Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits

Apple (Malus 7domestica Borkh) fruits are stored for long periods of time at low temperatures (1 \ub0C) leading to the occurrence of physiological disorders. 'Superficial scald' of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H2O2 homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H2O2 homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress

Olive ripening and oil flavour in Veneto: molecular and technological aspects

Extra-virgin olive oil is a very appreciated vegetable fat used mainly for food purposes due to its pleasant flavour and taste and to the demonstrated and well-known health promoting effects, which include anti-inflammatory and antioxidant functions and protection against cardiovascular diseases. Olive oil quality depends on many factors, among which environmental (climate and soil), genetic (variety), agronomic and technological factors play important roles. Among the agronomic factors, the maintenance and use of healthy fruits, and, most of all, the choice of the best ripening stage of olive fruits represent the factors having a prominent role in determining olive oil quality. The ideal cultivation area for olive tree is the Mediterranean region, which usually has mild winters and long dry summers. Despite these climatic limitations, olive trees grown in marginal areas such as Northern Italy are known to yield extra-virgin olive oils with a pleasant flavour and an overall high quality profile. However, the often unfavourable climatic conditions in this area make necessary optimising the choice of the best harvesting period and setting up technological factors in order to improve oil quality. Many different approaches have been suggested so far for the definition of a simple and reliable way to assess the right harvesting time and to maximise oil quality. More in detail, physiological and biochemical changes that occur throughout olive ripening have been taken into account, such as loss of firmness, skin colour changes, pigment content or ratio, sugar content, oil accumulation, decrease in phenolics and some others. Results were however often conflicting due to the great variability among cultivars, growing areas, and technological aspects. It is therefore necessary to study all these factors in every specific growing area. In this dissertation different approaches have been chosen to characterise a cultivar grown locally in Veneto, i.e. cv. Grignano, yielding a unique oil with lemon peel aromatic notes and to evaluate factors involved in the definition of oil quality also in comparison with Frantoio, a common Italian variety. First of all, a study on anoxic oil extraction has been performed in order to understand the influence of the absence of oxygen, which can oxidise olive oil components but is also a required reagent for the biosynthesis of flavour compounds, on the global volatile profile of oils extracted from olives harvested at four consecutive ripening stages across véraison. The analysis of the volatile components of oils has been performed by means of PTR-MS (Proton Transfer Reaction-Mass Spectrometry), a spectrometric technique that allows an evaluation of the global volatile fingerprint of samples without any preparatory step. This “real-time” evaluation of the spectral signature of extra-virgin olive oils from cv. Grignano coupled with unsupervised multivariate analyses, i.e. principal component analysis and hierarchical clustering, allowed to explore the complex spectra by means of the construction of a heat map. Results showed that the ripening stage has a predominant effect on the definition of the global volatile profile compared to anoxic extraction, and that the progression of ripening is characterised by the biosynthesis of specific groups of volatiles that seem to be “expressed” only transiently. Moreover, the progression of fruit ripening causes a saturation effect in the volatile profile. Consequently, given that ripening is predominant over technological factors, an in depth study regarding olive ripening in Veneto region has been carried out focusing on fruit firmness, oil accumulation, chlorophyll a content, colorimetric analyses (calculating colour indices), and gene expression profiles. Results demonstrated that in Grignano and Frantoio cvs. ripening-related events were different, since Grignano showed an earlier and faster ripening course, both in loss of firmness and in oil accumulation. These findings suggest that monitoring cv. Grignano ripening stages is more difficult, since changes in ripening-related physiological parameters occur very quickly. Moreover, differences among two consecutive harvest seasons were observed resulting in a complex reading of the overall ripening profile. Statistical analyses have been performed, and principal component analysis established that cultivar Frantoio shows a greater variability in oil accumulation data, confirming that cv. Grignano