Transcriptomic insights on the preventive action of apple (cv Granny Smith) wkin wounding on superficial scald development

Superficial scald is a post-harvest chilling storage injury leading to browning of the surface of the susceptible cv Granny Smith apples. Wounding of skins has been reported to play a preventive role on scald development however its underlying molecular factors are unknown. We have artificially wounded the epidermal and sub-epidermal layers of apple skins consistently obtaining the prevention of superficial scald in the surroundings of the wounds during two independent vintages. Time course RNA-Seq analyses of the transcriptional changes in wounded versus unwounded skins revealed that two transcriptional waves occurred. An early wave included genes up-regulated by wounding already after 6 h, highlighting a specific transcriptional rearrangement of genes connected to the biosynthesis and signalling of JA, ethylene and ABA. A later transcriptional wave, occurring after three months of cold storage, included genes up-regulated exclusively in unwounded skins and was prevented from its occurrence in wounded skins. A significant portion of these genes was related to decay of tissues and to the senescence hormones ABA, JA and ethylene. Such changes suggest a wound-inducible reversed hormonal balance during post-harvest storage which may explain the local inhibition of scald in wounded tissues, an aspect that will need further studies for its mechanistic explanatio

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

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

Apple Superficial Scald: Identification of physiological and molecular factors

Granny Smith and Red Delicious are sensitive cultivars to the insurgence of physiological disorders during low temperature storage. The occurrence of necrosis of epidermal cells is described as superficial scald and causes major economic losses to apple growers worldwide. It can arise after prolonged periods of storage at low temperature during shelf life at room temperature. The most common strategies adopted to inhibit scald development in use combine storage in control atmosphere (CA) with treatments with the antioxidant diphenylamine (DPA), or with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. The application of an initial low oxygen stress (ILOS), followed by CA storage is another tool for scald prevention since the use of DPA has been banned in Europe for health concerns in 2011. The high costs of 1-MCP and the request from the organic market sector of a suitable alternative requires more research for ILOS optimisation. Aim of this work is to provide a deeper characterization of the molecular factors putatively associated with superficial scald development. For this purpose, molecular responses to treatment with the ethylene inhibitor 1-MCP, as well as to the application of low and ultra low oxygen storage and wounding were studied to identify factors that may explain the mechanism of action of these preventive treatments in scald prevention. Wounding was applied as a methodological tool with the aim to identify the putative connections between scald incidence and molecular wounding responses as it had been previously shown that wounding inhibits locally apple superficial scald formation. Additionally, the metabolic and molecular responses to low and extreme low oxygen have been characterised and compared with those induced by 1-MCP to highlight ethylene-dependent and ethylene-independent and hypoxia-specific responses. Transcriptional data obtained in the wounding experiment, suggest the existence of at two waves of transcriptional regulation taking place in response to wounding: an early one, most likely linked to the effects of mechanical damage and a later one, taking place after three months of storage, possibly as a consequence of wounding effects. Through the evaluation of the combined effects of wounding and time of storage on transcriptional dynamics it was hypnotized that the priming effect of wounding against scald development may rely on a signalling effect specifically evoked by wounding on ethylene, JA and ABA biosynthesis and signalling pathways. In the time-course experiments exposing apple fruits to different oxygen conditions (normoxia, 21% O2, Nox; hypoxia, 0.8% O2; and extreme hypoxia, 0.4% O2) it was possible to show that the hypoxic stress induced the expression of specific components of the ROP-GAP rheostat in apples skins, in parallel to the up-regulation of NADPH oxidase activity, group VII ERF members and hypoxia marker gene ADH. These events coincided with a general down-regulation of ethylene biosynthetic genes suggesting the existence of a reciprocal regulatory loop involving a negative effect exerted by hypoxia on ethylene responses and signalling proportional to the degree of the