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

Abstract

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.

Long-read sequencing uncovers the adaptive topography of a carnivorous plant genome

“…This GO category included a gene encoding a class III chitinase (unitig_60.g25630.t1, showing >20-fold trap-enhanced expression) (SI Appendix, Dataset S8), representing one of the chitinase families [glycoside hydrolase (GH) family 18] active within the digestive fluid of both open and closed traps of various carnivorous plant species. In Nepenthes, the GH family 18 enzyme is encoded by a single-copy gene that is up-regulated in response to prey in both the pitted glands and surrounding tissues (54). Galactosidases and xylosidases (55) are also among the genes with the hydrolase annotation, and enzymes encoding both have been identified in the Nepenthes trap fluid proteome (56,57).…”

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

“…In addition to proteins identified in this study (Supplementary Tables 25-28), we obtained for phylogenetic analyses a number of previously published sequences of digestive fluid proteins 8,9,[72][73][74][75][76][77][78] (Supplementary Table 24). Although many protein and transcript sequences for possible digestive enzymes are available (for example, refs 17,79-84 ), we included only genes for which complete coding sequences were available and for which their presence in digestive fluid had been biochemically validated (Supplementary Table 24, last searched 20 January 2016).…”

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity

“…The fluid is composed of a mixture of enzymes generated by the plants themselves including aspartic proteases called nepenthesins [6,7] and those produced from inhabitant microorganisms [8]. In addition to proteases, the pitcher fluids of Nepenthes also contain esterases, phosphatases, ribonucleases, and chitinases [9][10][11][12][13] as well as different glycosyl hydrolases, e.g., β-D-glucosidases and β-D-glucosaminidases [8] which together act cooperatively on digesting the insect bodies. Starting from native microflora in unopened pitchers [14,15], the hydrolytic microcosms in pitcher fluid are hypothesized to be enriched by airborne microbes and those previously colonized on the insect carcasses.…”