A systems biology approach to shed light on apple fruit development

The research carried out and discussed in the present dissertation is positioned within the “TranscrApple” project (www.transcrapple.com), funded by the Provincia Autonoma di Trento (PAT) within the call “Grandi Progetti 2012”. The general objectives of this project, a significant part of which overlap with the present thesis, deal with the characterization, as wide as possible with the currently available technologies, of the transcriptional events, including those related to small RNAs (not discussed in the present dissertation), the metabolic changes, on a subset of primary and secondary metabolites, and hormones’ cross-talk, through a hormone profiling approach, occurring during apple fruit development. The present thesis is organized in different chapters, mirroring the experimental and temporal rationale effectively pursued to develop the research herein described. The main objective of the present work deals not only with providing an overview of transcripts, metabolites and hormones and their variations during fruit development, but also with the setting up of technical and experimental solutions aimed at using the achieved information within an integrative platform, according to a “systems biology view”. In model species, all this kind of studies are extremely easier, thanks to the availability of ready-to-use bioinformatics tools that are not flexible enough to be used in other species. However, especially in tree crops, this approach is still far from being defined and standardized. Chapter 1 introduces the theme “apple fruit development”, discussing the adoption of apple as a model system that, in the last decade, acquired great importance in terms of research among the fruit species thanks to the availability of its sequenced genome. After a brief introduction on the fundamental information available about the apple fruit growth, taking into account the technological and scientific points of view, few difficulties and gaps that hamper the achievement of a complete overview of the regulatory events coordinating the development and growth of the apple, are discussed in relation to the main quantitative and qualitative parameters characterizing the apple fruit production. Chapter 2 enters into the apple fruit research area; the preliminary phase and the multiple validations (concerning different cultivars and seasons) of transcriptional markers during the main apple developmental stages were shown to be fundamental for choosing, on the base of the expression profiles of these genes, the most representative samples, among those collected also in different seasons. Several markers have been identified, validated and employed, among those available from literature, allowing the selection of samples of cv Golden Delicious (herein considered as model) to be used for the subsequent transcriptional and metabolomic characterization carried out in the present research. Chapter 3 deals with the hormonal profiling survey carried out along the apple fruit development for the first time in this species. The results have allowed not only the achievement of brand new data related to the major hormonal classes, to be employed for further researches, but also the clarification and/or confirmation of new hormonal interactions connected to the fruit development stage or the transition between stages. Moreover, the relevance of this study consists in having achieved, for the first time in apple, quantitative data of an important set of hormones concurrently on the same samples.\ud Chapter 4 concerns the survey on the metabolites and their variations during the apple fruit development. A complete overview of the changes of the different classes of metabolites (mainly sugars, organic acids, aminoacids and polyphenols) is given during the apple developmental cycle. The acquired data have been derived from the same samples already analyzed in the previous chapters, and will be integrated with data of diverse nature, such as the RNAseq. Chapter 5 of the present thesis comes into the “system biology” area, initially among several technical difficulties, then partially solved, and gives an example of an alternative interpretation of the hormonal data put within a correlative network along with the RNAseq results achieved on the same samples

Candidate gene transcriptional signature unravels the reprogramming occurring in the peel of apple fruit of ‘Granny smith’ during postharvest storage

After harvest fruit are stored to preserve the quality features established during the on-tree development and maturation, ensuring thus a continuous availability of fresh fruit on the market. For certain fruit species like apple, storage can last for almost a year, especially when coupled with several strategies, such as the reduction of the oxygen concentration or the application of ethylene competitor molecules, like 1-methylcyclopropene (1-MCP). To guarantee the maintenance of the highest quality, the monitoring of the physiological processes ongoing during the postharvest ripening is compelling. For this purpose, 16 genes belonging to key fruit ripening pathways, such as the ethylene and the sugar/fermentation metabolism, have been chosen as potential markers for the molecular characterization of the major changes occurring in the fruit during storage. Among these genes, ACS, PPO, PG1, RAP2-like, and ADH exhibited the most significant differential expression across the various samples. Based on the transcriptional pattern, this set of genes constitutes a valuable molecular tool for a precise and reliable RNA-based monitoring of the postharvest ripening progression and fermentation process in apples. PPO, together with S6PDH, were furthermore employed to inspect the onset of the superficial scald in apple and resulted to correlate with the evaluation of the incidence of this disorder and the accumulation of the sugar alcohol sorbitol, known to play important protecting roles to chilling injuries. The assessment of the transcriptional signature of these elements can facilitate the development of gene expression markers suitable for a more informed investigation of the physiological progression of the postharvest ripening in apples, ultimately leading to the promotion of high-quality stored apples, extending storage time while minimizing postharvest disorders and fruit los