hypoxic stress, and, in turn, a repressive effect exerted by ethylene on hypoxic responses. Expression analysis in response to 1-MCP treatment showed that a large part of the hypoxia-dependent DEGs resulted to be significantly and co-ordinately up-regulated in hypoxic conditions and to be either slightly repressed or not regulated at all by 1-MCP treatment. It can be therefore hypothesised that hypoxia induces effects mediated ethylene biosynthesis repression inducing also a large number of hypoxia-specific and ethylene-independent processes. Ethylene and hypoxia were shown to share some regulatory processes acting in concert in the regulation of fermentative pathways, however hypoxia can directly interfere with ethylene’s action changing its signalling upstream of ethylene’s action.Granny Smith and Red Delicious are sensitive cultivars to the insurgence of physiological disorders during low temperature storage. The occurrence of necrosis of epidermal cells is described as superficial scald and causes major economic losses to apple growers worldwide. It can arise after prolonged periods of storage at low temperature during shelf life at room temperature. The most common strategies adopted to inhibit scald development in use combine storage in control atmosphere (CA) with treatments with the antioxidant diphenylamine (DPA), or with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. The application of an initial low oxygen stress (ILOS), followed by CA storage is another tool for scald prevention since the use of DPA has been banned in Europe for health concerns in 2011. The high costs of 1-MCP and the request from the organic market sector of a suitable alternative requires more research for ILOS optimisation. Aim of this work is to provide a deeper characterization of the molecular factors putatively associated with superficial scald development. For this purpose, molecular responses to treatment with the ethylene inhibitor 1-MCP, as well as to the application of low and ultra low oxygen storage and wounding were studied to identify factors that may explain the mechanism of action of these preventive treatments in scald prevention. Wounding was applied as a methodological tool with the aim to identify the putative connections between scald incidence and molecular wounding responses as it had been previously shown that wounding inhibits locally apple superficial scald formation. Additionally, the metabolic and molecular responses to low and extreme low oxygen have been characterised and compared with those induced by 1-MCP to highlight ethylene-dependent and ethylene-independent and hypoxia-specific responses. Transcriptional data obtained in the wounding experiment, suggest the existence of at two waves of transcriptional regulation taking place in response to wounding: an early one, most likely linked to the effects of mechanical damage and a later one, taking place after three months of storage, possibly as a consequence of wounding effects. Through the evaluation of the combined effects of wounding and time of storage on transcriptional dynamics it was hypnotized that the priming effect of wounding against scald development may rely on a signalling effect specifically evoked by wounding on ethylene, JA and ABA biosynthesis and signalling pathways. In the time-course experiments exposing apple fruits to different oxygen conditions (normoxia, 21% O2, Nox; hypoxia, 0.8% O2; and extreme hypoxia, 0.4% O2) it was possible to show that the hypoxic stress induced the expression of specific components of the ROP-GAP rheostat in apples skins, in parallel to the up-regulation of NADPH oxidase activity, group VII ERF members and hypoxia marker gene ADH. These events coincided with a general down-regulation of ethylene biosynthetic genes suggesting the existence of a reciprocal regulatory loop involving a negative effect exerted by hypoxia on ethylene responses and signalling proportional to the degree of the hypoxic stress, and, in turn, a repressive effect exerted by ethylene on hypoxic responses. Expression analysis in response to 1-MCP treatment showed that a large part of the hypoxia-dependent DEGs resulted to be significantly and co-ordinately up-regulated in hypoxic conditions and to be either slightly repressed or not regulated at all by 1-MCP treatment. It can be therefore hypothesised that hypoxia induces effects mediated ethylene biosynthesis repression inducing also a large number of hypoxia-specific and ethylene-independent processes. Ethylene and hypoxia were shown to share some regulatory processes acting in concert in the regulation of fermentative pathways, however hypoxia can directly interfere with ethylene’s action changing its signalling upstream of ethylene’s action

Effetti di 1-MCP e ILOS sul metabolismo redox e sui profili trascrizionali delle mele in conservazione

