Cloning and Characterization of a Ribonuclease, a Cysteine Proteinase, and an Aspartic Proteinase from Pitchers of the Carnivorous Plant Nepenthes ventricosa Blanco

Nepenthes ventricosa is a carnivorous plant with modified leaves, pitchers, that secrete hydrolases to digest trapped insects, thus obtaining supplemental nutrients. Using reverse transcriptase PCR (RT-PCR), three cDNAs encoding one putative ribonuclease (NvRN1) and two putative proteinases (NvAP1, NvCP1) were cloned from N. ventricosa pitcher tissues. The deduced amino acid sequences of NvRN1, NvAP1, and NvCP1 are homologous to the S-like RNase PD2 (Prunus dulcis), the aspartic proteinase NaAP3 (Nepenthes alata), and the cysteine proteinase CysP2 (Glycine max), respectively. NvRN1 and NvAP1 were expressed in pitcher tissues and male flowers. NvRN1 expression was also evident in female flowers. SDS-PAGE activity gels showed that pitcher fluid samples contained at least six proteinases of differing molecular masses (124, 65, 61, 49, 36, and 33 kD) and a single 21-kD RNase. Fluid samples from single pitchers showed no change in pattern of proteolytic activity over time, but samples from different pitchers exhibited differing activity patterns, indicating that separate pitchers may secrete proteinases of different molecular masses. Optimal proteinase activity at pH 3 and inhibition by treatment with pepstatin was consistent with previous studies showing that Nepenthes secrete aspartic proteinases. Proteinase activity was also inhibited by E-64, indicating that cysteine proteinases could be present in pitcher fluid.