Comparative analysis of antioxidant activity and capacity in apple varieties: Insights from stopped flow DPPH• kinetics, mass spectrometry and electrochemistry

The aim of this research was to investigate the antioxidant kinetics of different apple varieties, a red-flesh variety (‘R201’), a non-browning (‘Majda’), and a ‘Golden Delicious’. Kinetic approaches for antioxidant activity provide more detailed information than conventional assays by examining both the quantity and velocity of active molecules in their reaction with radicals. In this study, DPPH• stopped flow method was applied to study the antioxidant activity and capacity of the three apples varieties, allowing the determination of the reaction rates. The results show that the antioxidant activity of ‘R201’ was not significantly different from ‘Golden Delicious’. Instead, the activity of the non-browning variety was 20 times higher than the others according to the DPPH• kinetic approach, despite having the lowest phenolic content. To further understand their reactivity, the antioxidant molecules were identified using HPLC-HRMS/MS coupled with a coulometric array detector, which validated the DPPH• kinetics. This analysis also found differences in the phenolic profile of the three varieties, attributed to the enhanced antioxidant activity of ’Majda’ to its high content of ascorbic acid. Overall, the research highlights that the antioxidant behavior of apples primarily depends on the velocity of the antioxidants rather than the amount of phenolic compound

Flooding Responses on Grapevine: A Physiological, Transcriptional, and Metabolic Perspective

Studies on model plants have shown that temporary soil flooding exposes roots to a significant hypoxic stress resulting in metabolic re-programming, accumulation of toxic metabolites and hormonal imbalance. To date, physiological and transcriptional responses to flooding in grapevine are poorly characterized. To fill this gap, we aimed to gain insights into the transcriptional and metabolic changes induced by flooding on grapevine roots (K5BB rootstocks), on which cv Sauvignon blanc (Vitis vinifera L.) plants were grafted. A preliminary experiment under hydroponic conditions enabled the identification of transiently and steadily regulated hypoxia-responsive marker genes and drafting a model for response to oxygen deprivation in grapevine roots. Afterward, over two consecutive vegetative seasons, flooding was imposed to potted vines during the late dormancy period, to mimick the most frequent waterlogging events occurring in the field. Untargeted transcriptomic and metabolic profiling approaches were applied to investigate early responses of grapevine roots during exposure to hypoxia and subsequent recovery after stress removal. The initial hypoxic response was marked by a significant increase of the hypoxia-inducible metabolites ethanol, GABA, succinic acid and alanine which remained high also 1 week after recovery from flooding with the exception of ethanol that leveled off. Transcriptomic data supported the metabolic changes by indicating a substantial rearrangement of primary metabolic pathways through enhancement of the glycolytic and fermentative enzymes and of a subset of enzymes involved in the TCA cycle. GO and KEGG pathway analyses of differentially expressed genes showed a general down-regulation of brassinosteroid, auxin and gibberellin biosynthesis in waterlogged plants, suggesting a general inhibition of root growth and lateral expansion. During recovery, transcriptional activation of gibberellin biosynthetic genes and down-regulation of the metabolic ones may support a role for gibberellins in signaling grapevine rootstocks waterlogging metabolic and hormonal changes to the above ground plant. The significant internode elongation measured upon budbreak during recovery in plants that had experienced flooding supported this hypothesis. Overall integration of these data enabled us to draft a first comprehensive view of the molecular and metabolic pathways involved in grapevine\u2019s root responses highlighting a deep metabolic and transcriptomic reprogramming during and after exposure to waterlogging