ripening course is somehow more compact. One of the colour indices calculated in this study, namely CI, was suitable to distinguish drupe samples picked at different ripening stages. Then, to investigate on the peculiar flavour features of Grignano oil, a study on the terpenic metabolism in olive has been carried out isolating and characterising for the first time three enzymes responsible for the biosynthesis of terpenes (terpene synthases) in this species. To this end, a thorough search for putative terpene synthases in a published olive EST (expressed sequence tag) database was performed. Fragments have been then amplified and the full length genes have been used to deduce the amino acidic sequence to be heterologously expressed in E. coli. Expressed proteins have been checked for enzymatic activity and one of them turned out to be an active geraniol synthase, whilst the other two terpene synthases seemed inactive, a phenomenon not unusual in terpene metabolism. qPCR demonstrated that the three isolated terpene synthase genes have a ripening-dependent trend in transcript accumulation, suggesting a role in flavour development. Finally, gene expression analyses performed in the previous works emphasised the absence of validated reference genes suitable for gene expression studies in olive. Therefore, an extensive search for candidate reference genes to be used for olive gene expression studies has been performed using the above-mentioned EST database and retrieving studies regarding validated reference genes in other plant species. 13 candidate genes were identified for Olea europaea, which were tested both in olive drupes and leaves for their transcriptional stability. Stability analyses performed with genormPLUS and NormFinder software packages showed that 12 over the 13 genes in this work represented actually good reference genes and that, among these genes, the optimum number that must be used for good data normalisation was two. The best two reference genes identified in the study, specifically suitable for normalisation of gene expression data in fruit samples at different developmental stages, were GAPDH2 and PP2A1.L’olio extravergine di oliva è un olio vegetale molto apprezzato utilizzato principalmente per scopi alimentari a causa del profilo aromatico piacevole e a effetti salutistici noti e dimostrati, che includono proprietà antinfiammatorie e antiossidanti e una protezione nei confronti di alcune malattie cardiovascolari. La qualità dell’olio di oliva dipende da molti fattori, e tra questi fattori ambientali (clima e suolo), genetici (cultivar), agronomici e tecnologici hanno un ruolo importante. Tra i fattori agronomici, la gestione e l’uso di frutti sani e, soprattutto, la scelta dello stadio di maturazione adatto dei frutti rappresentano le variabili che hanno un ruolo fondamentale nella determinazione della qualità globale dell’olio di oliva. Le zone adatte per la coltivazione dell’olivo sono le regioni mediterranee, che sono caratterizzate solitamente da inverni miti ed estati lunghe e secche. Nonostante queste limitazioni climatiche, olivi cresciuti in zone marginali come l’Italia settentrionale conferiscono oli extravergini con profili aromatici gradevoli e di alta qualità. Le condizioni climatiche spesso avverse in queste aree rendono però necessarie un’ottimizzazione della scelta del miglior periodo di raccolta e l’implementazione di processi tecnologici allo scopo di migliorare la qualità dell’olio. Molti approcci diversi sono stati proposti finora per la definizione di un modo semplice e affidabile per valutare il periodo di raccolta corretto e massimizzare la qualità dell’olio estratto. Più in dettaglio, i cambiamenti fisiologici e biochimici che si verificano durante la maturazione dell’oliva sono stati presi in considerazione, come ad esempio la perdita di consistenza, cambiamenti del colore superficiale delle drupe, contenuto di pigmenti o loro rapporti, contenuto in zuccheri, inolizione, diminuzione di componenti fenoliche e altri. I risultati si sono dimostrati però molto spesso discordanti, a causa della grande variabilità presente tra diverse cultivar, aree di coltivazione e aspetti tecnologici. Si rende perciò necessario uno studio di questi fattori in ogni singola area di coltivazione. In questa tesi diversi approcci sono stati adottati per la caratterizzazione di una varietà coltivata localmente in Veneto, la cultivar Grignano, da cui si estrae un olio dal profilo sensoriale unico con note di buccia di limone, e per la valutazione di fattori coinvolti nella determinazione della qualità dell’olio anche attraverso un confronto con la cultivar Frantoio, una varietà diffusa comunemente in tutta Italia. Inizialmente è stato condotto uno studio che prevedeva l’estrazione di olio in atmosfera priva di ossigeno, che può essere responsabile dell’ossidazione di componenti dell’olio ma è anche un reagente richiesto nella biosintesi di composti aromatici, per valutare