Il ricorso all\u2019uso di inibitori dell\u2019azione dell\u2019etilene (1-MCP) e al bassissimo ossigeno (ILOS; Initial Low Oxygen Stress) consente di prolungare in modo significativo la conservazione delle mele, rallentandone il metabolismo primario, la sintesi e l\u2019azione dell\u2019etilene e i fenomeni di senescenza. Nonostante i progressi sulle conoscenze dei meccanismi d\u2019azione dell\u2019etilene poco si sa sulle interazioni (crosstalk) tra vie del segnale dell\u2019etilene e le risposte al bassissimo ossigeno (ILOS). In questo lavoro si \ue8 intrapresa una prima caratterizzazione di questi fenomeni mediante analisi molecolari (RNA-Seq e Real-Time RT-qPCR) ed analisi metaboliche delle principali molecole coinvolte nel mantenimento dell\u2019equilibrio redox cellulare (Glutatione, GSH e suoi metaboliti; Acido ascorbico, AsA; Cisteamina; Perossido di idrogeno, H2O2) su bucce di mele a diversi tempi e modalit\ue0 di conservazione. A questo scopo, sono state campionate bucce di mele dopo 0, 1 e 6 mesi di conservazione in atmosfera controllata (1 kPa O2; 1 kPa CO2) in assenza di trattamenti, o dopo trattamento con 1-MCP, oppure dopo applicazione di uno stress da bassissimo ossigeno (0,4 kPa O2). Sono state condotte analisi RNA-Seq che hanno permesso di identificare gruppi di geni del metabolismo redox che sono risultati differenzialmente espressi (DEGs) in risposta all\u2019inibizione dell\u2019etilene (1-MCP) e al bassissimo ossigeno (0,4 kPa). I Livelli di AsA, GSH e Cistein-glicina (prodotto del metabolismo di GSH) sono risultati inferiori nelle mele trattate con 1-MCP e in ILOS, mantenendosi inferiori per tutto il periodo di conservazione rispetto a quelli della altre tesi. Sorprendentemente, il contenuto di H2O2 \ue8 apparso superiore nelle bucce di mele trattate con 1-MCP. Questi dati potrebbero indicare un pi\uf9 rapido turn-over ,nelle mele trattate con 1-MCP o in ILOS, delle specie attive dell\u2019ossigeno, a scapito dei principali antiossidanti GSH e AsA per il mantenimento dell\u2019omeostasi redox delle cellule

Transcriptomic Insights on the Preventive Action of Apple (cv Granny Smith) Skin Wounding on Superficial Scald Development

none9noSuperficial scald is a post-harvest chilling storage injury leading to browning of the surface of the susceptible cv Granny Smith apples. Wounding of skins has been reported to play a preventive role on scald development however its underlying molecular factors are unknown. We have artificially wounded the epidermal and sub-epidermal layers of apple skins consistently obtaining the prevention of superficial scald in the surroundings of the wounds during two independent vintages. Time course RNA-Seq analyses of the transcriptional changes in wounded versus unwounded skins revealed that two transcriptional waves occurred. An early wave included genes up-regulated by wounding already after 6 h, highlighting a specific transcriptional rearrangement of genes connected to the biosynthesis and signalling of JA, ethylene and ABA. A later transcriptional wave, occurring after three months of cold storage, included genes up-regulated exclusively in unwounded skins and was prevented from its occurrence in wounded skins. A significant portion of these genes was related to decay of tissues and to the senescence hormones ABA, JA and ethylene. Such changes suggest a wound-inducible reversed hormonal balance during post-harvest storage which may explain the local inhibition of scald in wounded tissues, an aspect that will need further studies for its mechanistic explanation.openNadia Cainelli, Cristian Forestan, Dario Angeli, Tomas Roman Villegas, Fabrizio Costa, Alessandro Botton, Angela Rasori, Claudio Bonghi, Benedetto RupertiCainelli, Nadia; Forestan, Cristian; Angeli, Dario; Roman Villegas, Tomas; Costa, Fabrizio; Botton, Alessandro; Rasori, Angela; Bonghi, Claudio; Ruperti, Benedett