title: Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit.
abstract: The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions.
authors.affiliation: Istituto di Scienze della Vita, Scuola Superiore Sant'Anna Pisa, Italy.
authors.forename: Dubravka
authors.initials: D
authors.lastname: Cukrov
authors.affiliation: Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente, University of Padova Padova, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Zermiani
authors.affiliation: Istituto di Scienze della Vita, Scuola Superiore Sant'Anna Pisa, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Brizzolara
authors.affiliation: Centro Ricerca e Innovazione, Fondazione Edmund Mach di San Michele all'Adige Trento, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cestaro
authors.affiliation: Istituto di Scienze della Vita, Scuola Superiore Sant'Anna Pisa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Licausi
authors.affiliation: CERM, University of Firenze Firenze, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Luchinat
authors.affiliation: CERM, University of Firenze Firenze, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Santucci
authors.affiliation: Fondazione FiorGen Onlus Firenze, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Tenori
authors.affiliation: Istituto di Scienze della Vita, Scuola Superiore Sant'Anna Pisa, Italy.
authors.forename: Hans
authors.initials: H
authors.lastname: Van Veen
authors.affiliation: Istituto di Scienze della Vita, Scuola Superiore Sant'Anna Pisa, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Zuccolo
authors.affiliation: Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente, University of Padova Padova, Italy.
authors.forename: Benedetto
authors.initials: B
authors.lastname: Ruperti
authors.affiliation: Istituto di Scienze della Vita, Scuola Superiore Sant'Anna Pisa, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Tonutti
keywords: Malus domestica; cortex; ethylene-responsive factors; low oxygen stress; metabolic profiling; postharvest; pyruvate metabolism; storage