<p>The superficial scald is an important physiological disorder affecting apple fruit during postharvest storage. To date, the accumulation, and further oxidation, of α-farnesene was considered as the most probable cause for the development of this physiopathy. In order to perform a more broad investigation, a PTR-ToF&ndash;MS (proton transfer reaction&mdash;time of flight&mdash;mass spectrometry) was employed to monitor the volatile organic compounds (VOCs) production along with the progression of this disorder in fruit of &ldquo;Granny Smith&rdquo;, an apple variety known to be highly susceptible to scald. The untargeted metabolite investigation was performed on both skin and pulp, as well as comparing control versus treated tissues with 1-methylcyclopropene (1-MCP), an ethylene competitor widely used to prevent the development of this phenomenon. The rapid and non-destructive analysis of the VOC array carried out by PTR-ToF&ndash;MS identified three specific groups of metabolites in the skin, among which the 6-methyl-5-hepten-2-one (MHO) resulted significantly associated with the development of the superficial scald in apple. The results proposed in this work suggest the use of this novel equipment for an on-line monitoring of the VOCs released by the apple during the postharvest storage, as well as to use MHO as a possible biochemical marker for an early detection of the superficial scald symptoms.</p>

In the present study, the potential of PTR-ToF-MS for addressing fundamental and technical post-harvest issues was tested on the non-destructive and rapid monitoring of volatile compound evolution in three apple cultivars (‘Golden Delicious’, ‘Braeburn’ and ‘Gold Rush’) during 25 days of post-harvest shelf life ripening. There were more than 800 peaks in the PTR-ToF-MS spectra of apple headspace and many of them were associated with relevant compounds. Besides the ion produced upon proton transfer, we used the ion at mass 28.031 (C2H 4 +) produced by charge transfer from residual O 2 + as a monitor for ethylene concentration. ‘Golden Delicious’ apples were characterised by higher ethylene emission rates than ‘Gold Rush’ and ‘Braeburn’, and quantitative comparison has been supported by two segment piecewise linear model fitting. Ester evolution during post-harvest ripening is strongly dependent on endogenous ethylene concentration levels. For ‘Golden Delicious’ and ‘Braeburn’, sesquiterpenes (alpha-farnesene) exhibited a fast response to ethylene emission followed by a rapid decline after the endogenous ethylene maximum peak. Carbonyl compounds displayed a different time evolution as compared to esters and terpenes and did not show any evident relationship with ethylene. Methanol and ethanol concentrations during the entire storage period did not change significantly. We show how multivariate analysis can efficiently handle the large datasets produced by PTR-ToF-MS and that the outcomes obtained are in agreement with the literature. The different volatile compounds could be simultaneously monitored with high time resolution, providing advantages over the more established techniques for the investigation of VOC dynamics in fruit post-harvest storage trials.

The aroma trait in apple is a key factor for fruit quality strongly affecting the consumer appreciation, and its detection and analysis is often an extremely laborious and time consuming procedure. Molecular markers associated to this trait can to date represent a valuable selection tool to overcome these limitations. QTL mapping is the first step in the process of targeting valuable molecular markers to be employed in marker-assisted breeding programmes (MAB). However, a validation step is usually required before a newly identified molecular marker can be implemented in marker-assisted selection. In this work the position of a set of QTLs associated to volatile organic compounds (VOCs) was confirmed and validated in three different environments in Switzerland, namely Wädenswil, Conthey and Cadenazzo, where the progeny 'Fiesta×Discovery' was replicated. For both QTL identification and validation, the phenotypic data were represented by VOCs produced by mature apple fruit and assessed with a Proton Transfer Reaction-Mass Spectrometer (PTR-MS) instrument. The QTL-VOC combined analysis performed among these three locations validated the presence of important QTLs in three specific genomic regions, two located in the linkage group 2 and one in linkage group 15, respectively, for compounds related to esters (m/z 43, 61 and 131) and to the hormone ethylene (m/z 28). The QTL set presented here confirmed that in apple some compounds are highly genetically regulated and stable across environments.

Proton Transfer Reaction-Mass Spectrometry (PTR-MS) in its recently developed implementation based on a time-of-flight mass spectrometer (PTR-ToF-MS) has been evaluated as a possible tool for rapid non-destructive investigation of the volatile compounds present in the metabolome of apple cultivars and clones. Clone characterization is a cutting-edge problem in technical management and royalty application, not only for apple, aiming at unveiling real properties which differentiate the mutated individuals. We show that PTR-ToF-MS coupled with multivariate and data mining methods may successfully be employed to obtain accurate varietal and clonal apple fingerprint. In particular, we studied the VOC emission profile of five different clones belonging to three well known apple cultivars, such as ‘Fuji’, ‘Golden Delicious’ and ‘Gala’. In all three cases it was possible to set classification models which can distinguish all cultivars and some of the clones considered in this study. Furthermore, in the case of ‘Gala’ we also identified estragole and hexyl 2-methyl butanoate contributing to such clone characterization. Beside its applied relevance, no data on the volatile profiling of apple clones are available so far, our study indicates the general viability of a metabolomic approach for volatile compounds in fruit based on rapid PTR-ToF-MS fingerprinting.

Recently the coupling of proton transfer reaction ionization with a time-of-flight mass analyser (PTR-TOF-MS) has been proposed to realise a volatile organic compound (VOC) detector that overcomes the limitations in terms of time and mass resolution of the previous instrument based on a quadrupole mass analysers (PTR-Quad-MS). This opens new horizons for research and allows for new applications in fields where the rapid and sensitive monitoring and quantification of volatile organic compounds (VOCs) is crucial as, for instance, environmental sciences, food sciences and medicine. In particular, if coupled with appropriate data mining methods, it can provide a fast MS-nose system with rich analytical information. The main, perhaps even the only, drawback of this new technique in comparison to its precursor is related to the increased size and complexity of the data sets obtained. It appears that this is the main limitation to its full use and widespread application. Here we present and discuss a complete computer-based strategy for the data analysis of PTR-TOF-MS data from basic mass spectra handling, to the application of up-to date data mining methods. As a case study we apply the whole procedure to the classification of apple cultivars and clones, which was based on the distinctive profiles of volatile organic compound emissions.