Ethylene-auxin crosstalk regulates postharvest fruit ripening process in apple

The ripening of climacteric fruits, such as apple, is represented by a series of genetically programmed events orchestrated by the action of several hormones. In this study, we investigated the existence of a hormonal crosstalk between ethylene and auxin during the post-harvest ripening of three internationally known apple cultivars: 'Golden Delicious', 'Granny Smith' and 'Fuji'. The normal climacteric ripening was impaired by the exogenous application of 1-methylcyclopropene (1-MCP) that affected the production of ethylene and the physiological behaviour of specific ethylene-related quality traits, such as fruit texture and the production of volatile organic compounds. The application of 1-MCP induced, moreover, a de-novo accumulation of auxin. The RNA-Seq wide-transcriptome analysis evidenced as the competition at the level of the ethylene receptors induced a cultivar-dependent transcriptional re-programming. The DEGs annotation carried out through the KEGG database identified as most genes were assigned to the plant hormone signaling transduction category, and specifically related to auxin and ethylene. The interplay between these two hormones was further assessed through a candidate gene analysis that highlighted a specific activation of GH3 and ILL genes, encoding key steps in the process of the auxin homeostasis mechanism. Our results showed that a compromised ethylene metabolism at the onset of the climacteric ripening in apple can stimulate, in a cultivar-dependent fashion, an initial de-novo synthesis and de-conjugation of auxin as a tentative to restore a normal ripening progression

Comparative transcriptome and metabolite survey reveal key pathways involved in the control of the chilling injury disorder superficial scald in two apple cultivars, ‘Granny Smith’ and ‘Ladina’

The low temperature normally applied to prevent fruit decay during the storage of apples, can also triggers the onset of a chilling injury disorder known as superficial scald. In this work, the etiology of this disorder and the mechanism of action of two preventing strategies, such as the application of 1-MCP (1-methylcyclopropene) and storage at low oxygen concentration in ‘Granny Smith’ and ‘Ladina’ apple cultivars were investigated. The metabolite assessment highlighted a reorganization of specific metabolites, in particular flavan-3-ols and unsaturated fatty acids, while the genome-wide transcriptomic analysis grouped the DEGs into four functional clusters. The KEGG pathway and GO enrichment analysis, together with the gene-metabolite interactome, showed that the treatment with 1-MCP prevented the development of superficial scald by actively promoting the production of unsaturated fatty acids, especially in ‘Granny Smith’. ‘Ladina’, more susceptible to superficial scald and less responsive to the preventing strategies, was instead characterized by a higher accumulation of very long chain fatty acids. Storage at low oxygen concentration stimulated a higher accumulation of ethanol and acetaldehyde together with the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase and pyruvate decarboxylase in both cultivars. Low oxygen concentration, likewise 1-MCP, through a direct control on ethylene prevented the onset of superficial scald repressing the expression of PPO, a gene encoding for the polyphenol oxidase enzyme responsible of the oxidation of chlorogenic acid. Moreover, in ‘Granny Smith’ apple, the expression of three members of the VII subgroups of ERF genes, encoding for elements coordinating the acclimation process to hypoxia in plants was observed. The global RNA-Seq pattern also elucidated a specific transcriptomic signature between the two cultivars, disclosing the effect of the different genetic background in the control of this disorder

Flooding Responses on Grapevine: A Physiological, Transcriptional, and Metabolic Perspective

