http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-05-22T16:59:57Z http://hdl.handle.net/2067/1100 Title: Enhancement of the wheat defence response to fungal pathogens by limiting the degradation of the pectin component of the cell wall Authors: Volpi, Chiara Abstract: Fungal pathogens represent one of the most serious threats to agriculture production. The development of crop varieties that simultaneously control different pathogens is an attracting strategy to increase host plant resistance. This can be obtained by improving the pre-existing plant defence mechanisms such as the structure and composition of the plant cell wall, which is one of the first barriers encountered by the microbial pathogens during plant tissue colonization. To overcome this obstacle, most fungal pathogens produce a variety of enzymes that degrade the wall polysaccharides; among them, pectin degrading enzymes are among the first to be secreted by the pathogens to enter and spread into the plant tissue. Pectin in the plant cell wall is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The activity of PME is regulated by specific protein inhibitors (PMEIs). Since highly methylesterified pectin can be less susceptible to hydrolysis by pectic enzymes such as fungal endopolygalacturonases (endo-PGs), the inhibition of endogenous PME by PMEI might increase pectin resistance to degradation by fungal PGs. In order to verify the possibility to improve wheat resistance by modifying the degree of pectin methylesterification, a number of wheat Triticum durum cv. Svevo lines expressing the pectin methylesterase inhibitors either AcPMEI (from Actinidia chinensis) or AtPMEI-1 (from Arabidopsis thaliana) has been produced. Both AcPMEI and AtPMEI-1 endow wheat with a stronger inhibitory activity of endogenous PME. Only one line expressing AtPMEI-1 shows reduced endogenous PME activity while 12 lines expressing AcPMEI show different levels of reduced PME activity. Three Acpmei lines were further characterized on the level of expression and the activity of the transgenic protein. AcPMEI was found to be correctly targeted to the apoplast. The two Acpmei lines expressing high level of the inhibitor, MJ15-151 and MJ15-69, showed no obvious phenotypic differences with the control plants but a significant increase of the degree of methylesterification (DM) of about 20% and 32%, and a different methylester distribution compared to the wild-type plants, respectively. This increased DM and modified methyl distribution of pectin was correlated with a significant reduction of disease symptoms caused by the foliar fungal pathogen Bipolaris sorokiniana or the floral pathogen Fusarium graminearum. To further protect the cell wall pectic component, a pyramiding approach has been undertaken to combine PMEI and the polygalacturonase-inhibiting protein (PGIP). The cross between the Acpmei lines and a new developed durum wheat transgenic line expressing Pvpgip2 has been performed and the F1 progeny analyzed for the presence of both transgenes. Finally, to select additional fungal pathogens to be tested in the cross Acpmei/Pvpgip2 and in the parental lines, infection experiments with the biotrophic fungal pathogen Claviceps purpurea were performed on a stable transgenic bread wheat line expressing Pvpgip2 and a slight, even significant, reduction of symptoms in the transgenic line compared to the control plants was observed.; I patogeni fungini rappresentano una delle minacce più serie per la produzione agricola. La costituzione di varietà in grado di resistere all’aggressione di più patogeni rappresenta una delle strategie più promettenti nei programmi di miglioramento genetico per la resistenza. Ciò può essere ottenuto rafforzando i meccanismi di difesa pre-esistenti della pianta, come la struttura e la composizione della parete cellulare vegetale che è una delle prime barriere incontrate dai patogeni microbici durante la colonizzazione del tessuto ospite. Per superare questo ostacolo, la maggior parte dei patogeni fungini produce una varietà di enzimi che degradano i polisaccaridi della parete cellulare. Tra questi, gli enzimi pectici sono tra i primi