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Agricultural and Environmental Chemistry
Short-term responses of apple fruit to partial reoxygenation during extreme hypoxic storage conditions Stefano Brizzolara, Dubravka Cukrov, Massimo Mercadini, Federico Martinelli, Benedetto Ruperti, and Pietro Tonutti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00036 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019
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Short-term responses of apple fruit to partial re-oxygenation during extreme hypoxic storage
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conditions
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Stefano Brizzolara1, Dubravka Cukrov1, Massimo Mercadini2, Federico Martinelli3, Benedetto
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Ruperti4, Pietro Tonutti 1§
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1
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Italy
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2
Life Sciences Institute, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà, 33, 56127 Pisa,
Marvil Engineering, Zona produttiva SCHWEMM, 8, 39040 Magrè sulla strada del vino, Bolzano,
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Italy
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3
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Sesto Fiorentino, Firenze, Italy
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Padova, Viale dell’Università, 16, 35020 Legnaro, Padova, Italy
Department of Biology, University of Florence, Sesto Fiorentino, Via Madonna del Piano, 6, 50019
Department of Agronomy, Food, Natural resources, Animals and Environment, University of
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§
Corresponding Author. Email:
[email protected] 17 18
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Abstract
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The short-term (24h) responses of apple fruit (cv ‘Granny Smith’) to the shift of oxygen concentration
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from 0.4 to 0.8 kPa, a protocol applied in Dynamic Controlled Atmosphere (DCA) storage technique,
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have been studied. Metabolomics and transcriptomics analyses of cortex tissue showed an immediate
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down-regulation of fermentative metabolism and of the GABA shunt in parallel with the activation
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of several 2-oxoglutarate-dependent dioxygenases genes. A down-regulation of the free
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phenylpropanoid pathway genes and the diversion of propanoid synthesis towards the methyl-
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erythritol phosphate route was also observed. The partial re-oxygenation induced an increase of
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glyceric,
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phosphatidylethanolamines, and a decrease of specific amino acids (valine, methionine, glycine,
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phenylalanine, GABA), organic acids (arachidic and citric acid), and secondary metabolites (catechin
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and epicatechin). The oxygen shift also resulted in transcriptional re-wiring of several components of
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IAA and ABA regulation and signalling. These results provide novel insights on the complexity of
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the short-term physiological responses of apple fruit to partial re-oxygenation applied during DCA
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storage.
palmitic
and
stearic
acids
and
of
several
phosphatidylcholines
and
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Key-words: Dynamic Controlled Atmosphere (DCA), post-harvest, low oxygen, metabolomics,
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transcriptomics, Malus domestica
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Introduction
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Advancements in our knowledge of apple fruit postharvest physiology and technical improvements
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have recently led to the development of protocols based on the dynamic modulation of low oxygen
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concentrations during controlled atmosphere (CA) storage.1 The dynamic controlled atmosphere
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(DCA) technique is based on the use of oxygen levels that are kept at extremely low values (about
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0.3-0.4 kPa) close to the anaerobic compensation point (ACP). These hypoxic conditions -that are
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considered the lowest oxygen limit (LOL) tolerated by fruit- are in general effective in retaining
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firmness and acidity together with better scores for crispness and skin color if compared with oxygen
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concentrations above the LOL.2-7 In addition, a reduced incidence of cold storage disorders such as
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superficial scald has been reported in DCA stored apples.8 These results, which can vary greatly in
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relation to the genetic background, can be achieved if the DCA protocol is properly applied, through
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a strict control of the physiological responses of apples that may negatively react if extreme hypoxic
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stress conditions (at or slightly above LOL) are applied for too long. This brings to the accumulation
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of anaerobic metabolites and quality losses (e.g. off flavors, flesh browning). Based on specific
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parameters effective in assessing the stress level reached by the fruit (ethanol production, chlorophyll
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fluorescence, respiratory quotient), oxygen is adjusted (increased) to the considered “safe”
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concentration (about 0.8-0.9 kPa) that could be maintained till the end of the storage period or
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repeated adjustments of oxygen levels can be applied depending on the physiological and
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technological parameters. Plant tissues, including fruit, are highly sensitive and differently respond
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to slight changes in oxygen concentration, most likely through the modulation of the recently
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discovered oxygen sensing mechanism (N-end rule pathway) involving Ethylene Responsive Factors
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(ERFs).9, 10 Cukrov et al.11 reported that ‘Granny Smith’ apples differentially react, in terms of both
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gene expression and metabolic changes, when postharvest hypoxic conditions are set at 0.4 or 0.8
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kPa oxygen, and this occurs early after hypoxia is imposed. In fact, the accumulation of specific
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metabolites (e.g. alanine, GABA) and transcripts (e.g. alcohol dehydrogenase, ADH, pyruvate
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decarboxylase, PDC, alanine amino transferase, AlaAT, sucrose syntase, SuSy) shows significant ACS Paragon Plus Environment
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changes already after 3 days of incubation under the two different conditions. This indicates the
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presence of mechanisms highly sensitive and quickly reacting to even moderate changes in oxygen
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levels surrounding the fruit. Re-oxygenation after an anoxic stress has a profound and rapid impact
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as observed in model species. Anaerobically grown rice seedlings resulted in rapid transcriptomic
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changes in DNA methylation after re-oxygenation12 and Arabidopsis cell cultures immediately
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respond to anoxia and subsequent re-oxygenation activating the antioxidant machinery.13 Also fruit
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tissues appear to quickly react to changes in atmosphere composition. Trobacher et al.14 reported that
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removal of the stress conditions applied during static CA, rapidly (3h) resulted in a net decline in
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GABA level, in cv ‘Empire’ apples. No information is available concerning the short-term metabolic
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and molecular reactions of fruit tissues to slight changes in oxygen concentration as those used in
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DCA protocols that, depending on the cv and the storage duration, may apply one or repeated low
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oxygen fluctuations. Considering both fruit physiological and storage technology issues, the goal of
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this paper was to evaluate by metabolomic and transcriptomic profiling the responses of ‘Granny
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Smith’ apples after 24h from the oxygen concentration shift from 0.4 to 0.8 kPa.
