André de Oliveira Carvalho

Plant defensins - prospects for the biological functions and biotechnological properties.

Plant defensins are a prominent family of cationic peptides in the plant kingdom. They are structurally and functionally related to defensins that have been previously characterized in mammals and insects. They present molecular masses between 5 and 7kDa and possess a pattern of eight conserved Cys residues. The three-dimensional structure of plant defensins is small and globular. It has three anti-parallel beta-sheets and one alpha-helix that is stabilized by a structural motif composed of disulfide bridges. This motif is found in other peptides with biological activity and is called the Cys stabilized alphabeta motif (CSalphabeta). Based on the growing knowledge on defensin structure, gene expression and regulation, and also their in vitro biological activity, it has become clear that plant defensins are complex and sophisticated peptides whose function extends beyond their role in defense of plants against microbial infection. This review discusses recent data and will present comprehensive information regarding the study of defensins.

Role of plant lipid transfer proteins in plant cell physiology-a concise review.

Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides.

Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties.

BACKGROUND A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection. METHODS Peptides from Coffea canephora seeds were extracted in Tris-HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses. RESULTS The purified peptide of 9kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP(1) exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP(1) interfered in a dose-dependent manner with glucose-stimulated, H(+)-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP(1) family, represented here by Cc-LTP(1), was also able to inhibit mammalian α-amylase activity in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.

A 2S albumin-homologous protein from passion fruit seeds inhibits the fungal growth and acidification of the medium by Fusarium oxysporum.

Antimicrobial proteins have been isolated from a wide range of plant species. More recently, it has become increasingly clear that these types of proteins play an important role in the protection of plants. In this study, we investigate the presence of defense-related proteins from passion fruit (Passiflora edulis f. flavicarpa) seeds. Initially, seed flour was extracted for 2h (at 4 degrees C) with phosphate buffer, pH 5.5. The precipitate obtained between 0 and 70% relative ammonium sulfate saturation was re-dissolved in distilled water and heated at 80 degrees C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant (F/0-70) was extensively dialyzed. A Sephadex G-50 size exclusion column was employed for further separation of proteins. The fraction with antifungal activity was pooled and submitted to CM-Sepharose cation exchange. Two proteins, named Pf1 and Pf2, were eluted in 0.1 and 0.2M of salt, respectively, and submitted to reverse-phase chromatography in HPLC. This fraction inhibited the growth, in an in vitro assay, of the phytopathogenic fungi Fusarium oxysporum and colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae and strongly inhibited glucose-stimulated acidification of the medium by F. oxysporum in a dose-dependent manner. The molecular masses of these proteins, referred to now as Pf1-RP and Pf2-RP, were obtained by MALDI-TOF spectrometry and corresponded to 12,088 Da for Pf1-RP and 11,930 Da for Pf2-RP. These proteins were also subjected to automated N-terminal amino acid sequencing. Sequence comparisons for the heavy subunit of Pf2-RP showed the presence of a protein with a high degree of homology to storage 2S albumins.

Isolation, characterization and cloning of a cDNA encoding a new antifungal defensin from Phaseolus vulgaris L. seeds

The PvD1 defensin was purified from Phaseolus vulgaris (cv. Pérola) seeds, basically as described by Terras et al. [Terras FRG, Schoofs HME, De Bolle MFC, Van Leuven F, Ress SB, Vanderleyden J, Cammue BPA, Broekaer TWF. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 1992;267(22):15301-9], with some modifications. A DEAE-Sepharose, equilibrated with 20mM Tris-HCl, pH 8.0, was initially utilized for the separation of peptides after ammonium sulfate fractionation. The basic fraction (the non-retained peak) obtained showed the presence of one unique band in SDS-Tricine gel electrophoresis with a molecular mass of approximately 6kDa. The purification of this peptide was confirmed after a reverse-phase chromatography in a C2/C18 column by HPLC, where once again only one peak was observed and denominated H1. H1 was submitted to N-terminal sequencing and the comparative analysis in databanks revealed high similarity with sequences of different defensins isolated from other plants species. The N-terminal sequence of the mature defensin isolated was used to produce a degenerated primer. This primer allowed the amplification of the defensin cDNA by RT-PCR from mRNA of P. vulgaris seeds. The sequence analysis of the cloned cDNA, named PVD1, demonstrated 314bp encoding a polypeptide of 47 amino acids. The deduced peptide presented high similarity with plant defensins of Vigna unguiculata (93%), Cicer arietinum (95%) and Pachyrhizus erosus (87%). PvD1 inhibited the growth of the yeasts, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida guilliermondii, Kluyveromyces marxiannus and Saccharomyces cerevisiae. PvD1 also presented an inhibitory activity against the growth of phytopathogenic fungi including Fusarium oxysporum, Fusarium solani, Fusarium lateritium and Rizoctonia solani.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1) . Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the proteins biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1) , showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of α-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian α-amylase activity in vitro.

Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect α-amylase inhibitory activity

Plant defensins make up a family of cationic antimicrobial peptides with a characteristic three-dimensional folding pattern stabilized by four disulfide bridges. The aim of this work was the purification and functional expression of a defensin from cowpea seeds and the assessment of its alpha-amylase inhibitory activity. The cDNA encoding the cowpea defensin was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform Escherichia coli cells. The recombinant peptide was purified via affinity chromatography on a Ni Sepharose column and by reverse-phase chromatography on a C2/C18 column using HPLC. N-terminal amino acid sequencing revealed that the recombinant peptide had a similar sequence to that of the defensin isolated from seeds. The natural and recombinant defensins were submitted to the alpha-amylase inhibition assay. The cowpea seed defensin was found to inhibit alpha-amylases from the weevils Callosobruchus maculatus and Zabrotes subfasciatus. alpha-Amylase inhibition assays also showed that the recombinant defensin inhibited alpha-amylase from the weevil C. maculatus. The cowpea seed defensin and its recombinant form were unable to inhibit mammalian alpha-amylases. The three-dimensional structure of the recombinant defensin was modeled, and the resulting structure was found to be similar to those of other plant defensins.

The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells

Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles.

Characterization of Capsicum species using anatomical and molecular data.

Capsicum species are frequently described in terms of genetic divergence, considering morphological, agronomic, and molecular databases. However, descriptions of genetic differences based on anatomical characters are rare. We examined the anatomy and the micromorphology of vegetative and reproductive organs of several Capsicum species. Four Capsicum accessions representing the species C. annuum var. annuum, C. baccatum var. pendulum, C. chinense, and C. frutescens were cultivated in a greenhouse; leaves, fruits and seeds were sampled and their organ structure analyzed by light and scanning electronic microscopy. Molecular accession characterization was made using ISSR markers. Polymorphism was observed among tector trichomes and also in fruit color and shape. High variability among accessions was detected by ISSR markers. Despite the species studied present a wide morphological and molecular variability that was not reflected by anatomical features.

An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi

BACKGROUND The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses. METHODS In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement. RESULTS The purified peptide with a molecular weight of 7kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP-fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum. CONCLUSIONS AND GENERAL SIGNIFICANCE In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay.