Pin-II type proteinase inhibitor (PI) genes were cloned from fruit and stem tissues of Capsicum annuum L. var Phule Jyoti using primers designed from reported CanPI gene sequence (AF039398). In total, 21 novel CanPIs, members of the Pin-II PI family, were identified in the study, with three isoforms of 1-inhibitory repeat domain (IRD), eight isoforms of 2-IRD, three isoforms of 3-IRD, five isoforms of 4-IRD and two partial CanPI sequences. Most of the sequences showed variation (2 to 20%) in the deduced AA sequences which were pronounced close to the reactive site loop. Expression patterns of CanPIs in the fruit and stem tissues of mature C. annuum plants were shown to vary qualitatively and quantitatively using semi-quantitative RT-PCR expression analysis. In the fruit tissue, CanPIs with different IRDs (from 1 to 4) were expressed simultaneously. In stem tissue, 1- and 2-IRD CanPIs were strongly expressed along moderate expression of 3- and 4-IRD genes. Analysis of CanPI protein activity showed a range of active forms across the tissues. CanPI expression was differentially up-regulated upon wounding and insect attack. Although infestation by aphids (Myzus persicae) and lepidopteran pests (Spodoptera litura) specifically induced 4-IRD CanPIs, virus-infected leaves did not affect CanPI expression. Analysis of CanPI protein activity indicated that the up-regulation in CanPI expression was not always correlated with increase in PI activity. Our results demonstrated that CanPI expression is regulated spatially, temporally as well as qualitatively and quantitatively.

A cDNA clone encoding a protein (CaPI-2), which is highly homologous to proteinase inhibitor II (PI-2) from tobacco and tomato, was isolated from pericarp cDNA library of hot pepper. The 304 amino acid long primary structure contains a region of a putative signal peptide and three highly conserved repeated proteinase inhibitor domains with a trypsin- and two chymotrypsin-specific reactive sites. The mRNA encoding CaPI-2 was expressed at a steady-state level in leaves, stem, and roots. Mechanical wounding increased the CaPI-2 mRNA expression in the adjacent and remote site from wounded area. In addition to wounding, the effects of various chemical and physical stresses were investigated. Exogenous ABA, salt and electrical current was observed to increase the CaPI-2 expression, whereas acetylsalicylic acid gave an inverse effect on the wounding. In contrast, polyethylene glycol (PEG) and cold stress had no effects on the CaPI-2 expression level. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.