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ABSTRACT: Apple (Malus domestica L. Borkh) fruitlet abscission represents an interesting model system to study the early phases of the shedding process, during which major transcriptomic changes and metabolic rearrangements occur within the fruit. In apple, the drop of fruits at different positions within the cluster can be selectively magnified through chemical thinners, such as benzyladenine and metamitron, acting as abscission enhancers. In the present research, different abscission potentials were obtained within the apple fruitlet population by means of the above cited thinners. A metabolomic study was conducted on the volatile organic compounds (VOCs) emitted by abscising fruitlets, allowing to identify isoprene as an early marker of abscission induction. A strong correlation was also observed between isoprene production and abscisic acid (ABA) levels in the fruit cortex, which were shown to increase in abscising fruitlets with respect to non-abscising ones. Transcriptomic evidence indicated that abscission-related ABA is biologically active and its increased biosynthesis is associated with the induction of a specific ABA-responsive NCED (9-cis-epoxycarotenoid dioxygenase) gene. According to a hypothetical model, ABA may transiently cooperate with other hormones and secondary messengers in the generation of an intra-fruit signal leading to the downstream activation of the abscission zone. The shedding process therefore appears to be triggered by multiple interdependent pathways, whose fine regulation, exerted within a very short temporal window by both endogenous and exogenous factors, determines the final destiny of the fruitlets.
Plant physiology 02/2013; · 6.53 Impact Factor
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ABSTRACT: BACKGROUND: Auxins act as repressors of ripening inception in grape (veraison), while ethylene and abscisic acid (ABA) play a positive role as inducers of the syndrome. Despite the increasing amount of information made available on this topic, the complex network of interactions among these hormones remains elusive. In order to shed light on these aspects, a holistic approach was adopted to evaluate, at the transcriptomic level, the crosstalk between hormones in grape berries, whose ripening progression was delayed by applying naphtalenacetic acid (NAA) one week before veraison. RESULTS: The NAA treatment caused significant changes in the transcription rate of about 1,500 genes, indicating that auxin delayed grape berry ripening also at the transcriptional level, along with the recovery of a steady state of its intracellular concentration. Hormone indices analysis carried out with the HORMONOMETER tool suggests that biologically active concentrations of auxins were achieved throughout a homeostatic recovery. This occurred within 7 days after the treatment, during which the physiological response was mainly unspecific and due to a likely pharmacological effect of NAA. This hypothesis is strongly supported by the up-regulation of genes involved in auxin conjugation (GH3-like) and action (IAA4- and IAA31-like). A strong antagonistic effect between auxin and ethylene was also observed, along with a substantial 'synergism' between auxins and ABA, although to a lesser extent. CONCLUSIONS: This study suggests that, in presence of altered levels of auxins, the crosstalk between hormones involves diverse mechanisms, acting at both the hormone response and biosynthesis levels, creating a complex response network.
BMC Plant Biology 10/2012; 12(1):185. · 3.45 Impact Factor
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ABSTRACT: l-Aminoethoxyvinylglycine (AVG) and 1-naphthylacetic acid (NAA) are known to affect preharvest fruit drop, fruit quality and
fruit maturation in Golden Delicious apples (Malus×domestica Borkh). Experiments were carried out on GD/M9 trees treated at three different developmental stages (41, 28 and 17days before
the beginning of the commercial harvest) with AVG and NAA. Both chemicals significantly reduced fruit drop without significantly
affecting the fruit weight. Background colour development and ripening were both delayed by AVG, whereas NAA significantly
enhanced yellowing without affecting the evolution of ripening. Ethylene evolution and transcription profiles of genes involved
in ethylene biosynthesis (MdACS1 and MdACO1) and action (MdETR1, MdERS1 and MdCTR1) were monitored in cortex from the date of the first treatment until the beginning of fruit drop in the control trees (21days
after the beginning of commercial harvest). AVG blocked or efficiently reduced the ethylene evolution. This effect was paralleled
by a down-regulation of MdACS1, MdACO1, MdETR1 and MdERS1. NAA at the second and third date of application enhanced the onset of ethylene evolution, although, at the end of the experiment,
no difference was found between control and treated fruits. The chemical applied in the first date significantly down-regulated
the transcription of the genes at the end of the experiment. MdCTR1 expression, basically unaffected by AVG and NAA, appeared to be transiently down-regulated. The initial down-regulation is
under developmental control, whereas the late regain of transcript accumulation paralleled the ethylene evolution.
Plant Growth Regulation 04/2012; 56(3):317-325. · 1.60 Impact Factor
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ABSTRACT: Field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. In fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. Moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. Later in embryo development, the interactions between seed and fruit development become less strict. As there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the μPEACH 1.0 array platform and quantitative real time RT-PCR (qRT-PCR) was used to study this process.
A comparative analysis of the transcription profiles conducted in seed and mesocarp (cv Fantasia) throughout different developmental stages (S1, S2, S3 and S4) evidenced that 455 genes are differentially expressed in seed and fruit. Among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. Seed markers were a LTP1 (lipid transfer protein), a PR (pathogenesis-related) protein, a prunin and LEA (Late Embryogenesis Abundant) protein, for S1, S2, S3 and S4, respectively. Mesocarp markers were a RD22-like protein, a serin-carboxypeptidase, a senescence related protein and an Aux/IAA, for S1, S2, S3 and S4, respectively.The microarray data, analyzed by using the HORMONOMETER platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. Results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly (auxin) or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on.
In this research, genes were identified marking different phases of seed and mesocarp development. The selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits. Regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones.Further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk (i.e. hormone peptides and microRNAs).
BMC Plant Biology 06/2011; 11:107. · 3.45 Impact Factor
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Giulio Galla,
Sara Zenoni,
Gianpiero Marconi,
Giada Marino, Alessandro Botton,
Francesco Pinosa,
Sandra Citterio,
Benedetto Ruperti,
Klaus Palme,
Emidio Albertini,
Mario Pezzotti,
Martin Mau,
Timothy F Sharbel,
Nico De Storme,
Danny Geelen,
Gianni Barcaccia
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ABSTRACT: Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle.
Gene 05/2011; 484(1-2):1-12. · 2.34 Impact Factor
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Alessandro Botton,
Giulia Eccher,
Claudio Forcato,
Alberto Ferrarini,
Maura Begheldo,
Monica Zermiani,
Stefano Moscatello,
Alberto Battistelli,
Riccardo Velasco,
Benedetto Ruperti,
Angelo Ramina
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ABSTRACT: Apple (Malus × domestica) represents an interesting model tree crop for studying fruit abscission. The physiological fruitlet drop occurring in this species can be easily magnified by using thinning chemicals, such as benzyladenine (BA), to obtain fruits with improved quality and marketability. Despite the economic importance of this process, the molecular determinants of apple fruitlet abscission are still unknown. In this research, BA was used to obtain fruitlet populations with different abscission potentials to be analyzed by means of a newly released 30K oligonucleotide microarray. RNAs were extracted from cortex and seed of apple fruitlets sampled over a 4-d time course, during which BA triggers fruit drop, and used for microarray hybridization. Transcriptomic profiles of persisting and abscising fruitlets were tested for statistical association with abscission potential, allowing us to identify molecular signatures strictly related to fruit destiny. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via reactive oxygen species/sugar and hormones signaling cross talk, and followed by a block of embryogenesis and the consequent activation of the abscission zone.
Plant physiology 10/2010; 155(1):185-208. · 6.53 Impact Factor
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ABSTRACT: The fruits of Rosaceae species may frequently induce allergic reactions in both adults and children, especially in the Mediterranean area. In peach, true allergens and cross-reactive proteins may cause hypersensitive reactions involving a wide diversity 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 survey of the proteins determining the overall allergenic potential, their biological functions, and the factors affecting the expression of the related genes is still missing. In the present study, the expression profiles of some selected genes encoding peach allergen isoforms were studied during fruit growth and development and upon different fruit load and light radiation regimens. The results indicate that the majority of allergen-encoding genes are expressed at their maximum during the ripening stage, therefore representing a potential risk for peach consumers. Nevertheless, enhancing the light radiation and decreasing the fruit load achieved a reduction of the transcription rate of most genes and a possible decrease of the overall allergenic potential at harvest. According to these data, new growing practices could be set up to obtain hypoallergenic peach fruits and eventually combined with the cultivation of hypoallergenic genotypes to obtain a significant reduction of the allergenic potential.
