*3.2. Primary Metabolite Profiling Affected by Post-Harvest Hormone Treatment*

Primary metabolites are important components related to fruit quality. Additionally, they are considered crucial for plant growth and development. Thus, understanding the fruit metabolism can support developing future approaches for its manipulation [24]. In this work, a total of 46 primary metabolites were identified by GC-MS metabolomics analysis: 10 sugars (glucose, fructose, sucrose, allose, gulose, glucaric acid, myo-inositol, mannose, ribose, and arabinofuranose); 9 organic acids (oxaloacetic, citric, succinic, aconitic, malic, citraconic, fumaric, propanoic, and butanoic acids); 12 amino acids (proline, serine, valine, threonine, aspartic acid, glutamic acid, glutamine, γ-aminobutyric acid (GABA), asparagine, tryptophan, phenylalanine, and tyrosine); 12 saturated fatty acids (capric, lauric, myristic, palmitic, stearic, eicosanoic, docosanoic, tricosanoic, lignoceric, hyenic, cerotic, and montanic acids); 3 unsaturated fatty acids (oleic, linoleic, and linolenic acids) at 4, 10, and 21 DAH (Table 1). Table 1 shows the effects of methyl jasmonate and 1 methylcyclopropene on the accumulation or reduction of each metabolite at the three different maturation stages, indicated by the area normalized by the internal standard.

Moreover, a global overview of the metabolic changes occurring in tomatoes during ripening was obtained to evaluate significant differences among accumulated metabolites in treated fruits compared with the control group (Figure 2).

*Foods* **2021**, *10*, 877






Values were presented as normalized area by ribitol or n-tridecane (internal polar and non-polar standards, respectively). CTRL: Control fruits. Different superscript letters (a–g) indicate statistical significance(*<sup>p</sup>* < 0.05) at the same line (mean ± standard deviation, *n* = 4). GABA, γ-aminobutyric acid.

**Figure 2.** Global overview of metabolic changes occurring in tomato pericarp (*Solanum lycopersicum* L. cv. Grape) treated with 1-methylcyclopropene (MCP) and both hormones, 1-methylcyclopropene and methyl jasmonate (MCP + MeJA), compared to the control group (CTRL). Data were normalized to the CTRL. Metabolites showing up- or down-regulation in each treatment exceeding twofold compared to the CTRL are shown. The color scale displays the different amounts of metabolite in terms of proportional change relative to the level in the appropriate control. Suc, sucrose; Glc, glucose; Frc, fructose; Man, mannose; L-Asc, L-ascorbic acid; Arab, arabinofuranose; 6FP, fructose-6-phosphate; 3-GPA, glyceraldehyde-3- phosphate; Ser, serine; PEP, phosphoenolpyruvate; Trp, tryptophan; Phe, phenylalanine; Tyr, tyrosine; HGA, homogentisic acid; Pyr, pyruvic acid; IPP, isopentenyl pyrophosphate; GGPP, geranylgeranyl; PPP, phytyl pyrophosphate; Val, valine; AcCoA, acetyl-CoA; Oxaloacet, oxaloacetic acid; Cit, citric acid; Aco, aconitic acid; α-keto, α-etoglutaric acid; Succ, succinic acid; Fum, fumaric acid; Mal, malic acid; Glu, glutamic acid; GABA, γ-aminobutyric acid; Gln, glutamine; Pro, proline; Arg, arginine; Asp, aspartic acid; Thr, threonine; Asn, asparagine; FPP, farnesyl pyrophosphate.

A PCA was performed on primary metabolites at the 4th, 10th, and 21st ripening stages, confirming the high reproducibility among the four biological replicates and groups analyzed. Moreover, clear separation of the CTRL group and both treated groups was evidenced for the primary metabolites in the PCA score. Heatmap analysis was used to analyze the differences between treated and untreated groups regarding the metabolite changes on each day after harvest (Figures 3–5).

**Figure 3.** Relative contents of sugars and organic acids in tomato pericarp (*Solanum lycopersicum* L. cv. Grape) exposed to 1-methylcyclopropene (MCP) and both 1-methylcyclopropene and methyl jasmonate (MCP+MeJA) compared to the control group (CTRL). Unsupervised principal component analysis (PCA-score) and heatmap analysis represent the major sources of variability. Color scale represents the variation in the relative concentration of compounds, from low (green) to high (red) contents at 4, 10, and 21 days after harvest (DAH).

**Figure 4.** Relative contents of amino acids in tomato pericarp (*Solanum lycopersicum* L. cv. Grape) exposed to 1-methylcyclopropene (MCP) and both hormones, 1-methylcyclopropene and methyl jasmonate (MCP+MeJA), as compared to the control group (CTRL). Unsupervised principal component analysis (PCA-score) and heatmap analysis represent the major sources of variability. Color scale represents the variation in the relative concentration of compounds, from low (green) to high (red) contents at 4, 10, and 21 days after harvest (DAH).

**Figure 5.** Relative contents of fatty acids in tomato pericarp (*Solanum lycopersicum* L. cv. Grape) exposed to 1-methylcyclopropene (MCP) and both hormones, 1-methylcyclopropene and methyl jasmonate (MCP+MeJA), as compared to the control group (CTRL). Unsupervised principal component analysis (PCA-score) and heatmap analysis represent the major sources of variability. Color scale represents the variation in the relative concentration of compounds, from low (green) to high (red) contents at 4, 10, and 21 days after harvest (DAH).

Primary metabolism is essential for fruit quality. Sugars, organic acids, and amino acids are responsible for the taste of tomato fruits, facilitating sensory perception. Amino acids and fatty acids play important roles as precursors of aroma compounds [7]. Treatment with 1-methylcyclopropene impacted sugar and organic acids, inhibiting their production during ripening. Fruits treated only with 1-methylcyclopropene were most affected, showing a greater delay in accumulating sugars and organic acids than fruits treated with both 1-methylcyclopropene and methyl jasmonate (Figure 3). For instance, glucose showed a 22-, 13-, and 23-fold reduction at 4-, 10-, and 21 DAH, respectively, in MCP, as compared to the CTRL. Mannose, ribose, and malic and aconitic acids exhibited a 14-, 30-, 21-, and 20-fold decrease in levels at 4 DAH, respectively. Conversely, fructose, sucrose, and citraconic acid showed a 12-, 15-, and 27-fold decrease in levels at 10 DAH when MCP was compared with the CTRL (Table 1). Reductions in the levels of these metabolites in fruits treated with 1-methylcyclopropene are shown in (Figure 2), based on the fold-change analysis of the treated fruits and control group.

Exceptionally, glucose, glucaric acid, and mannose levels showed an increase at 10 DAH in MCP + MeJA, as compared to the CTRL. Similar behavior was observed for myo-inositol, propanoic, and butanoic acids at 21 DAH (Table 1). As shown in Figure 3, heatmap analysis demonstrated a tendency of these metabolites to increase at 10 DAH. As observed by ethylene emission, the minor impact on the production of sugars and organic acids observed for MCP + MeJA may suggest that methyl jasmonate plays an important role in ripening. This may act independently of endogenous ethylene, stimulate the synthesis of new receptors, or reverse the blockage of ethylene receptors after some time.

Amino acid profiling was also affected by the action of 1-methylcyclopropene. Inhibition of the production of amino acids during ripening was observed for both MPC and MCP + MeJA compared with the control (Figure 4). The most affected amino acids were aspartic acid at 4 DAH and GABA at 10 DAH, showing a 28- and 10-fold reduction in their levels with MCP, respectively. However, MCP + MeJA showed 11- and 14-fold decreases, respectively, as compared to the CTRL, as shown in (Figure 2). Conversely, tyrosine and phenylalanine showed levels two- and ninefold higher for MCP and MCP + MeJA at 4 DAH, as compared to the CTRL (Table 1, Figure 2). It is important to highlight that phenylalanine and tyrosine are aromatic amino acids, which participate in the shikimate pathway and are responsible for the aroma development of fruit. Table 1 shows that the total amino acid level was represented mostly by proline, glutamic, and aspartic acids, which are important to fruit quality, as they provide sweetness and umami flavor.

Additionally, fatty acid profiling was also affected by the post-harvest treatments. The action of 1-methylcyclopropene showed a greater impact on fatty acids such as oleic, capric, lauric, palmitic, stearic, and myristic acids at 10 DAH, as shown in (Figure 5), decreasing their levels by 17-, 10-, 14-, 17-, 14-, and 14-fold in the MPC group, respectively, and 7-, 6-, 9-, 11-, 1-, and 7-fold in the MCP + MeJA group, respectively, as compared to the CTRL (Table 1). The reduction in fatty acids by 1-methylcyclopropene was evident when the fold-change analysis was applicable (Figure 2). The MCP + MeJA group also showed a reduction in fatty acid levels; however, this was less impactful than the MCP group (Figure 5). The most impacted were the linoleic and myristic acids at 4 DAH with a reduction of 119- and 26-fold in MCP, respectively, and a 23- and 9-fold decrease in MCP + MeJA, respectively, as compared to the CTRL (Table 1).

Conversely, a tendency of increased levels of some fatty acids was also detected, as well as in lignoceric, cerotic, and α-linolenic acids at 4 DAH, and palmitic and linoleic acids at 21 DAH for the MCP and MCP + MeJA groups (Figure 5). In the MCP group, an increase was detected in the levels of lignoceric and α-linolenic acids at 4 DAH by 7- and 4-fold, respectively, while in MCP + MeJA, the increases were 28 and 3-fold, respectively. Moreover, palmitic and linoleic acids were increased by 2- and 10-fold, respectively, in MCP, and 3- and 10-fold, respectively, in MCP + MeJA at 21 DAH (Table 1). Interestingly, the MCP + MeJA group was less impacted when reductions were observed and more impacted when increases were observed compared with the MCP group. This behavior may indicate that methyl jasmonate can act as a stimulator in fatty acid production. Palmitic and eicosanoic acids contributed crucially to the total saturated fatty acid level, and oleic and linoleic

acids contributed to the total unsaturated fatty acid level, which is notable, as they play an important role in the fruit quality and nutritional value.

#### *3.3. Secondary Metabolite Profiling Affected by Post-Harvest Hormone Treatment*

The secondary metabolites identified in tomato fruits at 4, 10, and 21 DAH were lycopene, β-carotene, and lutein by HPLC analysis; α-tocopherol, β-tocopherol, γ-tocopherol, phytol, β-sitosterol, stigmasterol, and stigmastadienol were identified by GC-MS analysis.

Lycopene was the most affected by the action of 1-methylcyclopropene, reducing its level not only in MCP but also in MCP + MeJA by 29- and 25-fold, respectively, at 4 DAH. However, at 10 DAH, lycopene was reduced by eight- and sixfold, respectively, compared with the CTRL (Figure 6A); β-carotene and lutein showed a decrease less than threefold by 1-methylcyclopropene at the ripening stages (Table S1). These remarkable impacts on the synthesis of carotenoids are illustrated in (Figure 2), mainly at the onset of ripening.

However, the action of 1-methylcyclopropene had a lesser impact on lycopene accumulation at 21 days of hormone treatment, decreasing its production by 2.8-fold, and its action was completely reversed by the methyl jasmonate hormone. Fruits treated with methyl jasmonate showed an increase not only in lycopene production but also in β-carotene and lutein at 21 DAH, indicating the important role that methyl jasmonate plays in the synthesis of carotenoids (Figure 6A). Lycopene and β-carotene showed an increase of 10%, and lutein of 20%, as compared to the CTRL (Figure 6A, Table S1), which is considered relevant since these bioactive compounds have been associated with health benefits, leading to decreases in the occurrence of chronic non-communicable diseases [25]. The total carotenoid level was represented mainly by lycopene.

Tocopherol profiling showed similar behavior to carotenoids during ripening, decreasing its levels in both treated groups at 4 and 10 DAH (Figure 2). At 21 DAH, it presented a decrease, in the MCP group, and an increase, in MCP+MeJA, of tocopherols, as compared to the CTRL. The α-tocopherol levels showed a reduction in MCP and MCP + MeJA of 5- and 4-fold, respectively, at 4 DAH, while at 10 DAH, they decreased by 12 and 3-fold, respectively. The β-tocopherol levels showed a reduction of 14 and 12-fold at 4 DAH, and 23- and 9-fold at 10 DAH in MCP and MCP+MeJA, respectively. Additionally, γ-tocopherol was decreased by 6-fold at 4 and 10 DAH in both treatment groups, except for the MCP + MeJA at 10 DAH, which decreased 1.7 fold, as compared to the CTRL (Figure 6B, Table S2).

Conversely, at 21 DAH, tocopherol profiling was less affected by 1-methylcyclopropene and positively impacted by the concomitant treatment of 1-methylcyclopropene and methyl jasmonate, showing increases of 40% in the α-tocopherol and β-tocopherol levels and 21% in the γ-tocopherol levels, as compared to the CTRL (Figure 6B, Table S2). The total tocopherol level was characterized mainly by the α-tocopherol content. An acyclic diterpenoid identified was phytol, which presented a twofold reduction in MCP at 4 DAH and a twofold increase in MCP + MeJA at 10 DAH (Figure 6B, Table S2). The impact of these treatments at 4 and 10 DAH is also shown in (Figure 2).

Phytosterols were also affected by 1-methylcyclopropene, showing fivefold reductions in β-sitosterol levels in MCP at 4 and 10 DAH and threefold reductions in MCP + MeJA at 4 and 10 DAH, as compared to the CTRL. Stigmasterol exhibited four and sevenfold reductions in MCP at 4 and 10 DAH, respectively, while MCP + MeJA showed three and fivefold decreases at 4 and 10 DAH, respectively. Stigmastadienol was the phytosterol most affected by 1- methylcyclopropene, decreasing ninefold at 4 DAH (Figure 6C, Table S2); β-sitosterol and stigmasterol were the major sources of the total phytosterol level. Moreover, down-regulation exceeding twofold, as compared to the CTRL, is observed for the phytosterols in (Figure 2). Divergent behavior of phytosterols profiled at 4 and 10 DAH, β-sitosterol, stigmasterol, and stigmastadienol showed an increase in their levels by 42%, 34%, and 32%, respectively, in fruits treated with both 1-methylcyclopropene and methyl jasmonate at 21 DAH (Figure 6C, Table S2).

**Figure 6.** Secondary metabolites in tomato pericarp (*Solanum lycopersicum* L. cv. Grape) exposed to 1-methylcyclopropene (MCP) and both hormones, 1-methylcyclopropene and methyl jasmonate (MCP+MeJA), compared to the control group (CTRL) at 4, 10, and 21 days after harvest (DAH). Contents of carotenoids (**A**), normalized area of tocopherols and phytol (**B**), and phytosterols (**C**). Values are means ± SE of four biological replicates of 10 fruits each. Different letters indicate statistically significant differences (*p* < 0.05).
