Ricardo B. Ferreira

The role of plant defence proteins in fungal pathogenesis.

SUMMARY It is becoming increasingly evident that a plant-pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillion-year evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant-pathogen interaction is an essential goal for disease control in the future.

The wine proteins

Proteins are typically present in wines in low concentrations, contributing little to their nutritive value. However, they assume a considerable technological and economical importance because they greatly affect the clarity and stability of wines. Although exhibiting a large diversity, the majority of the wine proteins are structurally related and have been identified as pathogenesis related (PR) proteins. Thus, different wines are essentially composed by identical sets of polypeptides. They derive from the grape pulp, and survive the vinification process simply because they are highly resistant to proteolysis and to the low pH characteristic of wines. There is increasing evidence suggesting that although protein-dependent, the development of turbidity in wines is controlled by a number of factors of non-protein origin, such as polyphenols, the wine pH and the presence of polysaccharides. A variety of procedures has been developed and tested for the specific removal of proteins from wines. Even though bentonite fining is nonspecific and can impair the quality of wine, it remains the only effective method to stabilize wines.

Ion beam deposition and oxidation of spin-dependent tunnel junctions

Spin dependent tunnel junctions showing tunnel magnetoresistance (TMR) values of 39/spl sim/41% were fabricated using Ion Beam Deposition (IBD). Both the electrodes and the aluminum layer deposition were done by IBD. The aluminum oxidation was performed using the assist gun with an oxygen beam (+30 V acceleration voltage applied on the grids) using mixed O/sub 2//Ar plasma. The oxidation was monitored in real time with a residual gas analyzer (RGA). The junction area is defined by lithography, down to 3/spl times/2 /spl mu/m/sup 2/. As-deposited junctions with 15 /spl Aring/ of Al showed TMR of 27/spl sim/29%, independent of the junction area, with resistance-area products of 0.8/spl sim/1.6 M/spl Omega//spl times//spl mu/m/sup 2/. This TMR value reached 40% upon annealing at 290/spl deg/C, with resistance decreasing to 0.5/spl sim/0.8 M/spl Omega//spl times//spl mu/m/sup 2/.

The seed storage proteins from Lupinus albus

Abstract Studies on lupin seed storage proteins have been performed using purification techniques that are often time-consuming and lead to poor resolution between the individual globulins. In this paper we have utilized anion exchange chromatography on the MONO Q column of the FPLC as a high-resolution, easy and rapid method to fractionate the three main globulins from two cultivars of Lupinus albus , namely the sweet cv Multolupa and the bitter cv Torres Vedras. The structure of the purified globulins, designated by γ-, β- and α-conglutins, was studied by electrophoresis performed under non-denaturing conditions, under denaturing, non-reducing conditions and denaturing, reducing conditions, and by two-dimensional electrophoresis. We have also developed a rapid, easy and sensitive method to determine the polypeptide chain composition of proteins or protein subunits which is based on the lateral diffusion of 2-mercaptoethanol during electrophoresis. We propose a nomenclature for the polypeptide chains and individual subunits of L. albus globulins that can be extended to other seed storage proteins.

Fungal Pathogens: The Battle for Plant Infection

The attempted infection of a plant by a pathogen, such as a fungus or an Oomycete, may be regarded as a battle whose major weapons are proteins and smaller chemical compounds produced by both organisms. Indeed, plants produce an astonishing plethora of defense compounds that are still being discovered at a rapid pace. This pattern arose from a multi-million year, ping-pong−type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.

1∕f noise in linearized low resistance MgO magnetic tunnel junctions

Low RA MgO magnetic tunnel junctions prepared at Singulus (Ta3∕CuN30∕Ta5∕PtMn20∕CoFe2.5∕Ru0.7∕CoFeB3∕MgO1.2∕CoFeB3∕Ta5 (thickness in nanometers) were microfabricated at INESC-MN. The junctions were patterned into micron-sized sensors (5–20μm2) with controlled shape anisotropy (aspect ratio ranging from 2 to 20). A small external longitudinal bias field (15–30 Oe) was further used to improve sensor linearity. The MgO junctions have a resistance-area product of 150Ωμm2 and a maximum tunnel magnetoresistance of 130%. Noise measurements were done in linearized sensors, from dc to 500 kHz. The magnetic and nonmagnetic contributions to the 1∕f noise were determined. From the data fitting, Hooge parameters of ∼2.20×10−9μm2 were obtained for the nonmagnetic 1∕f noise. Analysis of direct experimental data revealed the possibility to detect variations of magnetic fields in the order of 10−10T∕Hz0.5 with these MgO junctions, demonstrating their potential for ultralow-field detection.