Journal of Agricultural and Food Chemistry 01/2009; 57(2):724-34. · 2.82 Impact Factor
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ABSTRACT: Aspergillus carbonarius is responsible for the majority of mycotoxin contaminations in grapes and its derivatives. 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 A. carbonarius, antagonists for the ability of producing OTA, allowing the identification of 119 differentially expressed sequences putatively involved in the regulation of OTA biosynthesis. 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.
International Journal of Food Microbiology 10/2008; 127(1-2):105-15. · 3.33 Impact Factor
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ABSTRACT: Freshly consumed apples can cause allergic reactions 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 environmental and endogenous factors affecting the allergenic potential of apples would provide 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 reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.
Journal of Agricultural and Food Chemistry 08/2008; 56(15):6707-16. · 2.82 Impact Factor
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ABSTRACT: After 10-year-use of AFLP (Amplified Fragment Length Polymorphism) technology for DNA fingerprinting and mRNA profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. AFLP marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cDNA-AFLP for transcriptome profiling and differentially expressed gene cloning. The evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. This may be achieved by means of the Gene Ontology (GO), consisting in three structured vocabularies (i.e. ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. In this paper, the functional annotation of about 8,000 AFLP-derived ESTs retrieved in the NCBI databases was carried out by using GO terminology.
Descriptive statistics on the type, size and nature of gene sequences obtained by means of AFLP technology were calculated. The gene products associated with mRNA transcripts were then classified according to the three main GO vocabularies. A comparison of the functional content of cDNA-AFLP records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ESTs of Arabidopsis and rice, respectively. On the whole, the statistical parameters adopted for the in silico AFLP-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable GO results. Such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species.
Reliable GO annotations of AFLP-derived sequences can be gathered through the optimization of the experimental steps and the statistical parameters adopted. The Blast2GO software was shown to represent a comprehensive bioinformatics solution for an annotation-based functional analysis. According to the whole set of GO annotations, the AFLP technology generates thorough information for angiosperm gene products and shares common features across angiosperm species and families. The utility of this technology for structural and functional genomics in plants can be implemented by serial annotation analyses of genome-anchored fragments and organ/tissue-specific repertories of transcriptome-derived fragments.
BMC Genomics 01/2008; 9:347. · 4.07 Impact Factor
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ABSTRACT: The influence of internal and external factors such as tree fruit load and elevation on ethylene biosynthesis and action was assessed during apple fruit development and ripening. Ethylene biosynthesis, as well as transcript accumulation of the hormone biosynthetic enzymes (MdACS1 and MdACO1), receptors (MdETR1 and MdERS1) and an element of the transduction pathway (MdCTR1), were evaluated in apples borne by trees with high (HL) and low (LL) fruit load. Orchards were located in two localities differing in elevation and season day degree sum. These parameters significantly affected the date of bloom and commercial harvest, and the length of the fruit developmental cycle. Trees from the low elevation (LE) bloomed and the fruit ripened earlier than those from the high elevation (HE), displaying also a shortened fruit developmental cycle. Dynamics of ethylene evolution was apparently not affected by elevation. The onset of ethylene evolution started 130 days after bloom (DAB) at both elevations. During early ripening, fruits from LL trees produced significantly more ethylene than those from HL trees. Expression analysis of MdACS1, MdACO1 and MdERS1 indicated that the transcript accumulation well correlated with ethylene evolution. MdCTR1 was expressed at constant level throughout fruit growth and development up to 130 DAB, thereafter, the transcript accumulation decreased up to commercial harvest, concurrently with the onset of ethylene evolution.
