Timing of Ethylene Inhibition Affects Internal Browning and Quality of 'Gala' Apples in Long-Term Low Oxygen Storage.

The objective of this study was to evaluate the timing of ethylene inhibition with preharvest and postharvest 1-methylcyclopropene (1-MCP) treatments on internal browning and quality of 'Gala' apples in long-term low O2 storage. 'Gala' apples were obtained from the same commercial orchard during their harvesting period for 2 years of study. Preharvest 1-MCP orchard spray (3.8% a.i) was applied at the label rate of 60 g 1-MCP per acre in the first year. Postharvest 1-MCP (1 µl L-1) treatments were made for 24 h at 0.5°C either at harvest time (1 day after harvest) or after storage in controlled atmosphere (CA) in both years. Apples were stored in 1.5 kPa O2 + 0.5 kPa CO2 or 0.6 kPa O2 + <0.5 kPa CO2 for 9 months in the first year and 1.5, 1.0, or 0.5 kPa O2 + 0.5 kPa CO2 for 8 months in the second year. Storage regimes with O2 concentrations less than 1 kPa were based on fruit respiration using SafePod™ technology. After removal from storage, all apples were then evaluated for internal browning and other quality attributes after 1, 7, and 14 days at room temperature (RT, 21-22°C). Internal browning developed in 'Gala' apples during both years of study, with up to 16% incidence across treatments in the first year and up to 84% in the second year. Apples stored in 0.5-0.6 kPa O2 had significantly less internal browning during both years of study, compared to apples stored in higher O2. The effect of 1-MCP on internal browning was negligible in 0.5-0.6 kPa O2 storage. 'Gala' stored in 1.5 kPa O2 and treated with postharvest 1-MCP after storage had significantly less internal browning with preharvest 1-MCP than those without preharvest treatment. Apples treated with postharvest 1-MCP at harvest time, instead of after storage, did not exhibit this same effect. Preharvest 1-MCP-treated fruit maintained greater firmness retention than those without preharvest 1-MCP, and this effect was further enhanced when 1-MCP was applied after storage. Postharvest 1-MCP had no effect on firmness retention in fruit without preharvest 1-MCP, but lower O2 maintained greater firmness in those apples. Preharvest 1-MCP had no significant effect on internal ethylene concentration, whereas it was reduced by postharvest 1-MCP at harvest time in the first year of study, regardless of storage regimes. However, internal ethylene was only affected by storage regime in the second year, with lower concentration in fruit from 0.5 kPa O2 than in those from higher O2. Greasiness developed only in the second year and postharvest 1-MCP consistently reduced it, regardless of treatment timing and storage regime. There was no greasiness in apples treated with postharvest 1-MCP at harvest and then held in 0.5 kPa O2 for 8 months plus 14 days at room temperature. Soluble solids concentration and malic acid content were slightly higher in 'Gala' apples with preharvest 1-MCP compared to those without, whereas there was little and inconsistent effect of postharvest 1-MCP on these attributes. Overall, storage regimes with less than 1 kPa O2 provided the least amount of internal browning and best quality attributes. Ethylene inhibition provided further benefits, but this was dependent on the timing of 1-MCP treatment.

Application of in vivo solid phase microextraction (SPME) in capturing metabolome of apple (Malus ×domestica Borkh.) fruit.

An in vivo direct-immersion SPME sampling coupled to comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GCxGC-ToFMS) was employed to capture real-time changes in the metabolome of 'Honeycrisp' apples during ripening on the tree. This novel sampling approach was successful in acquiring a broad metabolic fingerprint, capturing unique metabolites and detecting changes in metabolic profiles associated with fruit maturation. Several metabolites and chemical classes, including volatile esters, phenylpropanoid metabolites, 1-octen-3-ol, hexanal, and (2E,4E)-2,4-hexadienal were found to be up-regulated in response to fruit maturation. For the first time, Amaryllidaceae alkaloids, metabolites with important biological activities, including anti-cancer, anti-viral, anti-parasitic, and acetylcholinesterase (AChE) inhibitory activity, were detected in apples. Considering the elimination of oxidative degradation mechanisms that adversely impact the representativeness of metabolome obtained ex vivo, and further evidence that lipoxygenase (LOX) pathway contributes to volatile production in intact fruit, in vivo DI-SPME represents an attractive approach for global plant metabolite studies.

Targeted quantitative profiling of metabolites and gene transcripts associated with 4-aminobutyrate (GABA) in apple fruit stored under multiple abiotic stresses.