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Materials and Methods
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Plant material and treatments
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Apple (Malus domestica Borkh., cv ‘Granny Smith’) fruit were treated as reported in Cukrov et al.11
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Briefly, harvested fruit (starch index of 7, based on a 1 to 10 scale) were selected and immediately
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stored in three refrigerated (1 °C) experimental chambers under normoxia. After three days of
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acclimation of fruit to low temperature, 0.4 (0.4ox) and 0.8 (0.8ox) kPa oxygen atmospheres (in
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nitrogen and 0.9-1 kPa CO2) were applied in two different chambers. On day 30, when ethanol
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accumulation reached the threshold value of about 75 mg/L, oxygen concentration was increased
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from 0.4 kPa to 0.8 kPa. Fruit samples were collected as follows:
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Samples A After 24 DIA (days in atmospheres) of incubation at 0.4 kPa oxygen ACS Paragon Plus Environment
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Samples B After 31 DIA, 30 days at 0.4 + 1 day at 0.8 kPa oxygen (sampled 24h after the oxygen
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shift)
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Samples C After 31 DIA of incubation at 0.8 kPa oxygen
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Slices (about 1 cm thick) of cortex tissue (including both inner and outer parts), isolated from the
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equatorial part of three fruit collected at different sampling date, were immediately frozen in liquid
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nitrogen and stored at -80 °C.
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Metabolic profiling
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Sample analyses were performed at West Coast Metabolomics Center, UC Davis Genome Center
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(CA, USA). Six replicates (three biological replicates with two technical repetitions each) for
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treatment were analyzed. Cortex tissue was ground in liquid nitrogen and lyophilized. Samples
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extracts were prepared starting from 20 mg of lyophilized material, which were added to 1 mL of pre-
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chilled extraction solution and extraction procedure was performed as described by Fiehn.15 An
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aliquot of 20 μl of supernatant was completely dried in a SpeedVac concentrator. Derivatization was
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performed with methoxyamine and N-methy-N-(trimethylsilyl) trifluoroacetamide as described by
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Fiehn.15 GC-MS analysis was conducted with an Agilent 6890GC gas chromatographer (GC) coupled
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to a quadrupolar mass spectrometer (MS) and equipped with a 30 m long RTx-5Sil column (0.25 μm
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95 % dimethyl 5 % diphenyl polysiloxane film) with additional 10 m guard column (Restek,
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Bellefonte PA). Samples (1 μl) were introduced in both splitless and split modes. Temperature
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program was the following: from 50 °C to 275 °C increasing 12 °C/min and holding this temperature
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for 3 minutes. In splitless conditions, a helium (carrier gas) purge flow of 10.5 ml/min was applied
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for 1 min (8.2 psi). Split ratio was 1:10 and a split flow rate was 10.3 ml/min. Carrier gas was flowed
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constantly at 1 ml/min. HILIC-Q-TOF-MS analysis was conducted on an Agilent 1290 UHPLC
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equipped with a Waters Acuity 1.7 µm BEH HILIC 2.1 x 150 mm column for separation. An Agilent
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G6530A accurate-mass QTOF was used with an Agilent ESI Jet Stream ion source. Mobil phases
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were composed by solvent A (5 mM ammonium acetate with 0.2 % acetic acid) and solvent B (9:1 ACS Paragon Plus Environment
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acetonitrile:water with 5 mM ammonium acetate and 0.2 % acetic acid). Dry samples were re-
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suspended in 100 µl of solvent B and then column-injected following these conditions: from 0 to 4
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min, isocratic 100 % B, from 4 to 12 min, B linearly reduced to 45 %, from 12 to 20 min, isocratic
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45 % B. At the end 20 min re-equilibration phase was applied before injecting samples. Mass
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spectrometer was a Leco Pegasus IV time of flight controlled by a the Leco Chroma TF software
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2.31. Transfer line was held at 280 °C and ion source at 250 °C. Electron impact ionization was
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employed at 70 V with source temperature of 250 °C. The relative concentrations for each metabolite
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were obtained by peak areas. Identification of metabolites were performed using Agilent Fiehn GC-
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MS Metabolomics RTL Library as previously described by Martinelli et al.16
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All data analysis was performed using SAS software (SAS Institute). Normality test was conducted
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for each metabolite using Shapiro-Wilk test (P-value > 0.01). Only metabolites that passed normality
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test were analyzed using ANOVA (P-value < 0.05), and partial least squares discriminant analyses
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(PLSDA) were performed as described by Brizzolara et al.7
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RNA-seq analysis
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RNA extraction, RNA-seq analyses , data processing and fold change (FC) gene expression
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assessment of samples A (0.4ox at 24 DIA) vs B (0.4ox samples 24 h after the oxygen shift) have
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been performed as described in Cukrov et al.11 GO enrichment and changes in the pathway
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were analysed using Plant MetGenMAP analysis package considering identified DEGs
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(FDRA, B=C); in section II: BC; in section IV: B=A,
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BA, B>C, section VI: BC) 0.45 0.00 0.36 0.04 0.38 0.05 0.35 0.04 VI (B
