António Fernandes

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.

A Current Overview of the Papaya meleira virus, an Unusual Plant Virus

Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease, which is characterized by a spontaneous exudation of fluid and aqueous latex from the papaya fruit and leaves. The latex oxidizes after atmospheric exposure, resulting in a sticky feature on the fruit from which the name of the disease originates. PMeV is an isometric virus particle with a double-stranded RNA (dsRNA) genome of approximately 12 Kb. Unusual for a plant virus, PMeV particles are localized on and linked to the polymers present in the latex. The ability of the PMeV to inhabit such a hostile environment demonstrates an intriguing interaction of the virus with the papaya. A hypersensitivity response is triggered against PMeV infection, and there is a reduction in the proteolytic activity of papaya latex during sticky disease. In papaya leaf tissues, stress responsive proteins, mostly calreticulin and proteasome-related proteins, are up regulated and proteins related to metabolism are down-regulated. Additionally, PMeV modifies the transcription of several miRNAs involved in the modulation of genes related to the ubiquitin-proteasome system. Until now, no PMeV resistant papaya genotype has been identified and roguing is the only viral control strategy available. However, a single inoculation of papaya plants with PMeV dsRNA delayed the progress of viral infection.

Green coconut mesocarp pretreated by an alkaline process as raw material for bioethanol production.

Cocos nucifera L., coconut, is a palm of high importance in the food industry, but a considerable part of the biomass is inedible. In this study, the pretreatment and saccharification parameters NaOH solution, pretreatment duration and enzyme load were evaluated for the production of hydrolysates from green coconut mesocarp using 18% (w/v) total solids (TS). Hydrolysates were not detoxified in order to preserve sugars solubilized during the pretreatment. Reduction of enzyme load from 15 to 7.5 filter paper cellulase unit (FPU)/g of biomass has little effect on the final ethanol titer. With optimized pretreatment and saccharification, hydrolysates with more than 7% (w/v) sugars were produced in 48h. Fermentation of the hydrolysate using industrial Saccharomyces cerevisiae strains produced 3.73% (v/v) ethanol. Our results showed a simple pretreatment condition with a high-solid load of biomass followed by saccharification and fermentation of undetoxified coconut mesocarp hydrolysates to produce ethanol with high titer.

Comparison of biofilm and attachment mechanisms of a phytopathological and clinical isolate of Klebsiella pneumoniae Subsp. pneumoniae.

Some bacterial species can colonize humans and plants. It is almost impossible to prevent the contact of clinically pathogenic bacteria with food crops, and if they can persist there, they can reenter the human food chain and cause disease. On the leaf surface, microorganisms are exposed to a number of stress factors. It is unclear how they survive in such different environments. By increasing adhesion to diverse substrates, minimizing environmental differences, and providing protection against defence mechanisms, biofilms could provide part of the answer. Klebsiella pneumoniae subsp. pneumoniae is clinically important and also associated with fruit diseases, such as “pineapple fruit collapse.” We aimed to characterize biofilm formation and adhesion mechanisms of this species isolated from pineapple in comparison with a clinical isolate. No differences were found between the two isolates quantitatively or qualitatively. Both tested positive for capsule formation and were hydrophobic, but neither produced adherence fibres, which might account for their relatively weak adhesion compared to the positive control Staphylococcus epidermidis ATCC 35984. Both produced biofilms on glass and polystyrene, more consistently at 40°C than 35°C, confirmed by atomic force and high-vacuum scanning electron microscopy. Biofilm formation was maintained in an acidic environment, which may be relevant phytopathologically.

Fed-batch production of green coconut hydrolysates for high-gravity second-generation bioethanol fermentation with cellulosic yeast

The residual biomass obtained from the production of Cocos nucifera L. (coconut) is a potential source of feedstock for bioethanol production. Even though coconut hydrolysates for ethanol production have previously been obtained, high-solid loads to obtain high sugar and ethanol levels remain a challenge. We investigated the use of a fed-batch regime in the production of sugar-rich hydrolysates from the green coconut fruit and its mesocarp. Fermentation of the hydrolysates obtained from green coconut or its mesocarp, containing 8.4 and 9.7% (w/v) sugar, resulted in 3.8 and 4.3% (v/v) ethanol, respectively. However, green coconut hydrolysate showed a prolonged fermentation lag phase. The inhibitor profile suggested that fatty acids and acetic acid were the main fermentation inhibitors. Therefore, a fed-batch regime with mild alkaline pretreatment followed by saccharification, is presented as a strategy for fermentation of such challenging biomass hydrolysates, even though further improvement of yeast inhibitor tolerance is also needed.