l’effetto dell’estrazione in atmosfera controllata sul profilo volatile globale di oli ottenuti da olive raccolte in quattro stadi consecutivi di maturazione a cavallo dell’invaiatura. L’analisi delle componenti volatili è stata condotta mediante PTR-MS (spettrometria di massa con reazione di trasferimento di protoni), una tecnica di spettrometria che permette una valutazione del fingerprint globale dei volatili senza fasi preliminari o trattamento dei campioni. Questa valutazione “real-time” dei profili spettrali di oli extravergini da cultivar Grignano unita a analisi multivariata dei dati, mediante analisi delle componenti principali e clustering gerarchico, ha permesso di proiettare i complessi spettri ottenuti tramite l’analisi spettrometrica in una heat map. I risultati hanno dimostrato che lo stadio di maturazione ha un effetto predominante sulla definizione del profilo volatile rispetto all’estrazione in atmosfera controllata, e che il progredire della maturazione è caratterizzato dalla biosintesi di specifici gruppi di molecole volatili che sembrano quindi “espresse” in modo transiente. Inoltre, è stato osservato un effetto saturazione nel profilo volatile con il progredire della maturazione. Conseguentemente, dato che lo stadio di maturazione ha un effetto prevalente rispetto ai fattori tecnologici studiati, uno studio approfondito della fisiologia della maturazione in Veneto è stato condotto ponendo particolare attenzione a perdita di consistenza, inolizione, contenuto in clorofilla a, dati colorimetrici (calcolo di indici di colore) e dati di espressione genica. I risultati hanno dimostrato che gli eventi collegati alla maturazione sono differenti nelle cultivar Grignano e Frantoio, dato che Grignano manifesta una dinamica di maturazione più precoce e rapida, sia considerando la perdita di consistenza che l’accumulo di olio nella drupa. Questi risultati suggeriscono come monitorare la maturazione in Grignano sia più difficile, dato che i cambiamenti nei parametri di maturazione si verificano molto velocemente. Inoltre, sono state osservate differenze importanti nei parametri elencati in due stagioni successive, da cui consegue una notevole difficoltà nella comprensione del profilo globale di maturazione. Sono state condotte analisi statistiche sui campioni descritti e l’analisi delle componenti principali ha stabilito che la cultivar Frantoio mostra maggiore variabilità nei dati di inolizione, confermando che il decorso di maturazione della cultivar Grignano è in qualche modo più compatto. Tra gli indici di colore calcolati nello studio, uno, cioè CI, sembra appropriato per distinguere campioni raccolti in diversi stadi di maturazione. Successivamente, per approfondire le caratteristiche aromatiche tipiche della cultivar Grignano, è stato condotto uno studio riguardante il metabolismo terpenico in olivo, con l’isolamento e la caratterizzazione di tre enzimi responsabili della biosintesi di terpeni (terpene sintasi) per la prima volta in questa specie. A questo scopo è stata condotta una ricerca approfondita di putative terpene sintasi in un database di EST (expressed sequence tag) di olivo recentemente pubblicato. I frammenti selezionati sono stati amplificati permettendo di ottenere i geni full length, le cui sequenze sono state utilizzate per dedurre le sequenze aminoacidiche che sono state espresse in E. coli. Le proteine espresse sono state sottoposte a saggi enzimatici per determinarne l’attività e una di queste si è dimostrata una geraniolo sintasi attiva, mentre le altre due proteine non hanno dato prodotti anche se la presenza di terpene sintasi inattive non è così infrequente. Analisi qPCR hanno dimostrato come le tre terpene sintasi isolate abbiano un andamento legato alla maturazione nell’espressione genica, ed è possibile perciò un loro ruolo nella determinazione del profilo aromatico. Infine, l’utilizzo della qPCR come analisi di espressione genica nei lavori precedenti ha evidenziato come geni reference validati e adatti per studi di espressione genica in olivo non siano disponibili in letteratura. Una ricerca di geni reference candidati per questo scopo è stata quindi condotta utilizzando il database di EST citato sopra e cercando in letteratura studi riguardanti la validazione di geni reference in altre specie vegetali. Sono stati quindi identificati 13 geni candidati in Olea europaea, la cui stabilità dei trascritti è stata testata sia in drupe che in foglie di olivo. Analisi di stabilità condotte mediante gli strumenti genormPLUS e NormFinder hanno dimostrato che 12 tra questi geni sono effettivamente geni reference funzionali e utilizzabili per studi di espressione genica in olivo e che, tra questi, il numero di geni ottimale da utilizzare per una buona normalizzazione dei dati corrisponde a due. I migliori geni reference identificati in questo studio, adatti per analisi di espressione genica in frutti a diversi stadi di sviluppo, sono stati GAPDH2 e PP2A1

