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Pollen is a useful system for the studies of tip growth and cell wall deposition. For this reason, it was chosen as a promising model for studying how external stimuli are integrated within the cell and how several signalling molecules are interconnected. In particular, understanding how the overall shaping of the pollen tube tip and how several well-known factors are interconnected were the main goals of this study. As the proper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes its growth mechanism is controlled by several signaling molecules such as polyamines (PAs), which control different aspects of pollen tube germination. In pollen, the homeostasis of PAs is finely regulated and the perturbation of this balance has provided, over the years, interesting evidences about how PAs carry out some of their functions in the cell. For these reasons, PAs ware taken into consideration to be used as perturbing molecules, used to spread light in understanding not only the reciprocal interactions among factors, but also the timing of the events. Besides the great interest about pollen as a model of study, pollen is a relevant topic also because it is the main trigger of seasonal allergies. It often happens that the only “pollen count” does not completely mirror the potential allergenicity of the air. Here from, the necessity to integrate the forecasting of allergenic pollen in the atmosphere with the monitoring of airborne allergenic proteins. In these studies, the amount of airborne allergens were put in correlation not only with pollen counts but also with meteorological factors in order to obtain a broad panel of evidences about factors that may affect pollen and allergen dispersion. Finally, in vitro studies also focused on the characterization of allergens, deeply involved in pollen cell wall organization.

Gluten is the major factor involved in the structural properties of bakery products. Thanks to its ability to create aggregates, it gives dough viscosity, elasticity and cohesion, all features responsible for the baking performance. Gluten is composed of extensible, viscous gliadins and rigid, elastic glutenins. Moreover, the gliadin protein fraction is the main factor responsible for the development of celiac disease (CD) and other non-celiac gluten sensitivities. The only treatment for these people is lifelong adherence to a strict gluten-free diet. Nowadays, the development of new technologies in the food industry aims to both improve products already marketed, and to develop new gluten-free products. Cross-linking enzymes, able to organize and create protein networks, are suitable for the food industry as protein modifiers. Among those enzymes, Transglutaminase (TGase) due to its ability to improve the firmness, viscosity, elasticity and water-binding capacity of food products could be the key factor for manipulation in order to achieve higher food quality. The aim of this study was to evaluate the effects of addition of microbial transglutaminase from Streptoverticillium mobaraense, on protein aggregation in gluten free flours as well as in baked products. In our research we assessed five gluten free flours deriving from different plant sources such as cereals, pseudo-cereals and legumes, in particular corn, rice, amaranth, quinoa and lentil flours have been analyzed. By biochemical tests, flours’ total extracted proteins capacity to act as TGase substrate was evaluated. Since all tested flours showed considerable transamidase activity in microplate colorimetric assay, the possible formation of crosslinked products were analyzed by SDS-PAGE. For this purpose, micro-doughs prepared by adding the water, in 1:2 ratio (w/v), to different flours were treated with different amount of TGase enzyme (0.5; 0.75; 1; 2 Units). To clarify which protein fraction of the flours was the best substrate for the TGase, differential extractions were performed. Therefore, three sequential fractions, corresponding to albumins/globulins, prolamins and glutelins, were analyzed in relation to different enzyme amounts. Results showed that the microbial TGase has capacity to modify gluten free flour proteins and determine protein network formation. In particular, lowest enzyme concentration (0.5 U) was able to cause differences in protein bands between treated samples and their non-treated controls. These preliminary results give a perspective in the gluten-free research and suggest their possible use to create innovative products with improved texture.

Pollen tube growth is a rapid process restricted to the only tip region. Many factors cooperate to allow this apical growth, creating an intricate signalling network. The continuous rebuilding of the cell wall and apical migration of the cytoplasm sustained by cytoskeleton re-organisation are the most important driving forces needed for growth1, but many other factors are involved in this process, among which polyamines (PAs), that are essential during pollen tube emergence2 and ROS, that support the apical growth, at physiological concentration3. We investigated the effect of the natural PA spermine (Spm) and BD23, a synthetic aromatic derivative of Spm on the apical growth of Pyrus communis pollen tube and observed that both Spm inhibited the growth from 10 M onwards. Thanks to a FITC-labelled Spm, we were able to observe, that PAs enter through the pollen tube tip, then diffuse in the sub-apical region. The same region underwent drastic morphological changes, showing loss of polarity and enlarged tip when Spm and BD23 were supplied at 100 M or higher. The effects of PAs concern, at least in part, their ability to modulate ROS concentration. In fact the activity of NAD(P)H-oxidase (NOX), catalase (CAT) and superoxide dismutase (SOD) was drastically affected by PAs supplementation. Moreover, also the tip-focused Ca2+-gradient was suddenly altered. Interestingly, the actin cytoskeleton was affected by the treatment with 100 M of both the PAs causing its re-organization in thicker bundles that followed the swelling of the apex. Intriguing, the cell wall was enriched in both callose and cellulose exactly in the region where the swelling of the tip begins, probably to contrast the excess of ductility of the cell wall after treatment with PAs 100 M. The viability of pollen was slightly affected by 100 M treatment, and the degradation of nuclear DNA, was completely inhibited when pollen had been pre-treated with the caspase-3 inhibitor I peptide, Ac-DEVD-CHO (DEVD). Different was the scenario when the PAs were supplemented at 500 M, concentration that is far away from the physiological one. In this case, the effects on the pollen were more drastic, with a rapid drop of cell viability, actin depolymerisation, stimulation of DNA-laddering after 30 minutes incubation and the complete degradation of both vegetative and generative nuclei; these effects were only in part inhibited by the pre-treatment with DEVD. Thus, the present data may open new research avenues to understand how the diameter of the pollen tube is regulated and which role PAs could play in the puzzling process of apical growth.