High hydrostatic pressure upregulate central carbon metabolism genes in a distillery yeast strain

Methods In this study we performed a microarray analysis in a distillery Saccharomyces cerevisiae strain (BT0510) submitted to sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min at room pressure (0.1 MPa). The transcription of the genes involved in central carbon metabolism in response to HHP was investigated for bioinformatics tools.

Effect of high hydrostatic pressure on the biosynthesis of sulfur amino acids in Saccharomyces cerevisiae

High hydrostatic pressure (HHP) is successfully applied in several industrial segments, as in vaccine production and food conservation. The response of microorganisms to HHP treatment resemble the responses of other stresses with industrial relevance, like osmotic, temperature and ethanol, which make the HHP a valuable tool in biotechnology research, as in the ethanol production [1]. Amino acids play a key role in central metabolism besides being the building blocks of proteins, and they are important to the HHP stress response. In this study, the Saccharomyces cerevisiae BT0510 was exposed to 50 MPa for 30 min at room temperature, followed by incubation at room pressure with aeration for 15 min. Samples of total RNA were collected every 5 min for transcriptional analysis by DNA microarray technique. Bioinformatics analysis demonstrated the upregulation (≥ 2 fold) by HHP treatment of genes related to the sulfur amino acids synthesis, methionine and cysteine. The HHP treatment induced the genes MET3, MET10, MET14 and MET16, which are correlated with the conversion of intracellular sulfate in sulfide. MET2, related to the conversion of homoserine to O-acetylhomoserine, was also induced by HHP, as well as the gene that codes for Met17p, responsible for the incorporation of sulfide in O-acetylhomoserine to form homocysteine, that will be directed to methionine or cysteine synthesis. These amino acids are directly correlated with sulfur assimilation in yeast cells. Methionine is the S-adenosylmethionine precursor, which participates in the biosynthesis of lipids and polyamines, and is also involved in methylation reactions, being a methyl group donor [2,3]. Cysteine is part of iron-sulfur proteins and is the glutathione biosynthesis precursor. Glutathione maintain the redox state in cytoplasm, therefore, playing an important role in cell response to oxidative stress [2,3]. The key gene related to the biosynthesis of methionine (MET6) was upregulated by HHP, while the gene related to the biosynthesis of cysteine (CYS4) was unaffected. Five minutes after pressure release MET6 was repressed. The genes related to the conversion of methionine to S-adenosylmethionine, SAM1 and SAM2, were downregulated. Methionine residues are important against reactive oxygen species (ROS) [4], and genes associated with the reduction of methionine sulfoxide (MXR1 and MXR2) were induced by HHP treatment, suggesting that methionine plays an important role in the reduction of ROS resulting from stress caused by HHP [5]. Concerning the regulation of sulfur amino acids metabolism, MET28 was strongly induced during the entire HHP and post treatment. Other factors, such as the transcription factor encoded by MET4 were not affected by HHP, and also MET30 that negatively regulates Met4p. Met28p appears to play an important role in the biosynthesis of sulfur amino acids in response to HHP. It seems that this protein participates in the Met4 complexes-DNA stabilization. Methionine biosynthesis upregulation is not related to other stresses, such as heat and osmotic stresses, and appears to be specific to HHP, which reinforces the use of this treatment to study the stress response in microorganisms.