Studies on model plants have shown that temporary soil flooding exposes roots to a significant hypoxic stress resulting in metabolic re-programming, accumulation of toxic metabolites and hormonal imbalance. To date, physiological and transcriptional responses to flooding in grapevine are poorly characterized. To fill this gap, we aimed to gain insights into the transcriptional and metabolic changes induced by flooding on grapevine roots (K5BB rootstocks), on which cv Sauvignon blanc (Vitis vinifera L.) plants were grafted. A preliminary experiment under hydroponic conditions enabled the identification of transiently and steadily regulated hypoxia-responsive marker genes and drafting a model for response to oxygen deprivation in grapevine roots. Afterward, over two consecutive vegetative seasons, flooding was imposed to potted vines during the late dormancy period, to mimick the most frequent waterlogging events occurring in the field. Untargeted transcriptomic and metabolic profiling approaches were applied to investigate early responses of grapevine roots during exposure to hypoxia and subsequent recovery after stress removal. The initial hypoxic response was marked by a significant increase of the hypoxia-inducible metabolites ethanol, GABA, succinic acid and alanine which remained high also 1 week after recovery from flooding with the exception of ethanol that leveled off. Transcriptomic data supported the metabolic changes by indicating a substantial rearrangement of primary metabolic pathways through enhancement of the glycolytic and fermentative enzymes and of a subset of enzymes involved in the TCA cycle. GO and KEGG pathway analyses of differentially expressed genes showed a general down-regulation of brassinosteroid, auxin and gibberellin biosynthesis in waterlogged plants, suggesting a general inhibition of root growth and lateral expansion. During recovery, transcriptional activation of gibberellin biosynthetic genes and down-regulation of the metabolic ones may support a role for gibberellins in signaling grapevine rootstocks waterlogging metabolic and hormonal changes to the above ground plant. The significant internode elongation measured upon budbreak during recovery in plants that had experienced flooding supported this hypothesis. Overall integration of these data enabled us to draft a first comprehensive view of the molecular and metabolic pathways involved in grapevine’s root responses highlighting a deep metabolic and transcriptomic reprogramming during and after exposure to waterlogging