Spin dependent tunnel junctions for memory and read-head applications

Spin dependent tunnel junctions with TMR exceeding 30-40% can now be prepared with AlO/sub x/ and AlN barriers, with junction resistance tuned from 30-40 /spl Omega//spl times//spl mu/m/sup 2/ to 10/sup 8/ /spl Omega//spl times//spl mu/m/sup 2/. Thermal stability is better than 3000% for thicker barriers (>11 /spl Aring/) but is degraded to 220/spl deg/C for 6 /spl Aring/ barriers. A 9 bit MRAM cell is demonstrated using tunnel junctions and vertically integrated a:Si diodes. Junction switching is achieved with on chip 20 ns field pulses. Requirements for tunnel junctions for 100 Gb/in/sup 2/ read head applications are discussed. First prototypes of tunnel junction read heads with 600 /spl Aring/ read gaps were fabricated, where the TJ is at the air bearing surface. TMR loss was observed during the final head lapping steps.

Antioxidant Properties and Neuroprotective Capacity of Strawberry Tree Fruit (Arbutus unedo)

Berries contain significant amounts of phytochemicals, including polyphenols, which are reported to reduce cancer risk, coronary heart disease and other degenerative diseases. These effects are mainly attributed to the antioxidant capacity of polyphenols found in berries. Strawberry tree (Arbutus unedo) berries are used in folk medicine but seldom eaten as fresh fruits. Their phenolic profile and antioxidant capacity reveal a high potential, but they are not well characterized as a “health promoting food”. The aim of this study was to assess the antioxidant properties of the edible strawberry tree fruit in vitro and in a neurodegeneration cell model. Raspberry (Rubus idaeus), a well documented health-promoting fruit, was used as a control for comparison purposes. A. unedo yielded a similar content in polyphenols and a slightly lower value of total antioxidant capacity in comparison to R. idaeus. Although the chemically-measured antioxidant activity was similar between both fruits, R. idaeus increased neuroblastoma survival in a neurodegeneration cell model by 36.6% whereas A. unedo extracts caused no effect on neuroblastoma viability. These results clearly demonstrate that a promising level of chemically-determined antioxidant activity of a plant extract is not necessarily correlated with biological significance, as assessed by the effect of A. unedo fruit in a neurodegeneration cell model.

Transgenic plants with improved dehydration-stress tolerance: progress and future prospects

This review summarizes the recent progress made towards the development of transgenic plants with improved tolerance to water stress and salinity. Of the various strategies employed, emphasis has been given to the genes engineered for the biosynthesis of osmoprotectants and osmolytes. This review also briefly discusses the importance of the use of specific stress inducible promoters and the future prospects of transgenic plants with improved agronomic traits.

Antioxidant capacity of Macaronesian traditional medicinal plants.

The use of many traditional medicinal plants is often hampered by the absence of a proper biochemical characterization, essential to identify the bioactive compounds present. The leaves from five species endemic to the Macaronesian islands with recognized ethnobotanical applications were analysed: Apollonias barbujana (Cav.) Bornm., Ocotea foetens (Ainton) Baill, Prunus azorica (Mouill.) Rivas-Mart., Lousã, Fern. Prieto, E. Días, J.C. Costa & C. Aguiar, Rumex maderensis Lowe and Plantago arborescens Poir. subsp. maderensis (Dcne.) A. Hans. et Kunk.. Since oxidative stress is a common feature of most diseases traditionally treated by these plants, it is important to assess their antioxidant capacity and determine the molecules responsible for this capacity. In this study, the antioxidant capacity of these plants against two of the most important reactive species in human body (hydroxyl and peroxyl radicals) was determined. To trace the antioxidant origin total phenol and flavonoid contents as well as the polyphenolic profile and the amount of trace elements were determined. There was a wide variation among the species analysed in what concerns their total leaf phenol and flavonoid contents. From the High Performance Liquid Chromatography (HPLC) electrochemically detected peaks it was possible to attribute to flavonoids the antioxidant capacity detected in A. barbujana, O. foetens, R. maderensis and P. azorica extracts. These potential reactive flavonoids were identified for A. barbujana, R. maderensis and P. azorica. For R. maderensis a high content (7 mg g-1 dry weight) of L-ascorbic acid, an already described antioxidant phytomolecule, was found. A high content in selenomethionine (414.35 μg g-1 dry weight) was obtained for P. arborescens subsp. maderensis extract. This selenocompound is already described as a hydroxyl radical scavenger is reported in this work as also possessing peroxyl radical scavenging capacity. This work is a good illustration of different phytomolecules (flavonoids, organic acids and selenocompounds), presents in leaves of the five traditional medicinal plants endemic to Macaronesia, all exhibiting antioxidant properties.