A chimeric gene consisting of 1.3 kb of the 5′ regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β‐glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3′‐untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound‐inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non‐wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non‐wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis‐active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non‐wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.

The isolation and characterization of a genomic clone encoding proteinase inhibitor II of potato (Solanum tuberosum) is described. The structure of this gene was determined by sequencing a genomic fragment of about 2 kb containing the entire RNA coding as well as about 900 nucleotides of the 5'-upstream and 250 nucleotides of the 3'-downstream region. The transcription start site was determined by RNase protection experiments. The comparison of the genomic sequence with cDNA sequences reveals the presence of one intron with a length of 117 nucleotides. The genomic clone contains an open reading frame of 462 nucleotides allowing for a protein of 154 amino acids. The proteinase inhibitor II gene displays typical features of eucaryotic genes. The sequence TATAAA is found 26 nucleotides upstream of the transcription initiation site and the sequence CAAAT at position--103. In the 3'-region the sequence AATAA is found 33 nucleotides in front of the poly-A addition site.

A gene encoding an antifreeze protein (AFP) was isolated from carrot (Daucus carota) using sequence information derived from the purified protein. The carrot AFP is highly similar to the polygalacturonase inhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat (LRR) proteins. Expression of the AFP gene is rapidly induced by low temperatures. Furthermore, expression of the AFP gene in transgenic Arabidopsis thaliana plants leads to an accumulation of antifreeze activity. Our findings suggest that a new type of plant antifreeze protein has recently evolved from PGIPs.z 1999 Federation of European Biochemical Societies.

A potato proteinase inhibitor II gene was transferred into tobacco plants using Agrobacterium/Ti-plasmid-mediated gene transfer techniques. Whereas no or little expression of the proteinase inhibitor H gene could be detected in non-wounded leaves, high levels of proteinase inhibitor II mRNA were detected in leaves of several transgenic tobacco plants after mechanical wounding as well as after treatment of detached leaves with oligosaccharides. Wounding of a leaf also led to a systemic induction in non-wounded leaves as well as nonwounded stem, and roots. RNA-protection experiments showed that the transcription of the proteinase inhibitor gene in transgenic tobacco plants was initiated at the same nucleotide as that of the original gene. These observations demonstrate that although no proteinase inhibitor II homologous gene can be detected in tobacco, this plant nevertheless has the capacity to regulate the expression of the potato gene in the same complex manner as in the case of potato.