Plant Cell and Environment 12/2007; 30(11):1480-5. · 5.22 Impact Factor
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ABSTRACT: Mulberries are members of the genus Morus L., a taxonomic group showing a great genetic variability and adaptability to different environmental conditions. This study deals with the use of AFLP-based fingerprints as a tool for estimating genetic variability within as well as among three different mulberry species (i.e., M. alba L., M. latifolia Poir. and M. bombycis Koidz.). A high level of polymorphism (72.2) was found over all the 48 accessions analyzed. Genetic similarity (GS) within single Morus species ranged from 0.845 (M. bombycis) to 0.884 (M. alba) being intermediate in M. latifolia (0.869). The between-species mean genetic similarity estimates based on pair-wise AFLP marker fingerprint comparison were very similar ranging from 0.861 to 0.874. The partition of the genetic variation over the three Morus species was unexpected a proportion of the among-species genetic diversity as low as GST= 0.084 pointed out that about 92% of the total genetic diversity found among Morus accessions is due to DNA polymorphisms within a species, while only 8% of the total variation was highlighted among species. Our data indicate that some of the introduced accessions showing distinctive phenotypes, clearly differentiated from those revealed in the original habitat where they have been selected and adapted, hide an identical genotype.
Genetic Resources and Crop Evolution 02/2005; 52(2):181-192. · 1.55 Impact Factor
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ABSTRACT: Two cDNA clones (named Pp-LTP1 and Pp-LTP2) corresponding to different lipid transfer protein (LTP) genes have been isolated from peach (Prunus persica L. Batsch) epicarp and ovary, respectively. Sequence analysis revealed that the two fragments share 54% identity at nucleotide level and show common features of plant LTP genes, such as conserved cysteine residues and lipid-binding motifs. Phylogenetic analysis grouped Pp-LTP1 and Pp-LTP2 in two distinct clusters, the former with most of LTP genes sequenced in the Rosaceae family, the latter only with one almond LTP. Genomic Southern data indicated that a small LTP gene family is present in peach. Pp-LTP1 and Pp-LTP2 have been used as gene-specific probes to describe expression in flowers and fruits throughout development. In petals, sepals and stamen only Pp-LTP1 was expressed whereas transcripts of Pp-LTP2 strongly accumulated in non-pollinated and pollinated ovary with a decreasing trend in the period of four weeks after pollination. In fruits, a dramatic accumulation of Pp-LTP1 mRNA was detected in epicarp at all stages of fruit development and, with the exception of the early growth stage, no Pp-LTP1 transcripts have been detected in mesocarp. When Pp-LTP2 was used as a probe in the same fruit tissues, a faint hybridisation signal was observed only in epicarp of fruitlets collected at an early growth stage. Infection with Monilia induced only a slight increase of Pp-LTP1 transcript in epicarp of pre-climacteric and climacteric fruits. These results support the hypothesis of multiple roles played by LTPs and, considering that LTPs have been recognised as the major allergen of peach, indicate that Pp-LTP1 could be related to the allergenicity of peach.
Plant Science.
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ABSTRACT: 1-Methylcyclopropene (1-MCP) is an antagonist of ethylene for receptor binding sites and the effects of its application differ in relation to a number of factors including genotype and ripening physiology. Peach (Prunus persica L. Batsch cv. ‘Summer Rich’) and apple (Malus × domestica L. Borkh cv. ‘Golden Delicious’) fruits were incubated with 1-MCP (1 μL L−1) for 24 h at 20 °C and respiration rate, ethylene production and fruit firmness, together with ACC synthase, ACC oxidase, ETR1, ERS1, and CTR1 gene expression patterns were assessed throughout the post-treatment phase. 1-MCP was confirmed to be effective in delaying ripening in apples while in peaches only a limited effect of the chemical was observed. A dramatic inhibition of ethylene biosynthesis and ACS gene expression was induced by 1-MCP in apples whereas no marked difference was observed in peaches between the two controls (in air and in sealed jars without 1-MCP) and the treated fruit. In apples, Md-ETR1 and Md-ERS1 gene expression was down-regulated by 1-MCP starting from the end of the treatment, while Md-CTR1 appeared negatively affected by the chemical at a later stage. Transcription of Pp-ETR1, Pp-ERS1 and Pp-CTR1 genes appeared unaffected in 1-MCP treated peaches. Differences in receptor transcript levels between control fruit maintained in air and those enclosed in sealed jars without 1-MCP may be due to an effect of CO2 that rapidly accumulates following incubation of ripening peaches. Results support the hypothesis that the different behaviour of peaches and apples in response to 1-MCP application might be related to differences in terms of ratio, expression patterns and/or turn-over of the ethylene receptors.
Postharvest Biology and Technology.
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Alessandro Botton
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ABSTRACT: Chapter 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 A