Physical Characteristics of the Leaves and Latex of Papaya Plants Infected with the Papaya meleira Virus

Sticky disease, which is caused by Papaya meleira virus (PMeV), is a significant papaya disease in Brazil and Mexico, where it has caused severe economic losses, and it seems to have spread to Central and South America. Studies assessing the pathogen-host interaction at the nano-histological level are needed to better understand the mechanisms that underlie natural resistance. In this study, the topography and mechanical properties of the leaf midribs and latex of healthy and PMeV-infected papaya plants were observed by atomic force microscopy and scanning electron microscopy. Healthy plants displayed a smooth surface with practically no roughness of the leaf midribs and the latex and a higher adhesion force than infected plants. PMeV promotes changes in the leaf midribs and latex, making them more fragile and susceptible to breakage. These changes, which are associated with increased water uptake and internal pressure in laticifers, causes cell disruption that leads to spontaneous exudation of the latex and facilitates the spread of PMeV to other laticifers. These results provide new insights into the papaya-PMeV interaction that could be helpful for controlling papaya sticky disease.

Effect of high hydrostatic pressure on seeds Carica papaya

The breaking of seed dormancy induced by stress [1] is extremely important with regard to the acceleration of germination process [1,2]. Abiotic stress processes, just like high hydrostatic pressure (HHP), are applied in several biotechnological studies, mainly because HHP induces changes in physiological and biochemical processes [3]. Pretreatment with a stress induction tool can be lethal or induce the synthesis of protective factors. When the stress does not kill the organism, it is called sublethal stress, and it can even increase the resistance to other stresses. High hydrostatic pressure is an important model in stress response study, for it has the ability to change the plasma membrane fluidity so as to keep the organism alive, which allows studies on the metabolic changes. Water is used as the medium of fluid pressure transmission since it has lower compressibility and greater compatibility, resulting in less risk of contamination. In our experiment, Carica Papaya seeds were put into a polyethylene tube containing water, the system is then closed in a capsule of high resistance steel, and suffers pressures between 10 and 400 MPa. The seeds were set to germinate in vitro, while the pressure transfer fluid present in the capsule was used to analyze abscisic acid per HPLC. The HHP generated positive effects due the seed hydration, which reflected in the percentage and speed of germination [4]. The begin of the seeds germination subjected to HHP was anticipated to 70 hours due to greater water retention, characterized by the increasing of weight (about 2.8 times greater when compared to dry weight), followed by germination values between 70-90%, while the seeds subjected to ambient pressure (control group) showed hydration of about 2.4 times and germination around 55-65%. The ambient pressure (0.1 MPa) showed lower water retention, resulting in lower mean percentage and germination speed when compared to HHP treatments. The abscisic acid (ABA) is closely related to tolerance to stress conditions (HHP) and its relation to gibberellin (GA) is crucial in the process of germination. The pressure transfer fluid has proven useful for the detection of abscisic acid, characterized by the presence of chromatograph peak with retention time equals to analyses of samples fortified with industrial ABA. The analyses of the HPLC chromatograms demonstrate changes in the composition of the water used as medium of pressure transfer. The seeds undergoing HHP pressure showed through gas chromatogram, differences between the concentrations of protein, peptides and phenol, when compared to the seeds subjected to hydrostatic control group. The balance between the concentrations of abscisic acid and gibberelin is related to germination. The presence of ABA in fluid of pressure transfer indicates a factor of germination capacity of seeds subjected to HHP stress. HHP stress is an effective biotechnological tool in the search for new mechanisms of stimulation of Carica Papaya seeds in order to provide improved germination and breaking of dormancy. Some data obtained in this experiment were omitted because due to patent submission.

Monitoring expression of yeast cell wall protein-encoding genes in response to high hydrostatic pressure

Background The cell wall (CW) is one of the most important structures of the yeast cell, accounting for up to 30% of its dry weight. This organelle determines cellular morphology, affords mechanical protection and provides osmotic support. The yeast CW is a dynamic structure susceptible to many modifications, adjusting its composition and thickness to environmental changes. These responses usually involve changes in gene expression, increasing levels of proteins that have protective functions. High hydrostatic pressure (HHP) is a useful model of stress, which causes CW compression [1]. Exploring this process using the model organism Saccharomyces cerevisiae may allow us to understand the mechanisms of yeast stress tolerance in biotechnological processes and it may also helps in searching for effective antifungal drugs, since the CW is a desirable target of action.