Maura Da Cunha

Structural changes in radish seedlings exposed to cadmium

Radish (Raphanus sativus L. cv. Redondo Vermelho) seedlings were analysed by light and scanning electron microscopy to characterize the structural changes caused by the exposure to 0.5 or 1.0 mM cadmium chloride for 24, 48 and 72 h. The analyses showed changes in the anatomical and morphological characteristics of roots, stems and leaves of two-week-old seedlings. In all tissues, pressure potential was decreased. Premature death with the disintegration of the epidermis and an increase in the number of root hairs was observed in roots exposed to Cd. The stem of seedlings exposed to Cd exhibited more cells layers in the cambial region. The main effects observed in leaves in response to Cd were stomatal closure, lack of cell wall thickening and alterations in the shape of the chloroplasts. It is suggested that the structural changes observed in seedlings treated with Cd were mainly caused by a Cd-induced decrease in water uptake.

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.

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.

87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants

Transgenic tomato plants (Lycopersicon esculentum) overexpressing the prosystemin transgene have been shown previously to accumulate a soluble 87 kDa cystatin constitutively. We report here that this protein can be found in a crystalline form which can be purified using a glycerol/sucrose gradient. Midgut homogenate of third-instar larvae of two coleopteran pest insects, Callosobruchus maculatus and Zabrotes subfasciatus, had their proteolytic activity content significantly inhibited by tomato cystatin (TC). In leaves of wild-type tomato plants, cystatin mRNA accumulated systemically in response to wounding, treatment with methyl jasmonate (MJ) and when supplied with systemin, corroborating the anti-herbivorous activity. Accumulation of cystatin mRNA occurred when plants were supplied with chitosan and oligogalacturonic acid fragments (OGA), suggesting an effect of TC against pathogens. Moreover, this protein reduced the growth of two fungi, Fusarium solani and Trichoderma viride in vitro. Taken together, the data reinforce a role for TC in defense response against pests or pathogens.

Effects of the Papaya meleira virus on papaya latex structure and composition

Spontaneous latex exudation is the main symptom of papaya sticky (meleira) disease caused by the Papaya meleira virus (PMeV), a double-stranded RNA (dsRNA) virus. This paper describes different effects of PMeV on papaya latex. Latex samples were subjected to different histochemical tests to evaluate their chemical composition. Additionally, the integrity of the latex particles was assessed by transmission and scanning electron microscopy analysis. Biochemical and micro- and macro-element measurements were performed. PMeV dsRNA extraction was performed to evaluate the interaction of the virus with the latex particles. Sticky diseased latex was positive for alkaloid biosynthesis and showed an accumulation of calcium oxalate crystals. PMeV also increased H2O2 synthesis within sticky diseased laticifers. The protein, sugar and water levels were altered, probably due to chemical changes. The morphology of the latex particles was further altered; PMeV particles seemed to be bound to the latex particles. The alkaloid and H2O2 biosynthesis in the papaya laticifers indicate a papaya defense response against PMeV. However, such efforts failed, as the virus affected the plant latex. The effects described here suggest some advantages of the infection process, including facilitating the movement of the virus within the papaya plant.

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.

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.

Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)

