2.2. Volatilome and Profiling Techniques

VOCs are a major fraction of the plant metabolome, and being main contributors to fruit aroma and flavour, and thus to fruit quality, have proved to be of particular biological interest. Two lactones, γ- and δ-decalactone are considered to be the most relevant compounds to the final peach aroma ^[44][45][46][59,60,61]. Particularly, γ-decalactone is the major lactone compound of peach VOCs and it is widely used as a flavour and fragrance agent in the food industry ^{[2][47][48][49][50][51]}[2,62,63,64,65,66]. Another lactone showing a peach-like odour is γ -jasmolactone, first detected in peach juice ^[52][67]. A few other VOCs, including δ-octalactone, δ-dodecalactone and 6-pentyl-α-pyrone, are also major contributors of the overall peach aroma ^[49][64]. C6 aldehydes and alcohols, such as n-hexanal, (E)-2-hexenal and (E)-2-hexenol are also partly responsible for peach aroma, but not to the same extent. Similarly, esters, such as (Z)-3-hexenyl acetate, partially contribute to the flavour ^[47][62]. Most typical peach VOCs are produced during fatty acid biosynthesis and an involvement of LOX, ADHs and FAs has been proposed ^[53][68]. All lactones derive from saturated fatty acids, through several steps, including dehydrogenation, epoxidation, hydration, hydroxylation, shortening by β-oxidation and then internal esterification with hydroxyacetyl-coenzyme A ^[54][55][69,70]. Amongst the VOCs, only a small sub-set impacts the final aroma which increases during fruit ripening. However, VOC production in peach is not a static process, since their level changes dramatically during this stage and flavour compound patterns are different among tissues, species and cultivars, depending on post-harvest conditions ^{[4][5][56][57]}[4,5,71,72]. In particular, it has been demonstrated that the aromatic profile and organoleptic properties are influenced by both pre-harvest factors, such as growing conditions and maturity stage of the fruit, as well as post-harvest factors, including storage temperature, controlled atmosphere composition, ethylene modulation and the presence of wax coatings ^[58][59][60][16,73,74]. Overall, some broad trends in VOC changes have been observed. For example, aldehydes tend to decline, and esters to increase in peach fruit over their shelf-life ^[61][75]. Linalool is a terpene which is reported to be present in peaches at harvest and to rapidly decrease under cold storage ^[60][74]. CS induces the production of esters and lactones, two compounds typically related with advanced ripening stages [5], that increase during storage at 20 °C after cold treatment ^[61][62][75,76].

3. Peach Gene Expression and Correlation with VOCs

FADs are undoubtedly key enzymes influencing VOC production, but surprisingly their specific role in plant VOC synthesis has not yet been completely elucidated. In peach, ppFAD1 was reported to be involved in the formation of a precursor of lactones/esters ^[63][127]. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that two ω-3 FAD genes, PpFAD3-1 and PpFAD3-2, may be important in peach VOC biosynthesis since in ripe fruit PpFAD3-1 was high while expression of PpFAD3-2 was low. Instead, high PpFAD3-2 and low PpFAD3-1 transcript levels characterized young fruit ^[63][127]. The FAD gene family expression during peach ripening and in particular the transcript abundance of PpFAD1 seems to increase in the first days after harvest [5]. In contrast PpFAD2 is found at low levels for one or two days, and then increases together with ethylene and linolenic acid during post-harvest ripening [5]. FADs and their related genes also play a significant role in the changes in lipid membrane fluidity, which is typical of cold-responsive fruits ^[64][128]. The transcriptional regulation of these genes, in peach, has been associated with metabolic changes occurring during CI ^[11][19]. Both in several peach cultivars and nectarines, PpFAD4 gene expression was found to decrease under CS, showing, however, very distinctive differences among peach varieties before and after CS [7]. In peach PpLOX1 and PpLOX4, PpLOX2 and PpLOX3, are associated with the synthesis of lactones and of C6 aldehydes [5], and recently differences in expression of these genes amongst cultivars and in response to storage conditions have been shown [7]. Specifically, PpLOX1 expression increased following cold storage in three different nectarine cultivars, whereas in peach cultivars its gene expression fluctuated. Conversely, at the same temperature, PpLOX2, PpLOX3 and PpLOX4 showed a down-regulation with no significant differences among cultivars [7]. The expression of members of the epoxide hydrolase gene family, PpEPH2 and PpEPH3, were found to be involved in the formation of γ-decalactone ^[51][55][66,70] and to be down regulated during CS in different cultivars [7]. However, the alcohol acyltransferase PpAAT1, which catalyses the biosynthesis of this lactone ^[65][66][129,130] appeared significantly up regulated after cold exposure in different nectarine cultivars [7]. PpTPS1 terpene synthase, whose expression was found to decline significantly in different peach cultivars after CS [7] is localized in plastids and its expression during cold storage was correlated with the linalool production, while the isoform PpTPS2 was shown to be responsible for (E,E)-α-farnesene (a common biotic-stress-induced plant volatile) biosynthesis in the cytoplasm ^[67][132]. Under UV-B light treatments, RNA-Seq showed altered transcript levels for these two terpene synthases in peach ^[67][132], with a decrease of 86% of PpTPS1 and an 80-fold increase of PpTPS2. The reduction of the volatile linalool suggests that the levels of compounds contributing to flavour in peach fruits can be regulated by this ultraviolet treatment. In relation to the production of alcohol VOCs, in CS post-harvest peaches, PpADH2 gene expression seems to depend on the specific cultivar more than on the treatment [7], thus an examination of the other genes related to ester biosynthesis should be evaluated across different cultivars to assess whether they also show cultivar-specific responses. Therefore, VOC profile, gene expression and changes in their response to CS appear to be cultivar specific, and to obtain a complete picture there is the need to test these parameters across a wider range of cultivars.

