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Introduction
Due to the commercial and nutritional importance of tomato (Solanum lycopersicum) around the world, extensive research has been carried out to extend its shelf life and counteract postharvest losses for several years, finding solutions only to certain problems. With the advances in genetic engineering techniques, effective procedures have been developed to improve their properties during postharvest life and extend their shelf life by slowing their ripening rate (Isack and Lyimo, 2015; Asrey et al., 2021).
Among the chemical treatments applied to fruits intended to extend postharvest life is oxalic acid, which is an organic acid widely distributed in plants. Oxalic acid plays an important role in stress response and redox homeostasis in plant as well as exerts antisenescence effects. The presence of oxalic acid increases the activity of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase among others, and promotes the accumulation of antioxidants such as flavonoids, total phenolic compounds and carotenoids. Its functions include acting as an acid catalyst, metal ion chelator, as well as an electron donor (Shimada et al., 1997). It has been shown to delay the ripening process in fruits such as mango (Zheng et al., 2007a; Razzaq et al., 2015), banana (Huang et al., 2013), tomato (Kant et al., 2013; Li et al., 2016), pear (Tarabih, 2014), peach (Zheng et al., 2007b; Razavi et al., 2017), sweet cherry (Valero et al., 2011) and plum (Wu et al., 2011). These authors used oxalic acid solutions at milimolar concentrations, reporting favorable effects on the preservation of fruit attributes.
An unexplored field is the application of oxalic acid on GM fruits, and its putative effect on their postharvest physiology. There have been work about fruit softening such as: pectinmethyl esterase (PME) and polygalacturonase (PG) silencing (Pear et al., 1993), β-Galactosidase silencing (Smith et al., 2002), TomLoxA and B gene silencing (Griffiths et al., 1999; Bo et al., 2008; Hu et al., 2014).
León-García et al. (2017) silenced the TomLoxB gene in tomato (Solanum lycopersicum) fruits variety TA234, that resulted in increased firmness, reduced lipoxygenase activity and extended shelf life compared to control fruits. Studies have been conducted to learn about the physiology of the modified tomatoes (Velázquez-López et al., 2020; Mazón-Abarca et al., 2022a; Mazón-Abarca et al., 2022b); however, more work remains to be pursued. The objective of this study was to evaluate the response of tomato (Solanum lycopersicum) variety TA234 with silenced TomLoxB gene after the application of oxalic acid during ripening. Overall physiology, lipoxygenase activity, electrolyte leakage and total phenolics content were monitored.
Materials and methods
Plant material
In a prior investigation, León-García et al. (2017) conducted a study focused on the production of transgenic tomato plants with antisense constructs targeting tomato lipoxygenase (TomloxB) to carry out the genetic modification. The researchers employed Rhizobium radiobacter strain LBA4404 (previously known as Agrobacterium tumefaciens), containing the binary vector pCAMBIA 2301, which carried the CaMV 35S promoter. To effectively monitor and assess the success of the transformation, two specific genes were employed: neomycin phosphotransferase (NPTII) as a selection marker, and β-glucuronidase (UIDA) as a reporter gene. The plasmid used in the study contained the TomloxB sequence, which was 2745 base pairs long. This specific sequence, with its corresponding GenBank accession number U09025, was inserted into the plasmid in the antisense direction. Genetically modified (GM) tomatoes from the F1 lineage and unmodified or wild-type (WT) fruits from the same TA234 variety were included as a basis of comparison. Located at the Instituto Tecnológico de Veracruz , we find the postharvest handling laboratory, in whose greenhouse both, wild type and transgenic tomato fruits, were planted, grown and harvested. Four hundred and ninety-five tomatoes weighing between 18 and 20 g, at break maturity stage with no apparent skin damage were chosen. Subsequently, they were washed by immersion in a 500 mg/L sodium hypochlorite solution for 2 m and placed on a grid to remove surface water.
Chemical preparation and treatments
For the preparation of the oxalic acid (OA) solutions (Sigma-Aldrich®, Merck, St. Louis, MO) applied to tomatoes selecting different concentrations according to previous studies (Kant et al., 2013; Li et al., 2016); two concentrations were selected: 3 and 10 mM to carry out the study. Tomatoes were immersed in the respective solutions for 10 min at 25 °C. After treatment, they were placed in a plastic container for 3 h to remove the solution from the surface and subsequently stored at 25 °C with a relative humidity of 65-70 %.
Postharvest monitoring
Weight loss
Tomato weight was measured using an electronic balance (Sartorius, model BL 2100, Germany). Percent weight loss over time was reported.
External color
With the use of a Hunter lab colorimeter (model 4500L, Reston, VA), Lighness and Hue angle were measured at three different spots on the equatorial zone of the fruit (Kalantari et al., 2015).
Firmness
The force needed to puncture the tomato fruit was calculated in three equatorial spots with a fruit texture analyzer (model GS25, Geneq Inc., Montreal, Canada) fitted with an 8 mm diameter stainless steel cylindrical probe.
