Annalisa Tassoni

Chitosan treatment induces changes of protein expression profile and stilbene distribution in Vitis vinifera cell suspensions

Polyphenols, including stilbenes and flavonoids, are an essential part of human diet and constitute one of the most abundant and ubiquitous groups of plant secondary metabolites, and their level is inducible by stress, fungal attack or biotic and abiotic elicitors. Proteomic analysis of Vitis vinifera (L.) cultivar (cv.) Barbera grape cell suspensions, showed that the amount of 73 proteins consistently changed in 50u2005μg/mL chitosan‐treated samples compared with controls, or between the two controls, of which 56 were identified by MS analyses. In particular, de‐novo synthesis and/or accumulation of stilbene synthase proteins were promoted by chitosan which also stimulated trans‐resveratrol endogenous accumulation and decreased its release into the culture medium. No influence was shown on cis‐resveratrol. There was no effect on the accumulation of total resveratrol mono‐glucosides (trans‐ and cis‐piceid and trans‐ and cis‐resveratroloside). Throughout the observation period the upregulation of phenylalanine ammonia lyase, chalcone synthase, chalcone‐flavanone isomerase (CHI) transcript expression levels well correlated with CHI protein amount and with the accumulation of anthocyanins. Chitosan treatment strongly increased the expression of eleven proteins of the pathogenesis related protein‐10 family, as well as their mRNA levels.

Increasing sucrose concentrations promote phenylpropanoid biosynthesis in grapevine cell cultures.

Vitis vinifera cell suspensions are a suitable model system to study the metabolic regulation of a large range of high valuable polyphenols that are important in understanding the physiology of the plant and for nutraceutical, pharmaceutical and medical purposes. Increasing sucrose concentrations were found to promote cell growth and phenylpropanoid biosynthesis in grape cell cultures obtained from cv. Barbera immature berries. This led to an intracellular accumulation and/or release into the media of specific polyphenol families (in particular, anthocyanins, catechins and stilbenes). This effect was partially correlated with a sucrose modulation of the transcription of some key biosynthetic enzymes, such as phenylalanine ammonia lyase, chalcone synthase, chalcone-flavanone isomerase and stilbene synthase. Total catechin amounts, both endogenous and released in the media, were increased in proportion to the sugar concentration, as were anthocyanin and stilbene production. Sugar treatment notably improved the endogenous accumulation and release in the culture media of resveratroloside, a resveratrol mono-glycoside, which is the most abundant stilbene found in grape cultures, especially in cv. Barbera. We hypothesize that high sucrose concentrations (exceeding those naturally-occurring in ripe berries) could play a role in plant defense via the induction of secondary metabolites, such as stilbenes.

Polyamines and salt stress response and tolerance in Arabidopsis thaliana flowers.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) flowers and stalks collected from plants germinated and grown under increasing salt-stress conditions (0-75 mM NaCl). The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Spermidine synthase enzyme activity determined both in supernatant and pellet fractions, together with RT-PCR results, led us to hypothesize a different intracellular compartmentation of the isoforms of these enzymes. Free and conjugated polyamines (perchloric acid-soluble and -insoluble) were measured. Free spermidine was the most abundant polyamine and its levels, such as those of free spermine, increased with salt concentration, supporting the hypothesis for a specific role of those polyamines in the response and tolerance to salt stress of Arabidopsis thaliana flowers.

Comparison of biogenic amine and polyphenol profiles of grape berries and wines obtained following conventional, organic and biodynamic agricultural and oenological practices

The bio-active compounds present in food and beverages have a high potential influence on the future health of humans. The levels of biogenic amines, anthocyanins, polyphenols and antioxidant activity were measured in white (Pignoletto) and red (Sangiovese) grape berries and wines from the Emilia-Romagna region (Italy) obtained following conventional, organic and biodynamic agricultural and oenological practices. No significant difference was shown among the samples coming from different agricultural and winemaking practices. Principal Component Analysis was also performed. Biogenic amine amounts were higher in red than in white berries, while in the wines an opposite trend was observed, with histamine, tyramine and putrescine being the most abundant in Pignoletto wines. Red grapes and wines were richer in anthocyanins and showed higher antioxidant activity than white ones. The total level of polyphenols was similar in red and white berries, but with different metabolite profiles depending on the grape variety.

