects on activity. The two inactive analogs, des-Asp18 systemin and Ala17 systemin, were potent inhibitors of the inducing activity of the native systemin. These analogs, therefore, contain structural conformations sufficient for competition with systemin, but they are not competent for proteinase inhibitor gene induction. A synthetic COOH-terminal tetrapeptide, Met-Gln-Thr-Asp, retained low proteinase inhibitor inducing activity, but virtually any replacements with other amino acids either eliminated activity or reduced the activity to very low or nearly undetectable levels. These results indicate that residues near the COOH terminus of systemin are necessary for activity, possibly involving a phosphorylation at Thr17, and that other regions of the systemin sequence are important for interacting with a receptor(s) but are not sufficient to activate proteinase inhibitor gene expression.

Defensive genes in plants can be activated by several different types of nonpeptide signaling molecules. An endogenous polypeptide, consisting of 18 amino acids, was isolated from tomato leaves and was able at very low concentrations to induce the synthesis of two wound-inducible proteinase inhibitor proteins when supplied to young tomato plants. The sequence of the polypeptide was determined, and an identical polypeptide was synthesized that possessed full inducing activity. These data establish that a polypeptide factor can initiate signal transduction to regulate the synthesis of defensive proteins in plant tissues.