ad essere secreti dai patogeni per poter penetrare e colonizzare il tessuto ospite. La pectina è secreta nella parete cellulare in forma altamente metilesterificata ed in seguito viene deesterificata in muro dalle pectin metilesterasi (PME). L’attività delle PME è controllata dall’inibitore della pectin metilesterasi (PMEI). Poichè le pectine altamente metilesterificate sono meno suscettibili all’azione di enzimi pectici come le endo-PG, l’inibizione di PME endogene operata dalle PMEI può avere un ruolo indiretto nella difesa, limitando l’azione delle PG secrete da patogeni fungini. Per verificare l’ipotesi di incrementare la resistenza del frumento modificando il grado di metilesterificazione della pectina, sono state prodotte linee di frumento duro cv. Svevo esprimenti l’inibitore della pectin metilesterasi AcPMEI (da Actinidia chinensis) o AtPMEI-1 (da Arabidopsis thaliana). Entrambi gli inibitori, AcPMEI e AtPMEI-1, conferiscono al frumento una maggiore attività inibitoria della PME endogena. Soltanto una linea esprimente la AtPMEI-1 mostra una ridotta attività della PME endogena mentre 12 linee esprimenti la AcPMEI mostrano diversi livelli di riduzione dell’attività PME. Tre linee Acpmei sono state caratterizzate ulteriormente relativamente al livello di espressione e all’attività della proteina transgenica. La proteina AcPMEI è risultata correttamente localizzata nell’apoplasto. Le due linee Acpmei con un alto livello di espressione dell’inibitore, MJ15-151 e MJ15-69, non mostrano evidenti differenze fenotipiche rispetto alle piante di controllo nonostante presentino un significativo incremento del grado di pectin metilesterificazione di circa il 20% e 32% rispettivamente e una differente distribuzione dei metilesteri. L’incremento del grado di metilazione e la distribuzione modificata dei gruppi metilici della pectina è stata correlata con una significativa riduzione dei sintomi della malattia causati dai patogeni fungini Bipolaris sorokiniana e Fusarium graminearum, che infettano rispettivamente in modo prevalente le foglie o la spiga del frumento. Per proteggere ulteriormente la componente pectica della parete cellulare, è stato intrapreso un programma di piramidazione volto a combinare la PMEI e l’inibitore proteico della poligalatturonasi (PGIP). È stato effettuato l’incrocio tra le linee Acpmei e la nuova linea transgenica di frumento duro sviluppata esprimente il gene Pvpgip2 e la progenie F1 è stata analizzata per la presenza di entrambi i transgeni. Infine, per selezionare ulteriori patogeni fungini da analizzare nell’incrocio Acpmei/Pvpgip2 e nelle linee parentali, sono stati eseguiti esperimenti di infezione con il biotrofo fungino Claviceps purpurea su una linea transgenica stabile di frumento tenero esprimente il gene Pvpgip2. Una leggera, seppur significativa riduzione dei sintomi è stata riscontrata nella linea transgenica rispetto alle piante di controllo. Description: Dottorato di ricerca in Biotecnologie vegetali 2010-02-25T23:00:00Z http://hdl.handle.net/2067/1356 Title: Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion Authors: Lionetti, Vincenzo; Francocci, Fedra; Ferrari, Simone; Volpi, Chiara; Bellincampi, Daniela; Galletti, Roberta; D'Ovidio, Renato; De Lorenzo, Giulia; Cervone, Felice Abstract: Plant cell walls represent an abundant, renewable source of biofuels and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification) is their recalcitrance to enzymatic hydrolysis. We demonstrated that the structure of pectin affects the exposure of cellulose to enzymes and consequently the process of saccharification.; Le pareti cellulari vegetali rappresentano una abbondante e rinnovabile sorgente di biocarburanti ed altri prodotti utili. Una delle maggiori difficoltà nell’utilizzo delle pareti cellulari a livello industriale è la loro conversione in zuccheri semplici (saccarificazione) in quanto sono estremamente resistenti all’idrolisi enzimatica. In questo lavoro dimostriamo che la struttura della pectina influenza l’esposizione della cellulosa all’azione enzimatica e conseguentemente il processo di saccarificazione. 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