RSV, originating from the volcanic slopes of Vesuvius in the same region, produces small sweet pear-shaped fruits that are suitable for long term storage thanks to their texture. The ability to dissect genomic and metabolic responses to environmental cues is key to understanding the molecular basis of plasticity of fruit quality traits. In spite of the large amount of information on the genomic and metabolomic components controlling fruit quality traits, very little is known to date regarding the transcriptional dynamics controlling such traits. A systems biology approach, integrating different –omics datasets, is instrumental for elucidating complex mechanisms controlling organoleptic properties. The aim of this work was to quantify the extent of the transcriptional response to environmental cues, to measure the metabolic activity and to evaluate the organoleptic consequences of the genome variation identified. We used a multilevel approach combining genome, transcriptome, metabolome and sensorial data of three tomato varieties, Heinz 1706 , SM and RSV, grown in two different localities.To further explore the framework of relationships established and to analyze metabolic routes challenged, we extracted sub-networks of key genes and metabolites related to fruit quality attributes. In the SM sub-network related to appearance a contrasting regulation of carotenoids pigments with a red color attribute was evidenced .
The β-carotene hydroxylase transcript was expressed at low level in Sarno and was negatively correlated with β-carotene accumulation as expected by the challenging of related pathway . In fact, large square planting pots this gene encodes for an enzyme that, in the pathway of zeaxanthin biosynthesis, is involved in the conversion of β-carotene in β-criptoxanthin and then in zeaxanthin. Also the four XTHs, clustering on chromosome 3, negatively correlated with red color and three of these , expressed at the higher level in Acerra , are exclusive to Sa. Fruit texture attributes such as juiciness, hardness, skin resistance, flouriness and turgidity were highly influenced by the environment . Interestingly, the transcript levels of 4- coumarate CoA ligase-like protein in Sarno negatively correlated with the accumulation of caffeic acid, ferulic acid and glycosylated forms of coumaric acid and positively correlated with turgidity, skin resistance and hardness . Indeed, the downregulation of such enzyme, in suberin and flavonoid biosynthesis, promotes the conversion of caffeate in caffeoylCoA and ferulate in feruloylCoA. SM in Sarno showed a strong positive correlation between a down-regulated decarboxylase , involved into the 2- phenylethanol pathway, and two additional decarboxylase genes belonging to the same chromosome region .All three shared a positive correlation with arogenate/prephenate dehydratase and two carbohydrate genes and negative correlations with amino acids . Interestingly, in Sarno there was a direct relationship between the transcript levels of Solyc08g079750 confirmed by Real time PCR , with the L-aspartate content and saltiness and a negative relationship between tyramine accumulation and an N-acetyltransferase , involved in the pathway of tyramine degradation , present at 2 fold lower in Sarno .
At the same time the downregulation of glutamate decarboxylase correlated well with the high level of glutamic acid in Sarno . The enzyme is involved, in fact, in the degradation of glutamate in 4-aminobutyrate. The increase in arginine levels in Sarno is related to the down-regulation of two arginine decarboxylase transcripts involved inarginine degradation . Arginine decarboxylase had a negative correlation with sourness in Sarno and a positive correlation with flavor and a negative correlation with arginine in Acerra. . As for RSV, the red color resulted negatively correlated with kaempferol and naringenin in Sarno and positively correlated with two XTHs on chromosome 3 in Sarno and one in Acerra. Color uniformity correlated with phytoene and phytofluene, naringenins and chromosome 3 XTHs in Acerra. Negative relationships among XTHs, hardness and skin resistance in Acerra were evidenced . The last attribute was also positively correlated with raffinose, phenolic acids and caffeic acid present at higher levels in this environment. On the other hand, in Sarno such metabolites positively correlated with turgidity. In general, strong cell wall remodeling was evident in RSV with hardness and skin resistance more highly interconnected in Acerra and turgidity and juiciness in Sarno . In Acerra, flavor was negatively correlated with XTHs, most of the amino acids and with arginine decarboxylase , which also displayed a negative correlation with arginine accumulation and specific correlations with saltiness and sweetness in Sarno and with sourness and tomato odor in Acerra . A positive correlation with valine levels and branched chain amino acid aminotransferase , involved in valine biosynthesis, was found in Acerra. This transcript was also correlated with sweetness, saltiness and flavor, with organic acids and mevalonate in Acerra and with sweetness, sourness, odor and XTHs in Sarno.
