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A transcriptomic approach to food safety - Fruit allergens and mycotoxins
No full textChapter 1 - Food safety has become a public health priority, since food-related diseases affect up to one third of the population of the rich countries, and are spreading also in the developing ones. During the last decades, the integration of agricultural and food industries, and the food globalization have changed the patterns of food production and distribution, creating the ideal environment for the diffusion of new food-related disorders. Among the most important are the food allergies and the contamination of food and feed with mycotoxins such as aflatoxins or ochratoxins. The international community is aware of the problem and is trying to focus on a "farm to fork" approach operating in every step of the food chain with new regulations and preventive methods. In this context, nutritional genomics may represent a key scientific area, but its most important tool, which is transgenesis, is not well accepted by the consumers. Therefore, the biotechnologists are exploring alternative ways of using innovative genomic technologies to deliver crop improvements in a reasonable time. Marker assisted selection and functional genomics studies may allow an easier exploitation of natural variation, and to study the effective interactions existing between environment and genotype to set up innovative agricultural practices aimed at specifically modifying the expression of the genes of interest. In the present research, a transcriptomic approach was adopted for studying fruit allergens and mycotoxins, by using high-throughput techniques such as real-time PCR and cDNA-AFLP. The major findings of this study concern the factors affecting the allergenic potential in apple and peach, and the regulation of ochratoxin A biosynthesis in Aspergillus carbonarius, a filamentous fungus contaminating grapes and wines.
Chapter 2 - Apple consumption is highly recommended for a healthy diet because of its nutritional properties. However, freshly consumed apples can cause allergic reactions in the worldwide population because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, and their cross reactivity with sensitizing allergens of other species. Knowledge of the environmental and endogenous factors affecting the allergenic potential of apples would give important information to apple breeders, growers and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with no or reduced amount of allergens. In the present research, expression studies were performed for all the known genes encoding allergen isoforms belonging to the four classes. Fruit specimens collected from fifteen apple varieties were investigated by means of Real Time PCR, and Principal Component Analysis (PCA) was performed for the ordination and classification of varieties according to their total allergenic potentials. Three different trials were carried out to assess the effects of shadowing, elevation and storage, and water stress on the expression of allergen-related genes. Shadowing was shown to be effective in reducing and increasing the allergenic potential in cortex and epidermis, respectively, whereas elevation was found to increase the global allergenic potential, especially in the cortex. Storage of fruits harvested at low elevations enhanced the global allergen-related transcription in the cortex and decreased it in the epidermis. An opposite trend was determined by storage of fruits harvested at high elevations. In spite of the dramatic effect on both fruit size and vegetative growth of the trees, water stress was shown to slightly affect the expression of only two genes. The possible implications of the results for breeders, growers and consumers are critically discussed.
Chapter 3 - Section 1 - The full length cDNAs of Pp-LTP1 and Pp-LTP2, two members of the Lipid Transfer Protein multigene family in peach, have been isolated along with the proximal promoter and the complete genomic sequence of Pp-LTP1. As observed in other plant species, Pp-LTP1 is organized in two exons and, according to the deduced amino acid sequence of the secreted protein, it encodes the 9 kDa allergenic protein Pru p 3. Pp-LTP1 transcripts and Pru p 3 protein markedly accumulate in epicarp of ripe fruit in all of the peach and nectarine varieties examined (including "Royal Gem") with the exception of "Rita Star" that shows weak specific mRNA hybridization signal and no Pru p 3 accumulation. SDS-PAGE clearly displays a different banding pattern in correspondence of 9 kDa between "Rita Star" and "Royal Gem". In immunoblotting analysis, performed using sera of eight LTP positive patients, a strong band is present when using "Royal Gem" epicarp extract but not in the case of "Rita Star". According to these data, "Rita Star" appears an interesting model to better elucidate the role of LTP in fruit development and allergenic reactions.
Section 2 - Recombinant allergenic proteins represent an important tool in the diagnosis of allergic reactions to foods of plant origin, relying upon a deep and precise knowledge of the gene sequences encoding allergens. In the present study, the list of the known allergens of peach was implemented with candidate allergenic proteins identified by means of a bioinformatics approach. Possible implications for the development of new diagnostic tools are discussed.
Section 3 - In the Mediterranean area, the fruits of the Rosaceae species are most frequently involved in allergic reactions in the adult population and in patients older than 3-years of age. In peach, both true allergens and cross-reactive proteins cause hypersensitive reactions involving a wide variety of symptoms. Three known classes of allergenic proteins, namely Pru p 1, Pru p 3 and Pru p 4, have been reported to be mostly involved, but an exhaustive understanding concerning the proteins determining the overall allergenic potential, their biological function and the factors affecting the expression of the related genes is missing. In the present study, the expression profiles of some selected known and newly identified genes encoding allergen isoforms are characterized during fruit growth and development, and upon different fruit load and light radiation regimes. According to the results herein presented, enhancing light radiation and decreasing the fruit load achieved a reduction of the transcription rate of the majority of allergen-encoding genes.
