Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens.

AIM: This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. METHODS AND RESULTS: Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0.3 µmol l(-1) of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. CONCLUSIONS: The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection.

Cytology of Infection of Maize Seedlings by Fusarium moniliforme and Immunolocalization of the Pathogenesis-Related PRms Protein.

ABSTRACT We have investigated the histology of infection of maize seedlings by Fusarium moniliforme in association with a biochemical host defense response, the accumulation of the PRms (pathogenesis-related maize seed) protein. Light microscopy of trypan blue-stained sections and scanning electron microscopy revealed direct penetration by F. moniliforme hyphae through the epidermal cells of the seedling and colonization of the host tissue by inter- and intracellular modes of growth. Pathogen ingress into the infected tissue was associated with the induction of defense-related ultrastructural modifications, as exemplified by the formation of appositions on the outer host cell wall surface, the occlusion of intercellular spaces, and the formation of papillae. Cellular and subcellular immunolocalization studies revealed that PRms accumulated at very high levels in those cells types that represent the first barrier for fungal penetration such as the aleurone layer of germinating seeds and the scutellar epithelial cells of isolated germinating embryos. A highly localized accumulation of PRms within papillae of the inner scutellar parenchyma cells also occurred, suggesting that signaling mechanisms that lead to the accumulation of PRms in papillae of cell types that are distant from the invading pathogen must operate in the infected maize tissues. Our study also revealed the presence of a large number of fungal cells with an abnormal shape that showed PRms-specific labeling. PRms was found to accumulate in clusters over the fungal cell wall. Taken together, the occurrence of PRms in cell types that first establish contact with the pathogen, as well as in papillae, and in association with fungal cell walls suggests that PRms may have a function in the plant defense response.

Sequential expression and differential hormonal regulation of proteolytic activities during germination in Zea mays L.

We have characterized the proteolytic activities (proteases in cooperation with carboxypeptidases) involved in the different stages of germination of maize (Zea mays L.) grains. A sequential expression of different groups of proteases (aspartic, cysteine, serine and metallo-proteases) with pH optima in the acidic range has been found by using specific protease inhibitors. Pepstatin-sensitive proteolytic activity (aspartic-protease activity) is dominant in resting grains. Germination is accompanied by the appearance of a proteolytic activity which can be enhanced by low-molecular-weight thiol compounds and inhibited by thiol-protease inhibitors, which is indicative of the involvement of cysteine protease(s). This burst of cysteine-protease activity is coincident with the disappearance of the main storage-protein fractions. We conclude from this that cysteine protease(s), with an acid pH optimum, are good candidate(s) for the proteolytic attack of stored protein reserves in maize. After this stage, where cysteine-protease activity is dominant, a period with larger total proteolytic activity starts, coincidentally with the expression of the different types of other proteolytic activities (serine, aspartic and metallo proteases), in addition to the cysteine-protease activity above mentioned. When the development of carboxypeptidase activity during germination was analyzed, the highest activities were found during the earlier and later stages. This result is indicative of a cooperative interaction between carboxypeptidase and endoproteolytic systems in order to obtain a more effective mobilization of storage proteins in germinating maize grains. The phytohormones, gibberellic acid (GA3) and abscisic acid (ABA) which can stimulate or inhibit, respectively, the total proteolytic activity in extracts from germinating grains, exert a differential effect on the different proteolytic activities here detected.

The activation segment of procarboxypeptidase A from porcine pancreas constitutes a folded structural domain.

The controlled action of trypsin on porcine pancreatic procarboxypeptidase A releases a large activation peptide which contains the activation segment of the proenzyme. Circular dichroism studies indicate that the isolated activation peptide contains a high percentage of residues in ordered secondary structures (mainly alpha-helix). This result agrees with predictions of secondary structure carried out on the published amino acid sequence of the homologous rat proenzyme. Moreover, proton magnetic resonance spectroscopy shows that the peptide adopts a thermostable tertiary structure with characteristics typical of globular proteins. The results as a whole indicate that the activation segment of porcine pancreatic procarboxypeptidase A constitutes a folded structural domain.

The severed activation segment of porcine pancreatic procarboxypeptidase A is a powerful inhibitor of the active enzyme. Isolation and characterisation of the activation peptide.

The activation peptide of the monomeric procarboxypeptidase A from porcine pancreas was isolated by means of controlled trypsin digestion of the proenzyme followed by ion-exchange chromatography under dissociating conditions (7 M urea). The molecular weight of the isolated peptide was estimated to be around 11500-12000 (corresponding to approx. 100-103 residues) as judged by SDS electrophoresis and amino acid analysis, a figure that agrees with the differences between the corresponding values for procarboxypeptidase A and carboxypeptidase A (peptidyl-L-amino acid hydrolase, EC 3.4.17.1). The activation peptide has a high content of hydrophobic and acidic amino acids, and lacks cysteine. A remarkable feature is the strong competitive inhibitory action of the peptide on both porcine and bovine pancreatic carboxypeptidase A activity, with a Ki in the nanomolar range, and its null ability to inhibit porcine pancreatic carboxypeptidase B (EC 3.4.17.2). The above properties, and the fact that the peptide has the same N-terminal residue (lysine) as the parent procarboxypeptidase A, suggest that the isolated peptide contains most (if not all) of the activation segment of the proenzyme.