Brian Farneti

A Multidisciplinary Approach Providing New Insight into Fruit Flesh Browning Physiology in Apple (Malus x domestica Borkh.)

In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours. The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO). In the ‘Golden Delicious’ apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10). Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies (‘Fuji x Pink Lady’ and ‘Golden Delicious x Braeburn’). Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters. These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%. Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism. Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology. Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple.

Is there room for improving the nutraceutical composition of apple

In this study, we assessed the main bioactive compounds of a broad apple germplasm collection, composed by 247 accessions of wild (97) and domesticated (150) species. Among the stilbenes, trans- and cis-piceid were found to be ubiquitary components of both wild and cultivated apples. Apple was suggested to be the second dietary source of resveratrols. Results confirmed that the selection pressure of breeding and domestication did not uniformly affect all the phytochemicals contained in apples. For instance, organic acids (malic and ascorbic acid) and some phenolics (stilbenes, hydroxycinnamic acids, and dihydrochalcones) were significantly influenced by selection, while some relevant flavonoids (flavonols and flavan-3-ols) and triterpenoids (ursolic, oleanolic, and betulinic acids) were not. This comprehensive screening will assist in the selection of Malus accessions with specific nutraceutical traits suitable to establish innovative breeding strategies or to patent new functional foods and beverages.

Rapid Tomato Volatile Profiling by Using Proton-Transfer Reaction Mass Spectrometry (PTR-MS)

The availability of rapid and accurate methods to assess fruit flavor is of utmost importance to support quality control especially in the breeding phase. Breeders need more information and analytical tools to facilitate selection for complex multigenic traits such as flavor quality. In this study, it is shown that proton-transfer reaction mass spectrometry (PTR-MS) is a suitable method to monitor at high sensitivity the emission of volatiles determining the tomato aromatic profile such as hexanal, hexenals, methanol, ethanol, and acetaldehyde. The volatiles emitted by 14 tomato varieties (at red stage) were analyzed by 2 solvent-free headspace methods: solid-phase microextraction/gas chromatography MS and PTR-MS. Multivariate statistics (principal component analysis and cluster analysis) of the PTR-MS results allow an unambiguous separation between varieties, especially with a clear fingerprinting separation between the different tomato types: round truss, cocktail, and cherry tomatoes. PTR-MS was also successfully used to monitor the changes in volatile profiles during postharvest ripening and storage.

Comprehensive VOC profiling of an apple germplasm collection by PTR-ToF-MS

Fruit quality is generally represented by several components, among which aroma plays a fundamental role in determining the overall appreciation. To generate a comprehensive data inventory of aroma compounds in apple, a large collection represented by 190 apple accessions was characterized by a proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) instrument, a valid alternative to a gas chromatography-mass spectrometry (GS-MS) apparatus. The analytical performance of this instrument allowed to profile volatile organic compound (VOC) spectra of a portion of apple fruit flesh in a short time and efficient manner. Based on the VOC composition, the collection resulted grouped into six main clusters, mainly determined by ester and alcohols. These two VOC categories were also further exploited for the definition of an Alcohols/Esters index, which can be considered as a novel fruit quality descriptor useful for a further and more exhaustive characterization of several apple accessions. The distribution of these compounds and the possible further use of these information are discussed.

Untargeted metabolomics investigation of volatile compounds involved in the development of apple superficial scald by PTR-ToF-MS

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–MS (proton transfer reaction—time of flight—mass spectrometry) was employed to monitor the volatile organic compounds (VOCs) production along with the progression of this disorder in fruit of “Granny Smith”, 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–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.

Target metabolite and gene transcription profiling during the development of superficial scald in apple (Malus x domestica Borkh)

BackgroundFruit quality features resulting from ripening processes need to be preserved throughout storage for economical reasons. However, during this period several physiological disorders can occur, of which superficial scald is one of the most important, due to the development of large brown areas on the fruit skin surface.ResultsThis study examined the variation in polyphenolic content with the progress of superficial scald in apple, also with respect to 1-MCP, an ethylene competitor interacting with the hormone receptors and known to interfere with this etiology. The change in the accumulation of these metabolites was further correlated with the gene set involved in this pathway, together with two specific VOCs (Volatile Organic Compounds), α-farnesene and its oxidative form, 6-methyl-5-hepten-2-one. Metabolite profiling and qRT-PCR assay showed these volatiles are more heavily involved in the signalling system, while the browning coloration would seem to be due more to a specific accumulation of chlorogenic acid (as a consequence of the activation of MdPAL and MdC3H), and its further oxidation carried out by a polyphenol oxidase gene (MdPPO). In this physiological scenario, new evidence regarding the involvement of an anti-apoptotic regulatory mechanism for the compartmentation of this phenomenon in the skin alone was also hypothesized, as suggested by the expression profile of the MdDAD1, MdDND1 and MdLSD1 genes.ConclusionsThe results presented in this work represent a step forward in understanding the physiological mechanisms of superficial scald in apple, shedding light on the regulation of the specific physiological cascade.

Interference with ethylene perception at receptor level sheds light on auxin and transcriptional circuits associated with the climacteric ripening of apple fruit (Malus x domestica Borkh.)

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.

QTL analysis coupled with PTR-ToF-MS and candidate gene-based association mapping validate the role of Md-AAT1 as a major gene in the control of flavor in apple fruit

Volatile organic compounds (VOCs) are fundamental elements of flavor, one of the most important fruit-quality traits. Despite its importance, this aspect is still poorly considered in assisted breeding programs, due to the lack of suitable and fast detection systems as well as validated functional markers. In this work, a full-sib parental mapping population (‘Fuji × Delearly’) was initially employed to perform a comprehensive quantitative trait locus (QTL) survey, to assess the VOC segregation detected by a novel proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS) on fruit collected after a 2-month period of postharvest storage. Among this set of genomic regions, on chromosome 2 was also verified the coincident location between a group of QTLs, mainly associated to esters and alcohols, with a functional marker designed for Md-AAT1, a gene involved in the last step of the ester biosynthetic pathway. The allelic effect of this marker (here named Md-AAT1SSR) was further validated by candidate gene association mapping approach in a collection of 124 apple accessions. In this case, the volatile profiling was performed on peeled fruit flesh, as an important fraction of the aromatic blend of apple is released only after cutting. This work proposed a new and fast method for aroma phenotyping as well as a novel marker for an easy and widely applicable apple fruit quality advanced selection.

Genome-wide association study unravels the genetic control of the apple volatilome and its interplay with fruit texture

Highlight VOC production and fruit texture, assessed by multiple factor analysis, showed a contrasting behavior in apple. A GWAS approach dissected regulation of the volatilome and identified QTLs co-locating with important candidate genes.

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).