http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-05-20T19:51:22Z http://hdl.handle.net/2067/1413 Title: Increasing the amylose content of durum wheat through silencing of the SBEIIa genes Authors: Sestili, Francesco; Janni, Michela; Doherty, Angela; Botticella, Ermelinda; D'Ovidio, Renato; Masci, Stefania; Jones, Huw D.; Lafiandra, Domenico Abstract: Background: High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. Results: Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). Conclusion: We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable. Description: L'articolo è disponibile sul sito dell'editore: http://www.biomedcentral.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1700 Title: Comparative proteomic and transcriptional profiling of a bread wheat cultivar and its derived transgenic line over-expressing a low molecular weight glutenin subunit gene in the endosperm Authors: Scossa, Federico; Laudencia-Chingcuanco, Debby L.; Anderson, Olin D.; Vensel, William H.; Lafiandra, Domenico; D'Ovidio, Renato; Masci, Stefania Abstract: In this paper, a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that over-expresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding non-transformed genotype, has been carried out. Proteomic analyses showed that, during seed development, several classes of endosperm proteins were differentially accumulated in the transformed endosperm. As a result of the strong increase in the amount of the transgenic protein, the endogenous glutenin subunit, all sub-classes of gliadins, and metabolic as well as Chloroform/Methanol soluble proteins were diminished in the transgenic genotype. The differential accumulation detected by proteomic analyses, both in mature and developing seeds, was paralleled by the corresponding changes in transcript levels detected by microarray experiments. Our results suggest that the most evident effect of the strong over-expression of the transgenic glutenin gene consists in a global compensatory response involving a significant decrease in the amounts of polypeptides belonging to the prolamin superfamily. It is likely that such compensation is a consequence of the diversion of amino acid reserves and translation machinery to the synthesis of the transgenic glutenin subunit. 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1703 Title: A relaxed specificity in interchain disulfide bond formation characterises the assembly of a low-molecular-weight glutenin subunit in the endoplasmic reticulum Authors: Lombardi, Alessio; Barbante, Alessandra; Della Cristina, Pietro; Rosiello, Daniele; Castellazzi, Chiara Lara; Sbano, Luca; Masci, Stefania; Ceriotti, Aldo Abstract: Wheat (Triticum aestivum) grains contain large protein polymers constituted by two main classes of polypeptides: the highmolecular- weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation. Description: L'articolo è disponibile sul sito dell'editore: http://www.aspb.org 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1784 Title: Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress. Authors: Laino, Paolo; Shelton, Dale; Finnie, Christine; De Leonardis, Anna Maria; Mastrangelo, Anna Maria; Svensson, Birte; Lafiandra, Domenico; Masci, Stefania Abstract: In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterised by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The non-prolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of non-prolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress vs. control) during grain filling. The 2D patterns of non-prolamin proteins were monitored to identify polypeptides affected by heat stress. This study shows that heat stress alters significantly the durum wheat seed proteome, although the fold changes range only between 1.2 and 2.2. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI TOF and MALDI-TOF-TOF MS and included heat shock proteins, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress related proteins. Many of the heat induced polypeptides are considered to be allergenic for sensitive individuals. The differences observed with previously reported data regarding bread wheat may be explained by the absence of the D genome in durum wheat Description: L'articolo è disponibile sul sito dell'editore: www.interscience.wiley.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1706 Title: Characterisation of a specific class of typical low molecular weight glutenin subunits of durum wheat by a proteomic approach Authors: Muccilli, Vera; Cunsolo, Vincenzo; Saletti, Rosaria; Foti, Salvatore; Margiotta, Benedetta; Scossa, Federico; Masci, Stefania; Lafiandra, Domenico Abstract: Glutenin polymers are formed by high (HMW-GS) and low molecular weight glutenin subunits (LMWGS). The latter group of subunits has been less characterised compared to the former due to their great number and heterogeneity. In order to gain more information on the LMW-GS, we have used a durum wheat line carrying a 1BL.1RS translocation, in which the short arm of the chromosome 1B is replaced by the short arm of the chromosome 1R of rye. This line was obtained using the durum wheat cultivar Cando, in which the translocation is present, crossed and back-crossed four times with the Italian durum wheat cultivar Svevo. Comparative electrophoretic and mass spectrometric analyses carried out on LMW-GS prepared from the durum wheat cultivar Svevo and the line carrying the 1BL.1RS translocation have provided