A systems biology approach to shed light on apple fruit development

The research carried out and discussed in the present dissertation is positioned within the “TranscrApple” project (www.transcrapple.com), funded by the Provincia Autonoma di Trento (PAT) within the call “Grandi Progetti 2012”. The general objectives of this project, a significant part of which overlap with the present thesis, deal with the characterization, as wide as possible with the currently available technologies, of the transcriptional events, including those related to small RNAs (not discussed in the present dissertation), the metabolic changes, on a subset of primary and secondary metabolites, and hormones’ cross-talk, through a hormone profiling approach, occurring during apple fruit development. The present thesis is organized in different chapters, mirroring the experimental and temporal rationale effectively pursued to develop the research herein described. The main objective of the present work deals not only with providing an overview of transcripts, metabolites and hormones and their variations during fruit development, but also with the setting up of technical and experimental solutions aimed at using the achieved information within an integrative platform, according to a “systems biology view”. In model species, all this kind of studies are extremely easier, thanks to the availability of ready-to-use bioinformatics tools that are not flexible enough to be used in other species. However, especially in tree crops, this approach is still far from being defined and standardized. Chapter 1 introduces the theme “apple fruit development”, discussing the adoption of apple as a model system that, in the last decade, acquired great importance in terms of research among the fruit species thanks to the availability of its sequenced genome. After a brief introduction on the fundamental information available about the apple fruit growth, taking into account the technological and scientific points of view, few difficulties and gaps that hamper the achievement of a complete overview of the regulatory events coordinating the development and growth of the apple, are discussed in relation to the main quantitative and qualitative parameters characterizing the apple fruit production. Chapter 2 enters into the apple fruit research area; the preliminary phase and the multiple validations (concerning different cultivars and seasons) of transcriptional markers during the main apple developmental stages were shown to be fundamental for choosing, on the base of the expression profiles of these genes, the most representative samples, among those collected also in different seasons. Several markers have been identified, validated and employed, among those available from literature, allowing the selection of samples of cv Golden Delicious (herein considered as model) to be used for the subsequent transcriptional and metabolomic characterization carried out in the present research. Chapter 3 deals with the hormonal profiling survey carried out along the apple fruit development for the first time in this species. The results have allowed not only the achievement of brand new data related to the major hormonal classes, to be employed for further researches, but also the clarification and/or confirmation of new hormonal interactions connected to the fruit development stage or the transition between stages. Moreover, the relevance of this study consists in having achieved, for the first time in apple, quantitative data of an important set of hormones concurrently on the same samples. Chapter 4 concerns the survey on the metabolites and their variations during the apple fruit development. A complete overview of the changes of the different classes of metabolites (mainly sugars, organic acids, aminoacids and polyphenols) is given during the apple developmental cycle. The acquired data have been derived from the same samples already analyzed in the previous chapters, and will be integrated with data of diverse nature, such as the RNAseq. Chapter 5 of the present thesis comes into the “system biology” area, initially among several technical difficulties, then partially solved, and gives an example of an alternative interpretation of the hormonal data put within a correlative network along with the RNAseq results achieved on the same samples.Le ricerche illustrate nella presente tesi di dottorato si collocano nell’ambito del progetto “TranscrApple” (www.transcrapple.com), finanziato dalla Provincia Autonoma di Trento (PAT) nell’ambito del bando Grandi Progetti 2012. Gli obiettivi generali del progetto, che in larga parte si accomunano a quelli della presente tesi, prevedono di caratterizzare, nella maniera più ampia possibile con le tecnologie attualmente disponibili, gli eventi transcrizionali, compresi quelli relativi agli small RNA (non affrontati nella presente tesi), metabolici, su un subset di metaboliti primari e secondari, e ormonali, tramite un approccio di hormone profiling, che si verificano durante lo sviluppo della mela. La presente tesi è organizzata in diversi capitoli, riflettendo la logica sperimentale e temporale effettivamente seguita per sviluppare le ricerche. Lo scopo principale del lavoro qui presentato è quello non solo di fornire una panoramica di informazioni su trascritti, ormoni e metaboliti e le loro variazioni durante lo sviluppo del frutto, ma anche di proporre delle soluzioni tecniche e sperimentali per poter collocare le informazioni acquisite in una piattaforma integrativa, secondo la logica della systems biology. Nelle specie modello, tutto ciò è fortemente facilitato dall’ampia disponibilità di tool bioinformatici pronti all’uso ma non sufficientemente flessibili per poter essere adattati ad altre specie. Tuttavia, soprattutto per quanto riguarda soprattutto le specie arboree da frutto, questo tipo di approccio è ancora lontano dell’essere definito e standardizzato. Il Capitolo 1 introduce l’argomento “sviluppo della mela” in relazione all’adozione del melo come specie arborea modello, in misura sempre più crescente soprattutto nell’ultimo decennio grazie anche alla disponibilità della sequenza del genoma. Dopo un excursus sulle principali e più recenti acquisizioni relative allo sviluppo del frutto del melo, vengono discusse alcune delle principali criticità e lacune, sia dal punto di vista tecnologico che scientifico, che impediscono una visione completa degli eventi regolativi che coordinano lo sviluppo e la crescita della mela, anche in relazioni ai principali parametri qualitativi e produttivi. Nel Capitolo 2 si inizia ad entrare nel merito delle ricerche, illustrando la fase preparativa di ricerca e validazione multipla (tra cultivar diverse e annate diverse) di marcatori trascrizionali delle fasi di sviluppo del frutto indirizzate alla corretta selezione di campioni rappresentativi in serie temporali raccolte in annate differenti. Sono stati identificati, validati ed utilizzati diversi marcatori fra quelli proposti in letteratura, consentendo la selezione dei campioni di cv Golden Delicious (qui usata come modello) da utilizzare nelle successive fasi di caratterizzazione trascrizionale e metabolomica condotte nelle tesi. Nel Capitolo 3 si affronta il primo importante studio dei profili ormonali durante lo sviluppo del frutto. I risultati acquisiti hanno consentito non solo di acquisire dati relativi alla maggior parte degli ormoni da poter utilizzare in ricerche future, ma anche di chiarire, confermare e/o ipotizzare delle interazioni ormonali in funzione dello stadio di sviluppo o della transizione tra stadi diversi. La peculiarità di questo studio consiste nell’aver ottenuto, per la prima volta nel melo, dati quantitativi di un set importante di ormoni a partire dagli stessi campioni. Il Capitolo 4 affronta invece la questione relativa ai metaboliti e alle loro variazioni nel corso dell’intero sviluppo del frutto. Viene in questo modo fornita una visione complessiva di come variano le diverse classi di metaboliti (principalmente zuccheri, acidi organici, amminoacidi e polifenoli) durante lo sviluppo. Anche in questo caso i dati sono stati acquisiti dagli stessi campioni utilizzati negli altri capitoli per le altre tipologie di analisi e potranno essere impiegati in ricerche successive, ad esempio, in una logica integrativa, insieme a dati trascrittomici di diversa natura, epigenetici, ecc. Nel Capitolo 5, finalmente, la tesi si addentra fra mille difficoltà tecniche, poi in parte superate, nella giungla della cosiddetta systems biology, fornendo un esempio di come i dati ormonali possono essere valorizzati attraverso la loro integrazione con i dati trascrittomici ottenuti tramite RNAseq a partire dagli stessi campioni