4-Aminobutyrate accumulates in plants under abiotic stress. Here, targeted quantitative profiling of metabolites and transcripts was conducted to monitor glutamate- and polyamine-derived 4-aminobutyrate production and its subsequent catabolism to succinate or 4-hydroxybutyrate in apple (Malus x domestica Borkh.) fruit stored at 0 °C with 2.5 kPa O2 and 0.03 or 5 kPa CO2 for 16 weeks. Low-temperature-induced protein hydrolysis appeared to be responsible for the enhanced availability of amino acids during early storage, and the resulting higher glutamate level stimulated 4-aminobutyrate levels more than polyamines. Elevated CO2 increased the levels of polyamines, as well as succinate and 4-hydroxybutyrate, during early storage, and 4-aminobutyrate and 4-hydroxybutyrate over the longer term. Expression of all of the genes likely involved in 4-aminobutyrate metabolism from glutamate/polyamines to succinate/4-hydroxybutyrate was induced in a co-ordinated manner. CO2-regulated expression of apple GLUTAMATE DECARBOXYLASE 2, AMINE OXIDASE 1, ALDEHYDE DEHYDROGENASE 10A8 and POLYAMINE OXIDASE 2 was evident with longer term storage. Evidence suggested that respiratory activities were restricted by the elevated CO2/O2 environment, and that decreasing NAD+ availability and increasing NADPH and NADPH/NADP+, respectively, played key roles in the regulation of succinate and 4-hydroxybutyate accumulation. Together, these findings suggest that both transcriptional and biochemical mechanisms are associated with 4-aminobutyrate and 4-hydroxybutyrate metabolism in apple fruit stored under multiple abiotic stresses.

Metabolic Alterations in Postharvest Pear Fruit As Influenced by 1-Methylcyclopropene and Controlled Atmosphere Storage.

This study assessed the impact of 1-methylcyclopropene (1-MCP) and controlled atmosphere (CA) on the metabolism of targeted amino acids, organic acids, and antioxidants in stored 'AC Harrow Crisp' pears and their relationships to storage disorders. Pears were treated with 0 or 300 nL L-1 1-MCP and stored at 0 °C under ambient air or CA. Spectrophotometric assays demonstrated that glutathione levels fluctuated with storage and were most preserved by 1-MCP under ambient air. HPLC analysis revealed that ascorbate concentrations declined with storage and were little affected by 1-MCP and CA. Citrate, lactate, and fumarate accumulated with storage but were differentially affected by 1-MCP. Aspartate and glutamate concentrations were greater with 1-MCP; γ-aminobutyrate accumulated in disordered fruit. Principal component analysis demonstrated that alterations in citrate and fumarate were, respectively, correlated with internal breakdown and senescent scald. γ-Aminobutyrate and alanine were associated with internal cavities. All disorders were associated with antioxidant depletion.

Plant Glyoxylate/Succinic Semialdehyde Reductases: Comparative Biochemical Properties, Function during Chilling Stress, and Subcellular Localization.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2) are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh.) and rice (Oryza sativa L.) GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress.

Ancient Plant Glyoxylate/Succinic Semialdehyde Reductases: GLYR1s Are Cytosolic, Whereas GLYR2s Are Localized to Both Mitochondria and Plastids.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2) are considered to be involved in detoxifying harmful aldehydes, thereby preserving plant health during exposure to various abiotic stresses. Phylogenetic analysis revealed that the two GLYR isoforms appeared in the plant lineage prior to the divergence of the Chlorophyta and Streptophyta, which occurred approximately 750 million years ago. Green fluorescent protein fusions of apple (Malus x domestica Borkh.), rice (Oryza sativa L.) and Arabidopsis thaliana [L.] Heynh GLYRs were transiently expressed in tobacco (Nicotiana tabaccum L.) suspension cells or Arabidopsis protoplasts, as well in methoxyfenozide-induced, stably transformed Arabidopsis seedlings. The localization of apple GLYR1 confirmed that this isoform is cytosolic, whereas apple, rice and Arabidopsis GLYR2s were localized to both mitochondria and plastids. These findings highlight the potential involvement of GLYRs within distinct compartments of the plant cell.

Oxidative metabolism is associated with physiological disorders in fruits stored under multiple environmental stresses.

In combination with low temperature, controlled atmosphere storage and 1-methylcyclopropene (ethylene antagonist) application are used to delay senescence of many fruits and vegetables. Controlled atmosphere consists of low O2 and elevated CO2. When sub-optimal partial pressures are used, these practices represent multiple abiotic stresses that can promote the development of physiological disorders in pome fruit, including flesh browning and cavities, although there is some evidence for genetic differences in susceptibility. In the absence of surface disorders, fruit with flesh injuries are not easily distinguished from asymptomatic fruit until these are consumed. Oxidative stress metabolites tend to accumulate (e.g., γ-aminobutyrate) or rapidly decline (e.g., ascorbate and glutathione) in vegetative tissues exposed to hypoxic and/or elevated CO2 environments. Moreover, these phenomena can be associated with altered energy and redox status. Biochemical investigations of Arabidopsis and tomato plants with genetically-altered levels of enzymes associated with the γ-aminobutyrate shunt and the ascorbate-glutathione pathway indicate that these metabolic processes are functionally related and critical for dampening the oxidative burst in vegetative and fruit tissues, respectively. Here, we hypothesize that γ-aminobutyrate accumulation, as well energy and antioxidant depletion are associated with the development of physiological injury in pome fruit under multiple environmental stresses. An improved understanding of this relationship could assist in maintaining the quality of stored fruit.