Evaluation of RNA Extraction Methods and Identification of Putative Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Expression Studies on Olive (Olea europaeaL.) Fruits

Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated

Evaluation of RNA Extraction Methods and Identification of Putative Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Expression Studies on Olive (<i>Olea europaea</i> L.) Fruits

Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (<i>Olea europaea</i> L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated

Humic substances affect Arabidopsis physiology by altering the expression of genes involved in primary metabolism, growth and development

Humic substances (HS), as component of soil organic matter, have been widely studied. Their positive effects on plant physiology have been further demonstrated, but there are no clear evidences about the direct as well as indirect mechanisms regulating these processes. In this paper a transcriptomic approach based on the detection of cDNA-AFLP markers was used to identify candidate genes potentially involved in the regulation of the response to HS in Arabidopsis thaliana. Using 160 primer combinations, the cDNA-AFLP enabled to identify 133 genes putatively involved in plant-HS interaction. Sequence analysis and Gene Ontology classification indicated that a large number of genes involved in developmental and metabolic processes, as well as in transcription regulation or RNA metabolism were identified as HS-regulated. Real-Time PCR analyses confirmed transcription levels of 32 HS-regulated genes. This study demonstrates that HS exert their effects on plant physiology by means of complex transcriptional networks. From the overall transcriptomic results we may hypothesize that HS exert their function through a multifaceted mechanism of action, partially connected to their well demonstrated auxin activity, but involving also IAA-independent signalling pathways

Isolation and characterization of terpene synthases potentially involved in flavor development of ripening olive (Olea europaea) fruits

The flavor and taste of fruits are often determined by terpenes. We identified three cDNAs encoding putative terpene synthases from olive fruits of cv. Frantoio and Grignano. Heterologous expression in a bacterial system demonstrated that one of the terpene synthases, OeGES1, was an active monoterpene synthase that converted geranyl diphosphate to the monoterpene alcohol geraniol. The transcript accumulation pattern of this gene showed a peak during fruit ripening in both genotypes, indicating that the enzyme may be involved in the production of monoterpene flavor compounds in olive fruit. Although the putative terpene synthases OeTPS2 and OeTPS3 clustered with alpha-farnesene synthases and angiosperm monoterpene synthases, no detectable in vitro activity was found after expression in a bacterial system. Nevertheless, their transcripts sharply accumulated during fruit ripening starting from veraison

Influence of olive (cv Grignano) fruit ripening and oil extraction under different nitrogen regimes on volatile organic compound emissions studied by PTR-MS technique

Volatile organic compounds of extra virgin olive oils obtained from the local Italian cultivar Grignano were measured by proton transfer reaction\u2013mass spectrometry (PTR-MS). Oils were extracted by olives harvested at different ripening stages across veraison, performing each extraction step and the whole extraction process in nitrogen atmosphere to observe the changes in the volatile profiles of the oils. Principal component analysis carried out on the full spectral signature of the PTR-MS measurements showed that the stage of ripening has a stronger effect on the global definition of volatile profiles than the use of nitrogen during oil extraction. The fingerprint-like chemical information provided by the spectra were used to construct a heat map, which allowed the dynamical representation of the multivariate nature of mass evolution during the ripening process. This provided the first evidence that some groups of volatile organic compounds displayed a time course of regulation with coordinated increasing or decreasing trends in association with specific stages of fruit ripening

Influence of olive (cv Grignano) fruit ripening and oil extraction under different nitrogen regimes on volatile organic compound emissions studied by PTR-MS technique

Volatile organic compounds of extra virgin olive oils obtained from the local Italian cultivar Grignano were measured by proton transfer reaction–mass spectrometry (PTR-MS). Oils were extracted by olives harvested at different ripening stages across veraison, performing each extraction step and the whole extraction process in nitrogen atmosphere to observe the changes in the volatile profiles of the oils. Principal component analysis carried out on the full spectral signature of the PTR-MS measurements showed that the stage of ripening has a stronger effect on the global definition of volatile profiles than the use of nitrogen during oil extraction. The fingerprint-like chemical information provided by the spectra were used to construct a heat map, which allowed the dynamical representation of the multivariate nature of mass evolution during the ripening process. This provided the first evidence that some groups of volatile organic compounds displayed a time course of regulation with coordinated increasing or decreasing trends in association with specific stages of fruit ripening