Exploitation of starch industry liquid by-product to produce bioactive peptides from rice hydrolyzed proteins

Small peptides show higher antioxidant capacity than native proteins and may be absorbed in the intestine without further digestion. In our study, a protein by-product from rice starch industry was hydrolyzed with commercial proteolytic enzymes (Alcalase, Neutrase, Flavourzyme) and microbial whole cells of Bacillus spp. and the released peptides were tested for antioxidant activity. Among enzymes, Alcalase was the most performing, while microbial proteolytic activity was less efficient. Conversely, the antioxidant activity was higher in the samples obtained by microbial hydrolysis and particularly with Bacillus pumilus AG1. The sequences of low molecular weight antioxidant peptides were determined and analyzed for aminoacidic composition. The results obtained so far suggest that the hydrolytic treatment of this industrial by-product, with selected enzymes and microbial systems, can allow its exploitation for the production of functional additives and supplements rich in antioxidant peptides, to be used in new food formulas for human consumption.

Combined elicitation of methyl-jasmonate and red light on stilbene and anthocyanin biosynthesis.

Vitis vinifera cell suspensions are a suitable system to study the metabolic regulation of a large range of polyphenols, including flavonoids and stilbenes that play important roles in plant development. Grape cv. Barbera petioles cell cultures were treated with red light and 10 μM methyl-jasmonate (MeJA), alone or in combination, to investigate their influence and/or induction effect on the production of anthocyanins, catechins and free and mono-glucosylated stilbenes. The synthesis of total anthocyanins was slightly decreased by red light alone, while MeJA and MeJA plus red light increased the levels of these metabolites. When compared to the relative controls, the red light treatment decreased the amount of catechins and increased their release in the culture medium, while MeJA alone or in combination with red light increased their production. Red light treatment generally enhanced the amount of free and mono-glucosylated stilbenes during the entire observation period, as well as the percentage of their release in the media. Treatment with MeJA strongly promoted the production of total stilbenes, which was further elicited by the MeJA plus red light treatment. During the combined treatment, the presence of the light stimulus improved the effect of MeJA by anticipating the maximum increase of stilbenes which were also largely released (up to 90%). These results demonstrate that, in grapevine, as in other plant systems, the change of conditions in which the MeJA stimulus is perceived (e.g. going from total white to red light) drastically modifies the plant response to this hormone. The present paper confirms that the jasmonate transduction pathway is integrated into an elaborate signaling network that also comprehends the red light signaling pathway.

Wild food plants traditionally consumed in the area of Bologna (Emilia Romagna region, Italy)

BackgroundThis research was performed in an area belonging to the province of the city of Bologna (Emilia-Romagna region, Northern Italy). The purpose of the present survey was to record the local knowledge concerning traditional uses of wild food plants and related practices, such as gathering, processing, cooking, therapeutic uses, with the aim of preserving an important part of the local cultural heritage.MethodsThirty-nine people still retaining Traditional Local Knowledge (TLK) were interviewed between March-April 2012 and September - October 2013 by means of open and semi-structured ethnobotanical interviews. For each plant species mentioned, we recorded the botanical family, the English common name, the Italian common and/or folk names, the parts of the plant used, the culinary preparation, and the medicinal usage. The relative frequency of citation index (RFC), a tool that measures the local cultural importance of a plant species, was also included.ResultsThe folk plants mentioned by the respondents belonged to 33 botanical families, of which the Rosaceae (14 plants) and the Asteraceae (9 plants) were the most representative. The species with the highest RFC index (0.77) were Crepis vesicaria subsp. taraxacifolia (Thuill) Thell and Taraxacum officinale Weber. Eleven folk plants were indicated as having therapeutic effects. T. officinale Weber, C. vesicaria subsp. taraxacifolia (Thuill) Thell and Sonchus spp., which are used as food, were reported to be depurative, blood cleaning, refreshing, diuretic and laxative. The most commonly used species was Urtica spp, which was also the most frequently cited for medicinal uses.ConclusionsThe present survey documented the wild food plant traditional knowledge of an area belonging to the province of the city of Bologna (Emilia-Romagna region, Northern Italy). The general perception obtained is that on one side the TLK related to wild food plants has strongly been eroded, mainly due to immigration and urbanization phenomena, whereas on the other side these plants are revaluated today because they are perceived as healthy and also because they represent the preservation of biodiversity and a way of getting back to nature.