4. Multi-Omics Approaches and the Peach Post-Harvest Ripening Process

NGS technologies have revolutionized plant biology and have been extended widely to non-model systems with very low costs ^[68][30]. At the same time, combined approaches, based on the relationship amongst genomics, transcriptomics and metabolomics methods, have been developed to exploit these inter-related datasets. Multi-omics applications have been carried out for a wide range of different fruit species and have provided a powerful tool for identifying correlations between different biological components controlling plant functions and metabolic pathways. For instance, a combined analysis of metabolites and transcripts revealed the metabolic shifts underlying tomato fruit development and new associations between specific transcripts and metabolites were identified ^[69][133]. Similar approaches have been successfully applied to study candidate genes involved in tomato fruit ripening ^[70][71][134,135] and in other fruit e.g., grape berry development ^[72][73][74][136,137,138]. In peach, combined omics approaches were applied to identify relationships between fruit VOCs and QTLs for a better understanding of the gene regulation mechanisms behind the biosynthesis of the compounds. For instance, thanks to the availability of an annotated peach genome, QTLs were detected for 23 VOCs and associations between candidate genes and QTLs were established ^[75][139]. Furthermore, QTLs associated with characters of agronomic interest both to pre-harvest and post-harvest ripening across several Prunus species have been assembled through genotyping with many DNA markers distributed across the entire genome ^[76][140]. For peach this has been particularly challenging due to the restricted genetic diversity of cultivated peaches, but nevertheless hundreds of QTLs have been identified and related to fruit VOCs. Other combined genomics and metabolomics approaches have investigated pre- and post-harvest ripening processes and confirmed specific loci that control peach aroma ^[77][141]. By using GC-MS, compounds associated with aroma were also analysed and a correlation-based analysis of these datasets was developed, revealing that the peach volatilome is organized into modules composed of compounds from the same biological pathway or having similar chemical structures. A QTL approach was applied to a very heterogenous peach pedigree, always using the recently available reference peach genome ^[78][142]. Thanks to use of this genome, the intrachromosomal positions of several QTLs showed differences compared with those previously reported in peach and the mapping quality was generally enhanced. The results of this study provided new insights for a model study for pedigree-based analysis in several peach breeding programs. The validation of genes localized in QTLs through gene expression analysis (RNA-Seq and qPCR) has been recently tested as an approach in peach, in a study aimed at identifying candidate genes involved in fruit softening rate ^[79][143]. The results, suggesting that auxin may be important in rapid fruit softening, helped to improve our understanding of the genetic mechanisms involved in this process both in peaches and nectarines, and could lead to the identification of molecular markers associated with softening rate. Further multi-omics studies, based on transcriptomic and metabolomic analyses, identified new candidate genes impacting aroma volatiles in pre-harvest and post-harvest conditions in two peach cultivars ^[55][70]. In this investigation, datasets from microarrays and qRT-PCR analyses were combined with VOCs detected using HS-SPME-GC-MS. The combined dataset was analysed through a correlation-based approach (using CNA) to identify the genes showing a correlation with the major aromatic compounds, including lactones, esters and phenolics. The results showed a core set of genes, including alcohol acyl transferase, fatty acid desaturases and transcription factor genes, that are highly related with peach fruit VOCs and could be useful for future biotechnological activities.