Patricia Machado Bueno Fernandes

Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure

Gene expression patterns in response to hydrostatic pressure were determined by whole genome microarray hybridization. Functional classification of the 274 genes affected by pressure treatment of 200 MPa for 30 min revealed a stress response expression profile. The majority of the >2‐fold upregulated genes were involved in stress defense and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. Furthermore, uncharacterized genes were among the 10 highest expressed sequences and represented 45% of the total upregulated genes. The results of this study revealed a hydrostatic pressure‐specific stress response pattern and suggested interesting information about the mechanisms involved in adaptation of cells to a high‐pressure environment.

Antimicrobial activity and potential use of monoterpenes as tropical fruits preservatives.

Banana, mamao e abacaxi sao as frutas tropicais mais consumidas no mundo, sendo o Brasil um dos principais produtores. Os fungos Colletotrichum musae, Colletotrichum gloeosporioides e Fusarium subglutinans f.sp ananas sao os principais causadores de doencas e perdas em pos-colheita de frutas. A proposta deste estudo foi avaliar a eficacia de cinco monoterpenos em inibir o crescimento micelial e a germinacao dos conidios destes tres fitopatogenos. Os monoterpenos citral, citronelal, L-carvona, isopulegol e a-pineno foram diluidos em etanol a concentracao final de 0,2 a 1%. Todos os monoterpenos testados inibiram os tres fungos estudados de maneira dose-dependente. Citral foi o mais efetivo dos oleos testados e apresentou uma potente atividade fungicida em concentracoes acima de 0,5%. Mais ainda, avaliacao in vivo com estas frutas tropicais demonstrou a eficacia de citral como inibidor do crescimento fungico. Estes resultados indicam o uso em potencial de citral como um pesticida natural no controle das doencas em pos-colheita de frutas tropicais.Banana, papaya and pineapple are the most consumed tropical fruits in the world, being Brazil one of the main producers. Fungi Colletotrichum musae, Colletotrichum gloeosporioides and Fusarium subglutinans f.sp. ananas cause severe post harvest diseases and losses in fruits quality. The aim of this work was to evaluate the effectiveness of five monoterpenes to inhibit the mycelial growth and conidia germination of these three phytopathogens. The monoterpenes citral, citronellal, L-carvone, isopullegol and a-pinene were diluted in ethanol to final concentrations from 0.2 to 1%. All monoterpenes were found to inhibit the growth of the three studies fungi in a dose-dependent manner. Citral was the most effective of the oils tested and showed potent fungicidal activity at concentrations above 0.5%. Also, in vivo evaluation with these tropical fruits demonstrated the efficiency of citral to inhibit fungal growth. These results indicate the potential use of citral as a natural pesticide control of post-harvest fruit diseases.

Role of nitric oxide in the response of Saccharomyces cerevisiae cells to heat shock and high hydrostatic pressure

Nitric oxide (NO) is a simple and unique molecule that has diverse functions in organisms, including intracellular and intercellular messenger. The influence of NO on cell growth of Saccharomyces cerevisiae and as a signal molecule in stress response was evaluated. Respiring cells were more sensitive to an increase in intracellular NO concentration than fermentatively growing cells. Low levels of NO demonstrated a cytoprotective effect during stress from heat-shock or high hydrostatic pressure. Induction of NO synthase was isoform-specific and dependent on the metabolic state of the cells and the stress response pathway. These results support the hypothesis that an increase in intracellular NO concentration leads to stress protection.

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms

Papaya (Carica papaya L.) hosts the only described laticifer‐infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2‐DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI‐TOF‐MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2‐DE and DIGE followed by MALDI‐TOF‐MS/MS and LC‐IonTrap‐MS/MS, respectively. Conventional 2‐DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism‐related proteins were downregulated, and stress‐responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2‐DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.

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.

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis

INTRODUCTION A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. OBJECTIVE This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. METHODOLOGY Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. RESULTS Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. CONCLUSION The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.

High hydrostatic pressure and the cell membrane: stress response of Saccharomyces cerevisiae.

The brewing and baking yeast Saccharomyces cerevisiae is a useful eukaryotic model of stress response systems whose study could lead to the understanding of stress response mechanisms in other organisms. High hydrostatic pressure (HHP) exerts broad effects upon yeast cells, interfering with cell membranes, cellular architecture, and the processes of polymerization and denaturation of proteins. In this review, we focus on the effect of HHP on the S. cerevisiae cell membrane and describe the main signaling pathways involved in the pressure response.

Carica papaya microRNAs are responsive to Papaya meleira virus infection.

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya.

Cell wall alterations in the leaves of fusariosis-resistant and susceptible pineapple cultivars.

Fusariosis, caused by the fungus Fusarium subglutinans f. sp. ananas (Syn. F. guttiforme), is one of the main phytosanitary threats to pineapple (Ananas comosus var. comosus). Identification of plant cell responses to pathogens is important in understanding the plant–pathogen relationship and establishing strategies to improve and select resistant cultivars. Studies of the structural properties and phenolic content of cell walls in resistant (Vitoria) and susceptible (Perola) pineapple cultivars, related to resistance to the fungus, were performed. The non-chlorophyll base of physiologically mature leaves was inoculated with a conidia suspension. Analyses were performed post-inoculation by light, atomic force, scanning and transmission electron microscopy, and measurement of cell wall-bound phenolic compounds. Non-inoculated leaves were used as controls to define the constitutive tissue characteristics. Analyses indicated that morphological differences, such as cell wall thickness, cicatrization process and lignification, were related to resistance to the pathogen. Atomic force microscopy indicated a considerable difference in the mechanical properties of the resistant and susceptible cultivars, with more structural integrity, associated with higher levels of cell wall-bound phenolics, found in the resistant cultivar. p-Coumaric and ferulic acids were shown to be the major phenolics bound to the cell walls and were found in higher amounts in the resistant cultivar. Leaves of the resistant cultivar had reduced fungal penetration and a faster and more effective cicatrization response compared to the susceptible cultivar.

Crystal structure of a pro-inflammatory lectin from the seeds of Dioclea wilsonii Standl

The crystal structure and pro-inflammatory property of a lectin from the seeds of Dioclea wilsonii (DwL) were analyzed to gain a better understanding of structure/function relationships of Diocleinae lectins. Following crystallization and structural determination by standard molecular replacement techniques, DwL was found to be a tetramer based on PISA analysis, and composed by two metal-binding sites per monomer and loops which are involved in molecular oligomerization. DwL presents 96% and 99% identity with two other previously described lectins of Dioclea rostrata (DRL) and Dioclea grandiflora (DGL). DwL differs structurally from DVL and DRL with regard to the conformation of the carbohydrate recognition domain and related biological activities. The structural analysis of DwL in comparison to other Diocleinae lectins can be related to the differences in the dose-dependent pro-inflammatory effect elicited in Wistar rats, probably via specific interactions with mast cells complex carbohydrate, resulting in significant paw edema. DwL appears to be involved in positive modulation of mast cell degranulation via recognition of surface carbohydrates. Since this recognition is dependent on site volume and CRD configuration, edematogenesis mediated by resident cells varies in potency and efficacy among different Diocleinae lectins.