Global energy demand has been under intense pressure over the last few years, thus many research efforts have been made in many countries in order to evaluate and pose sustainable strategies. Sustainable biomass production by bioenergy crop on non-arable lands can play a crucial role not only in reducing global Greenhouse gas (GHGs) emission responsible for climate change but also providing a significant contribution to satisfy the increasing demand of sustainable energy production without any further risks on food security. The second generation of biofuels, which comes from living organisms, called bioenergy crops, have been considered better biomass producers than food crops, in fact they reach higher ethanol yields per unit of cultivated area. Among them, Arundo donax L. is the most promising species for bioenergy production in the Mediterranean basin due to its high yield, low input requirements and its capability to grow on marginal land and in adverse environmental conditions. Although the capability of A. donax to withstand a wide range of abiotic stresses condition has been reported worldwide, the investigation at molecular level is just at the beginning. Considering the impact of soil salinization on agricultural areas situated in the Mediterranean basin as well as the lack of information about the molecular mechanism involved in A. donax response to salt stress, we de novo sequenced, assembled and analysed the leaf transcriptome of two A. donax clones (G2 and G34) subjected to two levels of long-term salt stress treatment (namely, S3 severe and S4 extreme). The picture that emerges from the identified genes related to salt stress response in G2 is consistent with a dose-dependent response to salt, it also suggests a deep re-programming of the transcriptomic machinery in the case of S4 extreme salt stress condition, whereby a dramatic switch from C3 to C4 Calvin cycle likely occurred. Although A. donax propagates itself vegetatively by rhizomes and stem cutting nodes, variation in gene expression between G2 and G34 ecotypes occurred not only in salt treated but also in untreated samples. Indeed, the severe salt treatment in G34 ecotype resulted in a lower number of DEGs when compared to the same condition in G2 ecotype, indicating a lower re-adjusting of the gene expression. Nevertheless, the comparative analysis between S4 (extreme salt stress) G2 with S3 (severe salt stress) G34 conditions outlines a similar response and suggests that G34 ecotype tries to deal with stress condition as soon as S3 salt dose is applied. Moreover, given the ongoing increase of contaminated soils as well as the remarkable resistance of A. donax to heavy metals, we carried out a global de novo transcriptomic analysis in leaves and roots of G10 A. donax ecotype subjected to cadmium stress condition. By analysing the differential gene expression data, clear organ-specific differences emerged leading to the identification of specifically up-regulated genes in the Cd-treated roots compared to Cd-treated leaves. It is worthwhile to note that the obtained transcriptomic data indicate that after Cd uptake by plant roots, it is likely that only a small portion reaches the upper arial plant parts, since a low number of DEGs were retrieved in leaf tissue under cadmium treatment indicating a major role of roots in Cd detoxification. Based on transcriptomic data, the long-term exposure to Cd induced the expression of signaling molecules devoted to induce a downstream signal cascade activated by the phytohormone ethylene. In addition, the results showed a strong regulation of oxidative-responsive genes followed by the induction of transcripts involved in cell wall remodelling and lignification in Cd-treated roots. The identification of candidate genes involved in salt- and cadmium stress response constitute an important database resource towards the characterization of the molecular basis for the high resistance of A. donax to unfavourable environmental conditions. Many of the unigenes identified have the potential to be used for improving several important traits and for developing A. donax varieties with enhanced productivity and tolerance to different environmental scenarios. In addition, the identified SSRs addressed many challenges to discover molecular markers suitable for marked-assisted selection (MAS) in the breeding programs, to elucidate the interspecific phenotypic variation within A. donax ecotypes. Globally, these results support the cultivation of A. donax ecotypes in contaminated soils in order to only to avoid the competition with food crops but mainly to fulfil the sustainable energy demand.