Helianthus tuberosus and polyamine research: Past and recent applications of a classical growth model

The earliest studies concerning polyamines (PAs) in plants were performed by using in vitro cultured explants of Helianthus tuberosus dormant tuber. This parenchyma tissue was particularly useful due to its susceptibility to several growth substances, including PAs. During tuber dormancy, PA levels are too low to sustain cell division; thus Helianthus represents a natural PA-deficient model. When cultivated in vitro in the presence of auxins, Helianthus tuber dormant parenchyma cells at the G(0) stage start to divide synchronously acquiring meristematic characteristics. The requirement for auxins to induce cell division can be substituted by aliphatic PAs such as putrescine, spermidine or spermine. Cylinders or slices of explanted homogeneous tuber parenchyma were cultured in liquid medium for short-term studies on the cell cycle, or on solid agar medium for long-term experiments. Morphological and physiological modifications of synchronously dividing cells were studied during the different phases of the cell cycle in relation to PAs biosynthesis and oxidation. Long-term experiments led to the identification of the PAs as plant growth regulators, as the sole nitrogen source, as tuber storage substances and as essential factors for morphogenetic processes and cell homeostasis. More recently this system was used to study the effects on plant cell proliferation of platinum- or palladium-derived drugs (cisplatin and platinum or palladium bi-substituted spermine) that are used in human cancer cell lines as antiproliferative and cytotoxic agents. Cisplatin was the most active both in cell proliferation inhibition and on PA metabolism. Similar experiments were performed using three agmatine analogous. Different effects of these compounds were observed on cell proliferation, free PA levels and enzyme activities, leading to a hypothesis of a correlation between their chemical structure and the agmatine metabolism in plants.

Putative occurrence of lysine decarboxylase isoforms in soybean (Glycine max) seedlings.

The activity of lysine decarboxylase was studied in 3-day-old soybean (Glycine max (L.) Meer cv. Sakai) seedlings also in relation to light conditions. Lysine decarboxylase activity was mainly localized in the roots and to a lesser extent in the hypocotyls and was detectable in both the soluble and particulate fractions. The enzyme activity levels were similar during germination under light and dark conditions. With respect to lysine concentration, the initial decarboxylation rate of the soluble fraction showed a saturating curve. Conversely, the initial decarboxylation rate of the particulate fraction showed a sigmoidal curve. These results could suggest that at least two isoforms of lysine decarboxylase are present in different organs of soybean seedlings. In the root soluble fraction, the suicide inhibitor α-difluoromethyl-lysine suppressed the activity of lysine decarboxylase and of ornithine decarboxylase to the same extent, but had no effect on arginine decarboxylase activity.

Effects of chitosan on the protein profile of grape cell culture subcellular fractions

Grapevine is a large source of healthy polyphenols for human diet, and red table‐grapes and wines are the main source of stilbenes. These compounds are important both in the plant defence system and for human health. In the present study, Vitis vinifera cv. Barbera cell cultures were treated with 50 μg/mL chitosan and proteomic analyses on soluble and membrane subcellular fractions were performed against suitable controls. Three soluble stilbene synthase protein spots, four stilbene synthase spots in the microsomal fraction and four spots of membrane ATPase subunits were identified, the accumulation of which was modulated in response to chitosan treatment. Present proteomic and immunolocalisation data seem to provide evidence supporting the hypothesis that a stilbene biosynthetic multi‐enzyme complex is associated with the intracellular membrane. In addition, proteomic analyses showed a general decrease in the accumulation of proteins belonging to different primary metabolism pathways, both in the soluble and membrane fractions. In particular, energy, sugar and amino acid metabolisms were down‐regulated as a consequence of chitosan and acetic acid treatments. These metabolic modifications could lead to a consistent change in the profile and amount of metabolites stored in grape berries, with consequent effects on taste, flavour, organoleptic and nutraceutical properties of derived food products.