further information on these complex group of proteins. In particular, all the three types of typical LMWGS (LMW-s, LMW-m and LMW-i) were identified in Svevo, whereas the latter group is the only one present in the line with the 1BL.1RS translocation, thus confirming previous findings of the association of the LMW-i type subunits with genes present on chromosome 1A. Description: L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1783 Title: Influence of water deficit on durum wheat storage protein composition and technological quality Authors: Flagella, Zina; Giuliani, Marcella M.; Giuzio, Luigia; Volpi, Chiara; Masci, Stefania Abstract: Two durum wheat (Triticum durum Desf.) cultivars (Simeto and Ofanto) were grown in field trials in Foggia (Southern Italy), under two water regimes and in three cropping seasons, to evaluate the influence of water regime on grain protein composition in relation to technological quality. On grain samples, the following quality analyses were performed: protein and gluten content, gluten index and SDS test. Furthermore, the different protein fractions were evaluated: gliadins, glutenins, high molecular weight subunits (HMW-GS) and low molecular weight subunits (LMW-GS) of the larger glutenin polymers, and percentage of unextractable polymeric proteins (%UPP). Two-dimensional gel electrophoresis (2D) of glutenins was also performed on selected samples. A year-on-year variation in gluten index was observed and its values were positively correlated (r = 0.71**) with the number of days with maximum temperature ranging from 30 to 35 °C during grain filling. This was consistent with an increase in glutenin fractions. Between the investigated cultivars, Simeto showed the better technological performance and the higher glutenin content, HMW-GS/LMW-GS ratio and %UPP. Water deficit was found to affect technological quality and protein composition differently depending on the timing of stress occurrence. An increase in protein content and in HMW-GS/LMW-GS ratio, consistent with a decrease in grain yield was observed when water deficit occurred throughout the growing season. When a terminal water stress occurred in grain filling an improvement in gluten strength was observed consistently with an increase in the glutenins present in the larger glutenin polymers associated to a general modulation of HMW-GS and LMW-GS levels, as indicated by 2D analyses. Probably water deficit associated to high temperature stress which generally occurs under Mediterranean environments during grain filling, may have caused a higher aggregation level of glutenin subunits that was reflected in an improvement of technological quality. Changes in protein composition was found not to be related to total quantity of nitrogen accumulated during grain filling. Description: L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1701 Title: Analisi genomiche e proteomiche per il miglioramento della qualità e sicurezza delle produzioni agrarie Authors: Lafiandra, Domenico; Masci, Stefania; Reforgiato Recupero, Giuseppe; Terzi, Valeria; Carputo, Domenico; Odoardi, Miriam Abstract: Qualità e sicurezza alimentare sono le parole chiave ricorrenti sia nell’ambito del VII programma quadro dell’Unione Europea, sia nel PNR e nei piani di sviluppo delineati da diverse regioni italiane. In sostanza, l’innalzamento e la definizione della qualità dei prodotti agroalimentari rappresentano strumenti indispensabili per dare nuovo impulso e rinnovamento al settore. I recenti sviluppi della normativa europea ed italiana in questo settore mostrano una netta tendenza verso la necessità di fornire al consumatore, oltre ad una sicurezza microbiologica, anche chiare indicazioni su diversi aspetti qualitativi dei prodotti agro-alimentari. La possibilità di incrementare la quantità di metaboliti secondari (vitamine e antiossidanti) nelle colture risulta di particolare interesse per lo sviluppo di prodotti ad elevata connotazione salutistica (alimenti funzionali). Programmi di miglioramento genetico hanno già permesso di ottenere linee migliorate per tali caratteristiche. A questo si aggiunga la necessità di sviluppare strategie di valutazione che diano indicazioni scientificamente attendibili sulla tracciabilità e salubrità degli alimenti derivati da piante geneticamente modificate. La valutazione del rischio delle colture geneticamente modificate si basa principalmente sul cosiddetto "principio dell'equivalenza sostanziale" o della "sicurezza comparativa", basato sull'assunto che un genotipo transgenico non debba presentare modificazioni sostanziali rispetto al corrispondente genotipo non transgenico, che, nel caso delle varietà commerciali già presenti sul mercato, sono ritenute ovviamente salubri. Le nuove discipline “-omiche”, quali genomica, trascrittomica, proteomica e metabolomica, nate parallelamente e in seguito al sequenziamento completo di genomi modello, possono fornire una fotografia panoramica della composizione delle materie prime e degli alimenti, permettendo così di effettuare una valutazione complessiva della loro qualità e sicurezza. Tali innovativi e sofisticati strumenti d’indagine molecolari possono affiancare e rafforzare le metodiche comunemente usate, rispondendo in modo flessibile alle nuove esigenze di tracciabilità, rintracciabilità e certificazione di autenticità. Description: L'articolo è disponibile sul sito dell'editore: http://www.forumeditrice.it 2007-12-31T23:00:00Z