Profili trascrizionali dei geni coinvolti nella biosintesi e trasduzione del segnale etilenico durante l\u2019abscissione dei frutticini in melo

L\u2019abscissione dei frutticini di melo \ue8 preceduta da una stimolazione della biosintesi dell\u2019etilene e un aumento della sensibilit\ue0 della zona di abscissione (AZ) agli stimoli promossi dall\u2019ormone. Recentemente, \ue8 stato ipotizzato il ruolo dell\u2019etilene come \u201ccarrier\u201d del segnale abscissione che, generato nella cortex mediante l\u2019instaurazione di un cross-talk con ABA, zuccheri e ROS, viene trasferito al seme nelle fasi che precedono l\u2019attivazione dell\u2019AZ. Rimangono tuttavia ancora molti aspetti da chiarire. A tal proposito, \ue8 stata condotta un\u2019approfondita indagine a livello trascrizionale dei geni coinvolti nella biosintesi e trasduzione del segnale etilenico in cortex e seme di frutticini di melo trattati con benziladenina (BA). Sono stati identificati nella sequenza genomica 80 geni codificanti elementi della biosintesi (ACO e ACS), percezione (ETR, ERS, TPR e RTE) e trasduzione del segnale (CTR, EIN2 e EIN3/EIL), regolatori coinvolti nell\u2019ubiquitinizzazione e degradazione mediante proteasoma (EBF e ETP) e altri elementi noti (EIN5 e RAN1). Inoltre, 13 geni codificanti chinasi (MAPK e MAPKK), coinvolte nella presunta via trasduttiva del segnale coordinata da CTR, sono stati presi in esame in questo lavoro. Un elevato livello di ridondanza \ue8 stato riscontrato per la maggior parte dei geni, probabilmente dovuto all\u2019origine del genoma del melo e al recente evento di duplicazione a cui \ue8 andato incontro. In seguito, sono stati analizzati i profili trascrizionali dei geni identificati in cortex e seme di frutticini caratterizzati da diversi potenziali di abscissione. I risultati preliminari hanno evidenziato una notevole tessuto-specificit\ue0 di espressione dei geni analizzati e differenze trascrizionali che potrebbero coordinare l\u2019attivazione del segnale primario specifico dell\u2019abscissione. Questo studio \ue8 finanziato dal Progetto AGER, grant n\ub0 2010-2119

Thermographic imaging to identify abscising apple fruitlets after a thinning treatment

Metamitron is a widely used active ingredient in pome fruit thinning. Its inhibitory action on photosynthesis determines a nutritional stress that in turn stimulates the weakest fruitlets to abscise. However, a successful thinning treatment with metamitron, commercially known as Brevis®, relies upon several interacting factors, both endogenous and exogenous, and its effects are difficult to predict under certain specific conditions. For this reason, new predictors are desirable to assist the growers in applying the treatment at optimal time and dosage. Moreover, the physiology of metamitron action is in part still unknown, especially at the fruitlet level. In the present research, a thermographic assessment was carried out to verify the hypothesis that high abscisic acid levels together with photosynthesis block in the fruitlet tissues cause the temperature to raise to higher levels in treated fruitlets than in the untreated ones. A specific trial was carried out in ‘Golden Delicious’, pointing out that both leaves and fruitlets of thinned trees show higher temperatures, which can be used to identify abscising fruitlets. A working model is also proposed that summarizes the way of action of Brevis® on both the considered organ