In recent years, the application of isotopically labeled substrates has received extensive attention in plant physiology. Measuring the propagation of the label through metabolic networks may provide information on carbon allocation in sink fruit during fruit development. In this research, gas chromatography coupled to mass spectrometry based metabolite profiling was used to characterize the changing metabolic pool sizes in developing apple fruit at five growth stages (30, 58, 93, 121, and 149 days after full bloom) using 13C-isotope feeding experiments on hypanthium tissue discs. Following the feeding of [U-13C]glucose, the 13C-label was incorporated into the various metabolites to different degrees depending on incubation time, metabolic pathway activity, and growth stage. Evidence is presented that early in fruit development the utilization of the imported sugars was faster than in later developmental stages, likely to supply the energy and carbon skeletons required for cell division and fruit growth. The declined 13C-incorporation into various metabolites during growth and maturation can be associated with the reduced metabolic activity, as mirrored by the respiratory rate. Moreover, the concentration of fructose and sucrose increased during fruit development, whereas concentrations of most amino and organic acids and polyphenols declined. In general, this study showed that the imported compounds play a central role not only in carbohydrate metabolism, but also in the biosynthesis of amino acid and related protein synthesis and secondary metabolites at the early stage of fruit development.

Apples are predominantly stored in controlled atmosphere (CA) storage to delay ripening and prolong their storage life. Profiling the dynamics of metabolic changes during ripening and CA storage is vital for understanding the governing molecular mechanism. In this study, the dynamics of the primary metabolism of 'Jonagold' apples during ripening in regular air (RA) storage and initiation of CA storage was profiled. 1-Methylcyclopropene (1-MCP) was exploited to block ethylene receptors and to get insight into ethylene mediated metabolic changes during ripening of the fruit and in response to hypoxic stress. Metabolic changes were quantified in glycolysis, the tricarboxylic acid (TCA) cycle, the Yang cycle and synthesis of the main amino acids branching from these metabolic pathways. Partial least square discriminant analysis of the metabolic profiles of 1-MCP treated and control apples revealed a metabolic divergence in ethylene, organic acid, sugar and amino acid metabolism. During RA storage at 18°C, most amino acids were higher in 1-MCP treated apples, whereas 1-aminocyclopropane-1-carboxylic acid (ACC) was higher in the control apples. The initial response of the fruit to CA initiation was accompanied by an increase of alanine, succinate and glutamate, but a decline in aspartate. Furthermore, alanine and succinate accumulated to higher levels in control apples than 1-MCP treated apples. The observed metabolic changes in these interlinked metabolites may indicate a coordinated adaptive strategy to maximize energy production.

In developing apple fruit, metabolic compartmentation is poorly understood due to the lack of experimental data. Distinguishing subcellular compartments in fruit using non-aqueous fractionation has been technically difficult due to the excess amount of sugars present in the different subcellular compartments limiting the resolution of the technique. The work described in this study represents the first attempt to apply non-aqueous fractionation to developing apple fruit, covering the major events occurring during fruit development (cell division, cell expansion, and maturation). Here we describe the non-aqueous fractionation method to study the subcellular compartmentation of metabolites during apple fruit development considering three main cellular compartments (cytosol, plastids, and vacuole). Evidence is presented that most of the sugars and organic acids were predominantly located in the vacuole, whereas some of the amino acids were distributed between the cytosol and the vacuole. The results showed a shift in the plastid marker from the lightest fractions in the early growth stage to the dense fractions in the later fruit growth stages. This implies that the accumulation of starch content with progressing fruit development substantially influenced the distribution of plastidial fragments within the non-aqueous density gradient applied. Results from this study provide substantial baseline information on assessing the subcellular compartmentation of metabolites in apple fruit in general and during fruit growth in particular.

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