Transcriptome remodeling entailed the coordinate regulation of several hundreds of genes, with different genotypes showing a different responsiveness to different environments, suggesting a their specific sensitivity to external environmental inputs. The vast majority of transcriptional responses to the two environments were genotype-specific, indicating a strong G component in the responses to E. The two “local” genotypes showed a consistently higher numbers of genes which were differentially expressed between the two environments with respect to the reference one with a more asymmetric responses between the two environments. In addition, H showed a higher number of core expressed genes in both locations. Some processes were regulated independently of the genotype, whilst others were genotype-specific. In Sarno, genotype-independent over-represented categories included genes related to amino acid biosynthesis , while genes involved in cell wall, ethylene, methionine and aspartate metabolism were highly regulated in Acerra. Some of these amino acids are known to influence flavor, for instance, cysteine and methionine derivatives are essential flavor components in several plant matrixes while aspartate, together with glutamate, is a component of umami taste. Ethylene and cell wall metabolism, on the other hand, are well known to influence another important organoleptic characteristic of tomato fruits i.e. firmness. The effect of different environments on SM and RSV transcriptome is clearly stronger, with a larger number of transcripts being differentially expressed with a wider fold change. Indeed, genes that showed marked environmental changes are presumable important for overall fruit quality, since their enzymatic activities impact upon similar fruit quality attributes. The resilience of H to different environments can be attributed to a robust core gene set and an overall low number of DEG between the two environments, but also to the involvement of fewer trans-acting transcription factors in responses to Environment. Proper control of gene expression by transcription factors is critical for the capacity of an organism to respond to changing environments. This observation suggests that transcriptional regulatory cascades may be key components of differential resilience shown by different plant varieties to changing environmental conditions. Genome structure and gene organization have a pivotal role in shaping fruit metabolism and its regulation by endogenous and environmental stimuli, resulting in distinctive fruit quality attributes. Albeit some of the previously identified SM- and RSV-specific variants contributed to the differential expression, large square planter pots the vast majority of genes differentially expressed in the two environments were devoid of such variants, indicating that differential responses to environment were modulated by the overall genetic composition of each genotype, acting in trans, rather than by specific structural variants, acting in cis. Among the DEGs we found a large percentage of genes present in multiple copies. Differential expression of different “subsets” of multi copy genes can generate large changes in environmental sensitivity. The variable transcript accumulation of duplicated genes allows a vast diversity of metabolites to be generated, providing the opportunity for tuning fruit quality attributes by differential gene expression, to fulfill different requirements. The fact that not all annotated genes with the same functions are DEGs, indicates a possible different/essential role for these genes in response to environment.The cultivation of the three genotypes in different environments also led to a severe reprogramming of the fruit metabolite profile. Similar to what observed for transcriptional responses, also metabolic responses to different environments were highly genotype-specific. Metabolite composition is a key factor of fruit quality both with respect to flavor and nutritional quality, determining the palatability of the fruit.
Among all metabolites, amino acids, sugars and secondary metabolites proved to be more responsive to the environmental change. Free amino acids may play the role of taste enhancement since the concentration levels of these molecules may significantly affect tomato flavor. The major changes in SM in the level of glutamic acid together with glutamine, aspartic acid and γ-aminobutyrric acid between the two environments suggest a strong alteration in fruit taste. Glutamic acid, as well as other amino acids affecting the tomato taste, displayed higher concentration in SM fruits harvested in Sarno than in Acerra. Among sugars, raffinose, a sucrose derivate involved in cell wall component biosynthesis, was abundant in Acerra in all three genotypes. Raffinose is metabolized in sink tissues, such as fruit, to release sucrose used for cellulose and cell wall biosynthesis. Remodeling of cell wall metabolism could be related to the difference in texture observed between the two locations. The increase in sucrose observed in Acerra in H and RSV is related to the decrease in expression of β-fructofuranosidase insoluble isoenzyme 2 that is involved in the conversion of alpha-Dglucose and D-fructose. Changes in the levels of xanthophylls and carotenes between environments in there genotypes require a considerable modulation of the “carotenoid pathway”. Carotenoids are important not only for fruit color but also for flavor due to the fact that lycopene and β- carotene are the precursors of many important volatile compounds affecting aroma perception. Flavonoids and phenolic compounds also contribute to the determination of aroma, fragrance, and color. The increased abundance in Sarno of quercetins, kaempferols, naringenins and other phenolic compounds, particularly pronounced in SM, is due an enhancement of the flavonoids and phenylpropanoid pathway related to changes in texture attributes observed between the two fields. In Sarno SM also showed a relevant abundance of glycolakaloids, especially of tomatine and their modified forms, that is correlated with enhanced bitter flavor exhibited in such locations.SM showed an opposite gene expression trend compared to H and RSV, with fewer up-regulated genes in Sarno than in Acerra. By contrast, the accumulation of metabolites showed a completely different pattern with a higher number of abundant metabolites in Sarno than in Acerra. A possible explanation of these contrasting transcriptomic and metabolomic patterns could be related to a specific adaptation of this variety to a given environment. SM is well adapted to environmental conditions in Sarno, thereby expressing its maximum potential in the environment in question. SM genome showed an enhancement of transcriptional and post-translational events and the presence of a large number of DEIs involved in responses to environmental stimuli, in Acerra. This finding suggeststhat the SM genome requires greater adjustment to respond to new environmental stimuli. By contrast, in Sarno, the niche of origin for the SM ecotype, a low induction of transcription and post-translational events are required. In addition, various metabolic sensing and feedback mechanisms could be able to mediate appropriate responses in crucial cellular processes, such as cell signaling, chromatin structure/function and ultimately gene expression.To study the complexity of the concerted action of genes, metabolites and sensorial attributes in a broad perspective we analyzed in depth the framework of relationships established. The relational networks generated showed that several hubs maintained the same interactions in both environments while some edges are gained or lost by one environment to another. In order to better adapt to changing environments, gene expression led to gain or lose interactions and/or change in position in the network underlining different genome response. The gain of an edge between two nodes could be related to the appearance of new functionality. The loss of an edge can result in functional divergence, as when duplicated gene copies lose a subset of interactions. For instance, duplicated genes showing relationships present only in one environment underline the possibility that these genes acquired new functionalities in different environments or suggest a possible sub-functionalization of some duplicated genes. Gene duplication combined with linkage rewiring provides a fundamental engine for diversification of network nodes and edges, though we do not know yet what drives this engine and how the engine works. Indeed, about 50% of genes included in the networks are duplicated with a different degree among genotypes, including central hubs. Buffering and release of variation is a widespread phenomenon that is caused by incomplete functional redundancy at multiple levels. Unraveling the dynamics, mechanisms, and causes of gene architecture reorganization after duplication is a difficult task.