La domanda globale di fonti di energia sostenibili è fortemente aumentata nel corso degli ultimi anni. La produzione di biomassa derivante da colture bioenergetiche in suoli non utilizzabili per le coltivazioni può giocare un ruolo importante non solo nel ridurre le emissioni di gas a effetto serra (GHGs) responsabili del cambiamento climatico, ma anche nel soddisfare l’incremento della domanda di produzione di energia sostenibile, evitando ulteriori rischi sulla sicurezza alimentare. I biocombustibili di seconda generazione, i quali derivano dagli organismi viventi noti come colture bioenergetiche, sono stati considerati migliori produttori di biomassa rispetto alle colture alimentari, in quanto consentono di ottenere elevate rese di etanolo per unità di area coltivata. Tra le diverse colture bioenergetiche, Arundo donax L. è la specie vegetale più promettente nel bacino del Mediterraneo grazie al suo elevato rendimento in biomassa, al basso fabbisogno nutritivo e alla sua notevole capacità di crescere in suoli marginali, cosi come in condizioni ambientali sfavorevoli. Sebbene, la capacità di Arundo donax di tollerare un’ampia varietà di stress abiotici sia stata riportata in passato, lo studio a livello molecolare della risposta a condizioni ambientali avverse è in fase iniziale. Considerando l’impatto della salinizzazione dei suoli sulle aree agricole localizzate nel bacino del Mediterraneo, congiuntamente alla mancanza di informazioni sul meccanismo molecolare coinvolto nella risposta di A. donax allo stress salino, un’obiettivo di questa tesi è stato il sequenziamento del trascrittoma fogliare e l’assemblaggio de novo del trascrittoma di due ecotipi di A. donax (G2 e G34) sottoposti a due livelli di stress salino prolungato (S3, severo e S4, estremo). Il risultato che emerge dall’identificazione dei geni coinvolti nella risposta allo stress salino nel clone G2 è consistente con una risposta dipendente dalla concentrazione salina, suggerendo che il trattamento salino estremo (S4) comporta una profonda riprogrammazione del trascrittoma; in particolare si è osservata una modifica dell’espressione genica che testimonia la conversione dal ciclo fotosintetico C3 al C4. Sebbene A. donax si moltiplichi per via vegetativa da rizomi o segmenti di culmi, è stata osservata una differente espressione genica tra i cloni G2 e G34 non solo in campioni trattati con lo stress salino, ma anche nei campioni controllo. Difatti, il trattamento salino severo (S3) applicato sull’ecotipo G34 ha indotto un numero minore di geni differenzialmente espressi rispetto a quelli ottenuti nell’ecotipo G2 a parità di concentrazione salina. Inoltre, l’analisi comparativa condotta tra l’ecotipo G2 sottoposto al trattamento S4 (stress salino estremo) e l’ecotipo G34 sottoposto al trattamento S3 (stress salino severo) evidenzia una risposta simile, suggerendo che l’ecotipo G34 risponde a concentrazioni più basse di stress salino. Inoltre, visto l’incremento crescente di suoli contaminati da metalli pesanti, è stata effettuata un’analisi trascrittomica globale con assemblaggio de novo sia in foglia che radice dell’ecotipo G10 sottoposto a trattamento con cadmio. L’analisi dei dati di espressione genica ha condotto all’identificazione di geni specificatamente sovra-regolati nelle radici sottoposte al trattamento con il cadmio rispetto alle foglie. I dati di trascrittomica indicano che le radici svolgono un ruolo primario nell’interazione con il cadmio anche in funzione del fatto che solo un ridotto numero di geni differenzialmente espressi è stato riscontrato nel tessuto fogliare sottoposto al trattamento del cadmio. Inoltre, l’esposizione prolungata al metallo pesamte ha indotto nelle radici l’espressione di molecole segnale indirizzate ad attivare la via di segnalazione a cascata dell’etilene, di geni coinvolti nello stress ossidativo e nel rimodellamento e lignificazione della parte cellulare. L’identificazione di geni candidati coinvolti nella risposta allo stress salino e da cadmio costituisce un importante risorsa per la caratterizzazione delle basi molecolari della risposta di A. donax a condizioni ambientali sfavorevoli. Molti dei geni identificati potranno essere utilizzati per lo sviluppo di varietà di A. donax caratterizzate da una maggiore produttività e tolleranza agli stress. Infine, i marcatori microsatelliti SSR identificati in questo lavoro di tesi sono ideali per la selezione assistita da marcatori (MAS) in programmi di breeding e possono fare luce sulla variabilità fenotipica intraspecifica all’interno degli ecotipi della specie A. donax.