Chapter 4 - Aspergillus carbonarius is responsible for the majority of mycotoxin contaminations in grapes and its derivatives, infecting berries from the early developmental stages throughout the whole winemaking process. Most of A. carbonarius strains are ochratoxin A (OTA) producers, even though at very different levels. This broad variability was used to identify genes whose expression is linked with the ability of producing OTA. A cDNA-AFLP differential display screening was performed in two strains of Aspergillus carbonarius, antagonists for the ability of producing OTA, allowing the identification of 119 differentially expressed sequences putatively involved in the biosyntesis of OTA and its regulation. A likely connection was pointed out between the biosynthesis of the toxin, vegetative growth and sexual/asexual developmental progression, along with common signalling pathways involving G protein and Ca2+/calmodulin dependent phosphorylation and dephoshorylation cascades.
Chapter 5 - The transcriptomic approach adopted in the present research proved to be efficient, and allowed to achieve the following results:
1. The identification of putative hypoallergenic apple varieties
2. The identification of several factors affecting allergen-related gene expression in apple and peach
3. The identification of a peach variety with low content of Pru p 3 allergen
4. The classification of new candidate peach allergens
5. A model for OTA biosynthesis regulation in Aspergillus carbonarius
The present research put the basis for the future development of:
1. Breeding programmes for the constitution of hypoallergenic apple varieties
2. Agricultural practices reducing the allergenic potential of apple and peach fruits
3. Recombinant allergens of peach for diagnostic purpose
4. Biocompetition strategies to avoid the contamination of grapes and wines with ochratoxin
From the Field to the Lab: Establishing Different Abscission Potentials in Apple Fruitlets for Transcriptomic and Functional Studies
No full textPhysiological studies of the abscission process in non-model species require a profound knowledge of both the specific characteristics of the crop/variety and the multiple factors that are involved in the induction of the process. The main problem deals with the predictability of the destiny of the abscising organ, that is fundamental in comparative physiological studies aimed at identifying the causal factors preceding the activation of the abscission zones (AZs) and the detachment of the organ. Relevant progress has been made in the last decades regarding the abscission of the apple (Malus domestica L. Borkh) fruitlets, which were shown to be a good model system for these kinds of studies. The present method summarizes the most relevant knowledge regarding the following: (1) the establishment of differentially abscising fruitlet populations in apple; (2) the sampling techniques to adopt for successful collections of suitable samples; (3) a reference protocol for the isolation of total RNA from apple fruitlets’ tissues, and (4) the main bioinformatic steps to analyze RNA-Seq data. This method may represent a reliable knowledge base for developing of similar approaches in other crops in future
Aspetti nutraceutici ed allergenici legati al consumo di frutta
No full textLa conoscenza approfondita delle componenti funzionali della frutta rappresenta un requisito fondamentale per la messa a punto di forme ipoallergeniche biologicamente attive e di peptidi ricombinanti da impiegare nelle vaccinazioni. La ricerca in questo campo risulta estremamente utile per la messa a punto dei futuri strumenti diagnostici e terapeutici nell'ambito delle allergie alimentari. Transgenesi o valutazione della biodiversità disponibile accompagnata a nuove tecniche colturali? I pregi e i difetti insiti in ognuna delle tecnologie applicative
Impiego dei fitoregolatori in viticoltura: potenzialità e problematiche.
No full textThe use of plant bioregulators to regulate
physiological process occurring during plant growth
and development may represent an important tool for
growers. In viticulture, hormone treatments have been
mainly addressed to the reproductive developmental
cycle, taking into account that grapevine vegetative
activity can be successfully controlled by training
systems and agricultural practices. In this review we
discuss the role of various hormones in the control of
inflorescence development and berry growth and ripening.