(Ultraestrutura da fase secretora e da senescente dos coleteres de Bathysa gymnocarpa e B. stipulata (Rubiaceae)). Coleteres sao estruturas secretoras formadas por um eixo parenquimatico que inclui feixes vasculares, circundado por uma camada de celulas epidermicas secretoras em palicada. Em estudos sobre a estrutura dos coleteres tem sido observada a ultraestrutura das celulas secretoras, mas nao discriminam as fases secretora e senescente. Geralmente, em celulas secretores de mucilagem como os coleteres, o reticulo endoplasmatico e o complexo de Golgi estao envolvidos na producao e no transporte da secrecao. Neste estudo, foram determinadas duas fases baseadas na estrutura dos coleteres de Bathysa gymnocarpa K. Schum. and B. stipulata (Vell.) C. Presl. (Rubiaceae): a fase secretora e a fase senescente. Amostras foram coletadas e processadas utilizando tecnicas usuais de microscopia optica e eletronica. Os coleteres estudados sao constituidos por uma camada epidermica em palicada e um eixo central parenquimatico com tracos vasculares raros. Durante a fase secretora, as celulas epidermicas se apresentam com o citoplasma denso, pequenos vacuolos, reticulo endoplasmatico liso e rugoso evidente e complexo de Golgi proximo a grandes vesiculas. Durante a fase senescente, as celulas epidermicas apresentaram o sistema endomembranar desorganizado. Nenhuma organela intacta ou vesicula foi observada. A parede celular mais externa exibiu camadas similares as observadas durante a fase secretora. A fase senescente e facilmente definida pela morfologia do coleter, mas nao e bem definida em nivel subcelular nas celulas secretoras. Nossa investigacao sugere que a morte celular programada se inicia na fase secretora. Contudo, mais evidencias sao necessarias para avaliar esse fenomeno.

Photosynthetic performance and anatomical adaptations in Byrsonima sericea DC. under contrasting light conditions in a remnant of the Atlantic forest

A dinâmica fotossintetica da especie pioneira tropical Byrsonima sericea DC. foi estudada durante o processo de regeneracao de uma mata nativa por avaliacoes ecofisiologicas (trocas gasosas, fluorescencia da clorofila a e conteudo de pigmentos fotossinteticos) e parâmetros anatomicos das plantas em ambientes ensolarados e sombreados. As avaliacoes ecofisiologicas foram feitas mensalmente por um ano e os dados foram agrupados em estacao seca e chuvosa. Byrsonima sericea DC. apresentou plasticidade anatomica que a capacita a se estabelecer em ambientes com contrastantes condicoes luminosas. Nas condicoes ensolaradas, ela produz lâmina foliar mais espessa (cerca de 420 µm) e epiderme adaxial plana, enquanto em ambientes sombreados, as folhas apresentaram a epiderme convexa e lâmina mais delgada (cerca de 393 µm). Nao foram encontradas diferencas significativas na composicao de pigmentos em funcao do ambiente, no entanto, durante a estacao seca, as plantas apresentaram concentracoes significativamente maiores de pigmentos fotossinteticos. As plantas de ambientes ensolarados mostraram diminuicao na razao Fv/ FO (na estacao chuvosa) e NPQ (na estacao seca), mas nao foram observadas diferencas entre as plantas de sol ou sombra numa mesma estacao do ano. Taxas fotossinteticas significativamente maiores foram encontradas na estacao chuvosa para as plantas de sol quando comparadas com as plantas de sombra (9.9±0.8 e 7.4±0.3 µmol m-2s-1, respectivamente). Aumento significativo na transpiracao foi observado em plantas de sombra durante a estacao seca, mas sem afetar a eficiencia no uso da agua. Dados ecofisiologicos sugerem que, principalmente as plantas mais expostas ao sol sofreram limitacoes hidricas durante a estacao seca e que, na estacao chuvosa, estas plantas aumentaram sua taxa fotossintetica. Isso possivelmente foi devido a maior forca de dreno resultante do maior crescimento neste periodo. Os dados sugerem que alteracoes anatomicas, especialmente a epiderme adaxial convexa, estariam auxiliando na captacao de luz nas plantas sombreadas durante ambas as estacoes, prevenindo alteracoes nos pigmentos, como o aumento de clorofila b geralmente observado em plantas de sombra, mas nao detectado no presente estudo. Outra consequencia da maior quantidade de luz capturada por estas plantas de sombra foi que, durante a estacao seca sua taxa fotossintetica nao diferiu da das planas de ambientes ensolarados. No entanto, quando a agua nao foi um fator limitante, um melhor desempenho fotossintetico foi observado nesta especie pioneira, em ambientes abertos.