Gluten is the major factor involved in the structural properties of bakery products. Thanks to its ability to create aggregates, it gives dough viscosity, elasticity and cohesion, all features responsible for the baking performance. Gluten is composed of extensible, viscous gliadins and rigid, elastic glutenins. Moreover, the gliadin protein fraction is the main factor responsible for the development of celiac disease (CD) and other non-celiac gluten sensitivities. Nowadays, the development of new technologies in the food industry aims to both improve products already marketed, and to develop new gluten-free products. The purpose of our work was to set up a biotechnological process based on the combined use of cross-linking enzymes and selected microbial consortia to produce new gluten free bakery products with improved shelf-life, and sensory properties. Cross-linking enzymes, able to organize and create protein networks, are suitable for the food industry as protein modifiers. Among those enzymes, Transglutaminase (TGase) is surely a potentially interesting tool for its capacity to enhance the cohesiveness and elasticity of the dough, solving the problem of lack of consistency of gluten-free flours. Our research showed that the microbial TGase (from Streptoverticillium mobaraense) has the capacity to modify wheat flour proteins and determine protein network formation responsible for a major molecular structural stability of the product. The TGase effect on flour mainly involved gluten and globulin fraction modification. The results showed that the enzyme activity in the presence of a selected microbial consortium of lactic acid bacteria and yeasts (Lactobacillus sanfrancisciencis and Candida milleri) caused synergic effects on the sensory, rheological and shelf-life features of the products. These features resulted significantly affected also by formulation and process variables. Based on our results, we are evaluating the gluten-free flours of corn, rice, amaranth and lentil, to develop a new bakery product for people with gluten sensitivities.

Quinoa (Chenopodium quinoa Willd.) is a facultative halophyte and some varieties are able to cope with salinity levels as high as those present in sea water. Due to its traditionally broad cultivation area (from the salt flats of the Andes at 3,800 m above sea level to the seacoast and from Colombia down to southern Chile), there is a very wide range of quinoa cultivars adapted to specific conditions displaying a broad genetic variability in stress tolerance. For this reason, quinoa offers a valuable source of germplasm for breeding new cultivars adapted to stressful environments in diverse geographical conditions and for identifying mechanisms potentially useful in breeding tolerant glycophytes. In Chile, the genetic distance between highland (altiplano) and coastal cultivars of quinoa has been assessed but their responses to salinity are less known. In the present work, the response to salt treatments (0, 100 and 300 mM NaCl) was investigated in one altiplano landrace, R49, and two coastal ones, VI-1 and VR (from central and southern Chile, respectively). Salt treatment caused a significant increase in Na+ concentrations in all plant organs; the Na+ concentration accumulated in leaves under 300 mM salt was highest in VI-1 and was ca. 1.5- to 3-fold higher in leaves and roots of plants grown under 300 mM NaCl than 100 mM NaCl. Growth was significantly inhibited by the lower salt concentration only in R49, whereas 300 mM NaCl reduced growth also in VI-1 and VR, albeit to a lower extent than in R49. The concentrations of photosynthetic pigments, total phenolics and flavonoids were differentially affected by salt in leaves of the three cultivars. Seed production at harvest was unaffected or even enhanced (VR) by 100 mM NaCl relative to controls, while with 300 mM it was reduced in R49 and VI-1 (ca. 80% and 40% inhibition, respectively) but was not affected in VR. The germinability of seeds collected from plants grown in the presence (100 or 300 mM) or absence (0 mM) of NaCl was tested on agar plates containing 0-500 mM NaCl. Results showed that the response varied between landraces with VR exhibiting the highest sensitivity (germination was inhibited even by 100 mM NaCl). Seed protein profiles (SDS-PAGE of total proteins and the amount of the 11S seed storage protein chenopodin) were compared among different cultivars not treated with salt and the effect of salt treatments on protein profile were checked in the frame of the single cultivar; this allow to compare the differences due to the genotype and also the plant response to the salt treatments.