Tomato fruit synthesizes flavor and nutrients during its ripening, and ripening process is regulated by ripening-related TFs, such as AP2a and Rin TFs, so these TFs may also participate in the regulation of flavor synthesis. The majority of the amino acids were present at significantly lower levels in the AP2i fruits than in the wild type, and the most dramatic reductions were in β-Ala, Ile, Met, Phe, and Trp . Rin protein can target the promoter of TomloxC and ADH2 genes, which encode lipoxygenase and alcohol dehydrogenase, respectively, and are critical for the production of characteristic tomato aromas derived from LOX pathway . Phenylalanine is an important precursor of many aroma volatiles and flavonoids. For example, 2-phenylacetaldehyde and 2-phenylethanol are derived from phenylalanine ; both of these have pleasant fruity, floral odors, and important biological functions in plants . They attract mammals and other seed dispersers and exhibit antimicrobial properties . RNA-seq result show that two previously unreported genes , that encode prephenate dehydratase proteins, were significantly upregulated in TDR4-silenced fruit . These are probably involved in the first step of the sub-pathway that synthesizes L-phenylalanine or L-tyrosine, respectively, from L-arogenate.

A gene encoding a shikimate dehydrogenase appears to be downregulated in TDR4-silenced fruits , vertical farm tower which may contribute to a reduction in shikimate, an important precursor of L-phenylalanine and L-tyrosine. Benzoic acid, synthesized from trans-cinnamate, is hypothesized to be the functional group in salicylic acid, and its derivatives are assumed to be involved in inducing stress tolerance in plants . In our study, the content of benzoic acid increased  in TDR4– silenced fruit, along with the expression of two phenylalanine ammonia-lyase genes. PALs are key enzymes in plant metabolism, catalyzing the first step of the sub-pathway that synthesizes trans-cinnamate from L-phenylalanine. One of the two upregulated genes is PAL5 , which is strongly expressed in old leaves and flowers and may function in response to biotic and abiotic stresses . Our findings suggest that the TDR4 gene negatively regulates the expression of PALs to inhibit the synthesis of benzoic acid in tomato fruit. Glutamic acid is the most abundant amino acid in the diet, and a high level of free glutamate in some foods results in an umami taste . During tomato ripening, the glutamic acid content rises dramatically . In our study,the expression levels of five related genes were significantly different in TDR4-silenced and control tomato fruit, and four of these were upregulated, including glutamate dehydrogenase , glutamine synthetase , glutamate decarboxylase 1 , and a gene encoding isocitrate dehydrogenase .

GDH1, which acts in the mitochondria, catalyzes the reversible amination of 2-oxoglutarate to L-glutamic acid . GS is a chloroplast glutamine synthetase that assimilates ammonia into glutamine , which is a metabolic intermediate in the synthesis of other nitrogen-containing compounds in plants . GAD3 converts L-glutamic acid to γ-aminobutanoic acid . The increased transcript abundances of these genes indicate the acceleration of glutamate metabolism in TDR4-silenced fruit. Glutathione is a tripeptide that exists in a broad range of organisms, from bacteria to humans . In humans, GSH plays an important role in the metabolism and detoxification of cytotoxic and carcinogenic compounds and reactive oxygen species . In plants, GSH is crucial for plant development and the plant response to the abiotic and biotic environment, and it is also involved in the detoxification of xenobiotics . In our analysis of tomato fruit, TDR4 silencing resulted in a significant reduction in glutathione . KEGG pathway analysis showed that the GST gene, encoding glutathione S-transferase, was upregulated, which would promote the conversion of glutathione to glutamate. Eriodictyol chalcone is a type of flavonoid. It has been reported that eriodictyol chalcone accumulates predominantly in the tomato peel and exhibits the highest accumulation at the breaker stage, gradually decreasing during ripening .

Tomato SlAN11 regulates flavonoid biosynthesis . In our study, the content of eriodictyol chalcone was significantly increased in TDR4-silenced fruit , compared to that in controls. KEGG pathway analysis showed that flavonoid biosynthesis was altered in TDR4– silenced fruit, which was consistent with the observation that chalcone synthase 1 and chalcone synthase 2 were upregulated .5-Caffeoylquinic acid is one of the most abundant and widespread soluble phenolics among vascular plants . Evidence suggests that it can protect plant cells against oxidative stress, and plays a role in resistance to phytopathogens . The existence of another route involving the direct 30 -hydroxylation of p-coumaryol quinic acid was first suggested in carrot cell cultures, and studies of the impact of the expression of the hydroxycinnamoyl quinic acid gene in tobacco and tomato plants demonstrated that this route may be predominant in the Solanaceae family . Analysis of TDR4-silenced tomato fruit revealed a significant reduction in 5-caffeoylquinic acid levels . α-Tomatine is an anti-nutritional factor for humans . In tomato, α-tomatine is present at high concentrations during the mature green stage and dramatically decreases during fruit ripening . In the present study, levels of α-tomatine were significantly elevated in TDR4-silenced fruit .

At the same time, the expression level of GAME11 was increased in TDR4-silenced fruit . It was reported that using VIGS technology to silence GAME11a putative dioxygenase in the cluster resulted in a significant reduction in α-tomatine levels and accumulation of several cholestanol-type steroidal saponins in tomato leaves , which was consistent with our results. Putatively, GAME11 catalyzes the closure of the E-ring of 22,26-dihydroxycholesterol to form the furostanol-type aglycone . In summary, the silencing of TDR4 promotes α-tomatine biosynthesis by enhancing the expression of GAME11 and TDR4 is a negative regulator of GAM11 gene.The aim of this study was to determine the subcellular distribution of ADP-Glc pyrophosphorylase in the starch-accumulating tissues of developing tomato fruit. The activity of this enzyme is likely to be an important factor in determining the starch content of the tomato fruit . The high starch content of fruit of a line of tomato developed from a cross between L. hirsutum and a low starch, commercial line of tomato was shown to be associated with a considerably higher AGPase activity than the parental tomato, and the possession of an L. hirsutumderived allele encoding the large subunit of AGPase. AGPase is confined to the plastid in many organs , but there are plastidial and extra-plastidial isoforms in the developing endosperm of maize, barley, and other cereals . Chen et al. have proposed, on the basis of immunogold labeling experiments, that plastidial and cytosolic isoforms of AGPase also occur in the columella and placenta of young tomato fruit. The existence of cytosolic as well as plastidial AGPase would have important consequences for the regulation of starch synthesis in the fruit. However, we have discovered that the ratio of ADP-Glc to UDPGlc in the developing fruit is very low—a feature that is consistent with a primarily or exclusively plastidial location for AGPase activity . To resolve these apparently contradictory findings we prepared plastids from the starch-accumulating tissues of developing fruit and used these to gain direct, quantitative information about the subcellular location of enzyme activity and protein.To select an appropriate stage of fruit development for preparation of plastids we initially studied changes in starch content and AGPase activity through development. The starch content of the fruit increased on a fresh weight basis between 8 and about 15 d post-pollination and declined thereafter, vertical plant tower reaching undetectably low levels at maturity . Measurements on separate samples of pericarp and of columella plus placenta revealed net rates of starch accumulation between 4 and 12 DPP of 4.4 and 7.4 nmol Glc equivalents min2 1 g2 1 fresh weight for the pericarp and the columella, respectively . For fruits of between 8 and 11 DPP, activity of AGPase in the pericarp and the columella was 100 and 220 nmol min2 1 g2 1 fresh weight, respectively . Activity then fell and was below the level of detection at about 30 DPP in pericarp and columella .Plastids were prepared from columella and from pericarp during the linear phase of starch accumula-tion. A homogenate made by chopping the tissues in the presence of 0.5 m sorbitol was centrifuged to yield supernatant and plastid-enriched pellet fractions. The following is evidence that the preparations were suitable for determining enzyme localization. First, for AGPase and for the plastidial and cytosolic marker enzymes , the sum of the activities in the supernatant and pellet fractions was between 91% and 113% of the activity in the original homogenate . Thus, there was no serious loss of enzyme activity during preparation of the fractions. Second, the yield and purity of plastids indicated by the proportion of activity of plastidial and cytosolic marker enzymes in the pellet was adequate to allow robust interpretations of data from these preparations.

An approximate 10% and 17% of the total activity of the plastidial marker enzymes was recovered in the pellet fractions from columella and pericarp tissues, respectively . For both tissues, less than 0.5% of the total activity of cytosolic marker enzymes was recovered in the pellet. This value is much lower than, and significantly different from that of the plastidial marker enzymes, indicating that the level of contamination of plastids by cytosol was low. To determine the subcellular distribution of AGPase activity we compared the proportion of the total AGPase activity recovered in the pellet with that of the plastidial and cytosolic marker enzymes. An approximate 10% and 19% of the AGPase activity in the columella and pericarp tissues, respectively, was recovered in the pellet . There is no significant difference between these values and those for the plastid marker enzymes . The values are strikingly different from those for cytosolic marker enzymes. This indicates that most or all of the AGPase activity is associated with plastids. The association of AGPase activity with the pellet fraction does not necessarily mean that the activity was contained within the plastid. AGPase in the pellet could, for example, be attached to the surface of some component of the pellet. To discover whether AGPase activity in the pellet fraction was actually inside a membrane-bound organelle, the latency of AGPase was compared with the latency of a plastidial marker enzyme, alkaline pyrophosphatase, and a cytosolic marker enzyme, alcohol dehydrogenase. In these experiments, the activity of an enzyme is compared in assays containing intact organelles or organelles that have been deliberately lysed. An enzyme within an organelle will not be accessible to substrates in the assay, and thus its activity will be lower in the assay with intact organelles than in the assay with lysed organelles. The difference in activity between the intact and lysed samples is expressed as a percentage of the activity in the lysed assay and is described as the latency of the enzyme. As expected, the plastidial marker enzyme was substantially latent , and the cytosolic marker enzyme was not . The latency values for AGPase were essentially the same as those for the plastidial marker enzyme . This indicates strongly that most or all of the AGPase activity in the pellet is contained within plastids, rather than simply attached to the surface of a component of the pellet. Further evidence about the location of AGPase activity in the pellet fractions was provided by experiments in which samples of homogenate were treated with Triton X-100 prior to centrifugation. This detergent lyses the plastids, releasing plastidial enzymes so that they no longer appear in the pellet after centrifugation. As expected, treatment with Triton reduced the yield of plastidial marker enzymes in the pellet from the usual value of about 17% to only 1% to 5%. The yield of AGPase in the pellet was reduced by Triton treatment in exactly the same way . This result is again consistent with the idea that most or all of the AGPase activity in the pellet fraction is contained inside plastids.The above results indicate that most or all of the AGPase activity is plastidial in the starch-storing tissues of tomato fruit. The difference between these results and those of Chen et al. , who suggested from immunogold labeling experiments that there is a cytosolic form of the enzyme in tomato fruit, might be explained if the cytosol contains AGPase protein, which is inactive in our assays. Therefore, we investigated the distribution of AGPase protein in the homogenate, pellet, and supernatant fractions from columella and pericarp by immunoblotting with an antiserum raised against the major small subunit of AGPase from maize endosperm, and with an antiserum raised against AGPase from spinach leaf.