Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes

Carnivory in plants is an adaptation strategy to nutrient-poor environments and soils. Carnivorous plants obtain some additional mineral nutrients by trapping and digesting prey; the genus Nepenthes is helped by its specialized pitcher traps. To make the nutrients available, the caught prey needs to be digested, a process that requires the concerted activity of several hydrolytic enzymes. To identify and investigate the various enzymes involved in this process, fluid from Nepenthes traps has been analysed in detail. In this study, a novel type of Nepenthes endochitinase was identified in the digestion fluid of closed pitchers. The encoding endochitinase genes have been cloned from eight different Nepenthes species. Among these, the deduced amino acid sequence similarity was at least 94.9%. The corresponding cDNA from N. rafflesiana was heterologously expressed, and the purified protein, NrChit1, was biochemically characterized. The enzyme, classified as a class III acid endochitinase belonging to family 18 of the glycoside hydrolases, is secreted into the pitcher fluid very probably due to the presence of an N-terminal signal peptide. Transcriptome analyses using real-time PCR indicated that the presence of prey in the pitcher up-regulates the endochitinase gene not only in the glands, which are responsible for enzyme secretion, but at an even higher level, in the glands’ surrounding tissue. These results suggest that in the pitchers’ tissues, the endochitinase as well as other proteins from the pitcher fluid might fulfil a different, primary function as pathogenesis-related proteins.