Dinamica trascrizionale degli ecotipi di Arundo donax L. ai trattamenti a lungo termine con sale e cadmio mediante l'analisi basata sul RNA-Seq / Santoro, DANILO FABRIZIO. - (2022 Apr 08).
Dinamica trascrizionale degli ecotipi di Arundo donax L. ai trattamenti a lungo termine con sale e cadmio mediante l'analisi basata sul RNA-Seq
SANTORO, DANILO FABRIZIO
2022-04-08
Abstract
Global energy demand has been under intense pressure over the last few years, thus many research efforts have been made in many countries in order to evaluate and pose sustainable strategies. Sustainable biomass production by bioenergy crop on non-arable lands can play a crucial role not only in reducing global Greenhouse gas (GHGs) emission responsible for climate change but also providing a significant contribution to satisfy the increasing demand of sustainable energy production without any further risks on food security. The second generation of biofuels, which comes from living organisms, called bioenergy crops, have been considered better biomass producers than food crops, in fact they reach higher ethanol yields per unit of cultivated area. Among them, Arundo donax L. is the most promising species for bioenergy production in the Mediterranean basin due to its high yield, low input requirements and its capability to grow on marginal land and in adverse environmental conditions. Although the capability of A. donax to withstand a wide range of abiotic stresses condition has been reported worldwide, the investigation at molecular level is just at the beginning. Considering the impact of soil salinization on agricultural areas situated in the Mediterranean basin as well as the lack of information about the molecular mechanism involved in A. donax response to salt stress, we de novo sequenced, assembled and analysed the leaf transcriptome of two A. donax clones (G2 and G34) subjected to two levels of long-term salt stress treatment (namely, S3 severe and S4 extreme). The picture that emerges from the identified genes related to salt stress response in G2 is consistent with a dose-dependent response to salt, it also suggests a deep re-programming of the transcriptomic machinery in the case of S4 extreme salt stress condition, whereby a dramatic switch from C3 to C4 Calvin cycle likely occurred. Although A. donax propagates itself vegetatively by rhizomes and stem cutting nodes, variation in gene expression between G2 and G34 ecotypes occurred not only in salt treated but also in untreated samples. Indeed, the severe salt treatment in G34 ecotype resulted in a lower number of DEGs when compared to the same condition in G2 ecotype, indicating a lower re-adjusting of the gene expression. Nevertheless, the comparative analysis between S4 (extreme salt stress) G2 with S3 (severe salt stress) G34 conditions outlines a similar response and suggests that G34 ecotype tries to deal with stress condition as soon as S3 salt dose is applied. Moreover, given the ongoing increase of contaminated soils as well as the remarkable resistance of A. donax to heavy metals, we carried out a global de novo transcriptomic analysis in leaves and roots of G10 A. donax ecotype subjected to cadmium stress condition. By analysing the differential gene expression data, clear organ-specific differences emerged leading to the identification of specifically up-regulated genes in the Cd-treated roots compared to Cd-treated leaves. It is worthwhile to note that the obtained transcriptomic data indicate that after Cd uptake by plant roots, it is likely that only a small portion reaches the upper arial plant parts, since a low number of DEGs were retrieved in leaf tissue under cadmium treatment indicating a major role of roots in Cd detoxification. Based on transcriptomic data, the long-term exposure to Cd induced the expression of signaling molecules devoted to induce a downstream signal cascade activated by the phytohormone ethylene. In addition, the results showed a strong regulation of oxidative-responsive genes followed by the induction of transcripts involved in cell wall remodelling and lignification in Cd-treated roots. The identification of candidate genes involved in salt- and cadmium stress response constitute an important database resource towards the characterization of the molecular basis for the high resistance of A. donax to unfavourable environmental conditions. Many of the unigenes identified have the potential to be used for improving several important traits and for developing A. donax varieties with enhanced productivity and tolerance to different environmental scenarios. In addition, the identified SSRs addressed many challenges to discover molecular markers suitable for marked-assisted selection (MAS) in the breeding programs, to elucidate the interspecific phenotypic variation within A. donax ecotypes. Globally, these results support the cultivation of A. donax ecotypes in contaminated soils in order to only to avoid the competition with food crops but mainly to fulfil the sustainable energy demand.File | Dimensione | Formato | |
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Tesi di dottorato - SANTORO DANILO FABRIZIO 20220221115547.pdf
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