Capturing Plant Metabolome with Direct-Immersion in Vivo Solid Phase Microextraction of Plant Tissues.

For the first time, an in vivo sampling mode of direct immersion-solid phase microextraction (DI-SPME) was employed to capture the metabolome of living plant specimens, using apple (Malus × domestica Borkh.) as a model system. Metabolites were extracted from apple tissues and introduced by thermal desorption into a comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry instrument. The feasibility of this sampling approach, based on exploitation of microextraction principles, including negligible depletion of free analyte concentrations, solventless sampling and sample preparation, and on-site compatibility, was determined in global metabolite analysis. Rather than adopting an approach of traditional sample preparation, requiring metabolism quenching and laborious sample preparation, the objective of the study was to capture the metabolome in vivo, evaluate the feasibility of the approach to provide unbiased extraction coverage, and compare analytical precision when different SPME sampling modes are employed. The potential of in vivo DI-SPME in quantitative plant metabolomics was assessed by evaluating changes in metabolic fingerprints in response to fruit maturation. The in vivo SPME sampling approach has been demonstrated as capable of sampling living systems with high reproducibility, considering that nearly 50% of hundreds of evaluated compounds included in the determination of analytical performance met the 15% RSD FDA criterion. Esters were extracted with high repeatability (% RSD for hexyl butanoate and butyl butanoate of 16.5 and 5.9, respectively, from 9 determinations in 3 apples) and found to be upregulated in response to apple fruit maturation.

Solid phase microextraction coupled with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for high-resolution metabolite profiling in apples: implementation of structured separations for optimization of sample preparation procedure in complex samples.

Metabolomics currently represents one of the fastest growing high-throughput molecular analysis platforms that refer to the simultaneous and unbiased analysis of metabolite pools constituting a particular biological system under investigation. In response to the ever increasing interest in development of reliable methods competent with obtaining a complete and accurate metabolomic snapshot for subsequent identification, quantification and profiling studies, the purpose of the current investigation is to test the feasibility of solid phase microextraction for advanced fingerprinting of volatile and semivolatile metabolites in complex samples. In particular, the current study is focussed on the development and optimization of solid phase microextraction (SPME) - comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-ToFMS) methodology for metabolite profiling of apples (Malus × domestica Borkh.). For the first time, GC × GC attributes in terms of molecular structure-retention relationships and utilization of two-dimensional separation space on orthogonal GC × GC setup were exploited in the field of SPME method optimization for complex sample analysis. Analytical performance data were assessed in terms of method precision when commercial coatings are employed in spiked metabolite aqueous sample analysis. The optimized method consisted of the implementation of direct immersion SPME (DI-SPME) extraction mode and its application to metabolite profiling of apples, and resulted in a tentative identification of 399 metabolites and the composition of a metabolite database far more comprehensive than those obtainable with classical one-dimensional GC approaches. Considering that specific metabolome constituents were for the first time reported in the current study, a valuable approach for future advanced fingerprinting studies in the field of fruit biology is proposed. The current study also intensifies the understanding of SPME-GC×GC-ToFMS hyphenation and outlines the benefits of facilitating GC×GC for SPME method optimization. The obtained results clearly illustrate that acquisition of a more complete metabolome snapshot is only attainable under optimized conditions for both techniques.

Postharvest variation in apple (Malus x domestica Borkh.) Flavonoids following harvest, storage, and 1-MCP treatment.

The impact of 1-methylcyclopropene (1-MCP) on the synthesis and retention of flavonoid compounds during storage and ripening of red Delicious (Malus x domestica Borkh.) apples was investigated. Numerous anthocyanins, flavonols, flavan-3-ols, and a hydroxycinnamic acid from three different fruit harvest maturities were monitored after a 120 day storage and 1 week shelf life period using high-performance liquid chromatography/diode array detector analysis. The total flavonoid concentration was 5% greater in fruit treated with 1-MCP, whereas chlorogenic acid levels were 24% lower. All compounds analyzed increased in concentration during fruit harvest; however, the anthocyanins generally declined after storage, while chlorogenic acid levels increased. 1-MCP treatment resulted in the retention of anthocyanins in the latter stages of storage but did not affect the flavonols and flavan-3-ols. Chlorogenic acid biosynthesis from early and optimal fruit harvest maturities was greatly inhibited by 1-MCP during storage and the 1 week shelf life period. However, 1-MCP did not affect chlorogenic acid concentrations in late-harvested fruit. Results suggest that 1-MCP may inhibit the activity of phenylalanine ammonia-lyase and subsequent biosynthesis of flavonoid compounds. However, because very little postharvest biosynthesis of flavonoids occurs in apples, 1-MCP treatment may be useful for maintaining some of the intrinsic flavonoid levels of red Delicious apples, if applied at the proper harvest maturity.