The control of these processes may have relevant
implications for disease control in the vineyard
considering that the susceptibility of different grape
cultivars to Botrytis bunch rot, powdery mildew and
downey mildew attacks is closely correlated with
bunch architecture. The timing and the extent of ripening
is of considerable scientific interest, but has also
implications for the various grape industries (fresh
market, winery logistic and processing, as well as
grape withering). In viticulture several bioregulators
can be used to regulate events of reproductive developmental
cycle. However, the most important applications
regard the use of gibberellins to modify inflorescence
length and bunch architecture as well as
berry size in seedless varieties, and abscissic acid,
ethylene and brassinosteroids to improve quality traits
of berries. Auxins can be mainly used as inhibitors of
ripening; therefore, they can be applied to delay the
vintage without significant changes of global quality of
berries. A delayed ripening can be also achieved by
spraying 1-methylcyclopropene, an inhibitors of ethylene
action. The knowledge of these effects has been
significantly improved in the last years thanks to the
increasing availability of information concerning the
molecular basis of hormone action. In the future, this
information could be used to develop new strategies
in the control of reproductive developmental cycle. In
addition, crystallography X-ray is an important tool to
elucidate the structure of plant hormone receptors
and thus to select, from chemical libraries, small biomolecules
able to interact with them. This is a crucial
point to develop new molecules with a lower synthesis
cost, which is often the main constraint on the introduction
of new plant bioregulators
Thinning in peach: Past, present and future of an indispensable practice
No full textPeach (Prunus persica L. Batsch) trees need to be thinned in order to guarantee yield of fruits of marketable size and a sufficient return to bloom in the following season. In this fruit crop, however, thinning is often complicated by several endogenous as well as exogenous factors, such as genotype, orchard structure, environmental conditions and agronomic management, and the current thinning methods (manual, mechanical or chemical), taken singularly, present more disadvantages than advantages. Therefore, the only possible option is currently represented by the adoption of a “thinning strategy”, consisting in subsequent interventions at different phenological stages, to finely tune the final fruit load along its physiological evolution. Such strategies must also consider the ongoing climate changes, which further complicates the thinning interventions also due to the lack of Decision Support Systems (DSSs), and the current trends of the global market. The present review takes a picture of the current situation of peach thinning, with a particular focus on the current development of new chemical thinners, and its future perspectives. Some general remarks are made considering the evolution of the global market and the obstacles found in peach research in some of the most traditional peach areas of the world
Gli allergeni nelle specie fruttifere
No full textSe da un lato il consumo di frutta fresca rappresenta il punto fermo di una dieta bilanciata, in grado di proteggere l’organismo da tutta una serie di sindromi degenerative e tumorali, esso comporta anche alcuni rischi per una sempre più crescente fetta di popo- lazione, soprattutto nei paesi più sviluppati, rispetto alla possibilità di presentare reazioni allergiche. Tali reazioni, che talvolta pos- sono provocare sintomi anche mortali (es. shock anafilattico) con un’incidenza pressoché doppia nei bambini rispetto agli adulti, si manifestano in seguito al contatto con sostanze di origine solitamente proteica che, entrando nell’organismo per via respiratoria (allergeni inalanti) o tramite l’apparato digerente (allergeni alimentari), causano la reazione del sistema immunitario e la conseguen- te manifestazione sintomatica. Le specie arboree coltivate possono quindi rappresentare una sorgente di allergeni durante tutto il ciclo di produzione, ossia da quando in piena fioritura disperdono il proprio polline nell’ambiente circostante a distanze di alcuni chilometri dal sito di coltivazione, fino al momento del consumo del prodotto finale, fresco o trasformato. Le porzioni di sequen- za amminoacidica responsabili della reazione allergica (epitopi) possono essere presenti in forma conservata sia in allergeni inalanti che alimentari, causando le cosiddette reazioni crociate, ovvero la possibilità di sviluppare allergie ad alimenti in seguito alla sensi- bilizzazione generata da pollini
I geni omeotici che controllano la differenziazione del fiore
No full textLa differenziazione delle strutture fiorali è controllata da fattori di trascrizione codificati da una vasta famiglia di geni del tipo MADS- box, coinvolti nella regolazione di funzioni biologiche importanti in animali, funghi e piante, anche se non tutte le sottofamiglie sono presenti nei tre regni. L’acronimo MADS deriva dalle iniziali dei primi quattro geni identificati in tale famiglia: MCM (MINICHROMO- SOME MAINTENANCE1 di lievito), AGAMOUS (gene di classe C di Arabidopsis thaliana), DEFICIENS (gene di classe B di Antirrhynum majus) e SRF (SERUM RESPONSE FACTOR, gene umano coinvolto nelle risposta all’oncogene c-fos). Sulla base della sequenza, si posso- no distinguere MADS di tipo I e di tipo II, che si diversificano nella porzione carbossi-terminale per la presenza, nei MADS di tipo I, di sequenze altamente simili a SRF, ed in quelli di tipo II, dei domini I, K e C (MIKC). Il dominio I conferisce specificità nell’interazione con il DNA, il dominio K media la formazione di dimeri MADS-box, mentre il dominio C funziona come attivatore della trascrizione
The Yes and No of the Ethylene Involvement in Abscission
Full text linkAbscission has significant implications in agriculture and several efforts have been addressed by researchers to understand its regulatory steps in both model and crop species. Among the main players in abscission, ethylene has exhibited some fascinating features, in that it was shown to be involved at different stages of abscission induction and, in some cases, with interesting roles also within the abscising organ at the very early stages of the process. This review summarizes the current knowledge about the role of ethylene both at the level of the abscission zone and within the shedding organ, pointing out the missing pieces of the very complicated puzzle of the abscission process in the different species
Accumulo dei trascritti di Pp-LTP1 e Pp-LTP2, gli allergeni della pesca, durante la maturazione e la fase postraccolta
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