Lipoxygenase activity
Lipoxygenase (LOX) activity was assayed according to Velázquez-López et al. (2020), using linoleic acid as substrate. Tomato juice was extracted and centrifuged for 5 min at 8765xg in an Eppendorf centrifuge model 5415R (Hamburg, Germany). The supernatant was filtered with a 0.45 µm pore diameter Millipore membrane disc filters. Forty µL of the sample were taken, mixed with 1360 µL of 0.2 M sodium phosphate buffer and 2.64 mL of substrate consisting of 42 µL of linoleic acid (Sigma-Aldrich ®), 39 µL of Tween 20 (Sigma-Aldrich ®) and 250 µL of 1 N NAOH (Golden Bell®); subsequently the volume was made up to 100 mL with sodium phosphate buffer and kept at 37 ºC for 10 m. To stop the reaction, the samples were cooled in ice water. A UV-Vis spectrophotometer with diode array (Agilent Technologies®, model 8453, Waldbronn, Germany) was used to measure the absorbance at 234 nm.
Electrolyte leakage
Electrolyte leakage was estimated according to the method of Li et al. (2016) with some changes. Two 6-mm diameter discs per tomato were removed with a scalpel from the equatorial and opposite region of the fruit. Five tomatoes were used for each treatment. Fruits were then immersed in 40 mL of distilled water and incubated for 3.5 h at 25 ºC. Electrical conductivity was measured using a benchtop conductivity meter (Thermo Scientific Orion 1114000-WA, 3star, Madrid, Spain), then the samples were placed on a stirring hot plate (Corning PC-420D, Somerville, MA) for 20 m at 230 ºC and incubated for 3.5 h at 25 ºC. Electrical conductivity was measured after the incubation time was completed. The percentage of total electrolyte leakage was defined as the ratio between the initial and final conductivity, multiplied by 100.
Total phenolics content
Total phenolics content was analyzed according to the method reported by Domínguez et al. (2016): 200 µL filtered tomato juice was blended with 1 mL of Folin-Ciocalteau reagent (Sigma-Aldrich®), shaken vigorously and after 3 min, 1.5 mL of 7 % sodium carbonate (J.T. Baker, Xalostoc, Mexico) and 800 µL of Milli-Q water were added, then incubated for 1 h at 25 °C. The absorbance was measured at 765 nm using a UV-Vis diode array spectrophotometer (Agilent Technologies, model 8453, Waldbronn, Germany). Total phenolics were reported as mg gallic acid equivalent (GAE) per 100 g FW (Fresh Weight). Based on a calibration curve with different concentrations of gallic acid and performed in duplicate, with a R2 of 0.9997.
Experimental design and statistical analysis
A completely randomized design (CRD) was used. Analysis of variance (one-way ANOVA) and Tukey’s rank test (p < 0.05) were employed to investigate the difference between treatments. The analyses were computed in Minitab v. 18 (LLC, State College, PA). All treatments were made in triplicate.
Results
Weight loss
The data obtained for weight loss were as follows: GM-3mM tomatoes showed a significantly (p < 0.05) smaller weight loss of 8.51 ± 0.30 % compared to the rest of the treated tomatoes during the entire 30-days storage period (Figure 1). WT-C tomatoes showed the highest weight loss (19.57 ± 0.39 %) during the same period. The 3 mM concentration allowed lower weight losses than the 10 mM treatment. Likewise, GM fruits were superior in response to wild-type fruits.
Lightness
Lightness in GM-3 mM tomatoes, recorded a significantly lower decrease of 45.07 ± 0.30 L* (p < 0.05), compared to GM-C treatments with 42.92 ± 0.83 L* and GM-10 mM with 38.96 ± 0.59 L*. WT-3 mM tomatoes recorded values of 40.47 ± 0.68 L*, being significantly higher (p < 0.05) than WT-C tomatoes with 36.51 ± 0.53 L* and WT-10 mM tomatoes with 37.61 ± 0.31 L* at 15 days of storage; with GM-3 mM tomatoes having the highest values, among all treatments (Figure 2).
Hue angle
The Hue angle value for the tomatoes analyzed at the break stage was 88.76 ± 1.90 °; when they reached the red color, an indication of fruit ripening; their values were 41.32 ± 1.68 hº. All the treated tomatoes gradually decreased the Hue angle values throughout the storage period. GM-3 mM tomatoes showed a significantly (p < 0.05) lower decrease in Hue angle with a value of 43 ± 1.38 °, compared to GM-C tomatoes with 39 ± 1.53 ° and GM-10 mM tomatoes with 37 ± 1.39 °, even to WT-3 mM tomatoes with 37 ± 1.38 °, and to both WT-10 mM and WT-C tomatoes with a value of 35 ± 1.99 ° on day 24 of storage (Figure 3).
Firmness
A decrease in firmness was observed during storage for both types of tomatoes (Figure 4). No significant differences (p < 0.05) in firmness were found between GM-C (6.3 ± 0.25 N) and GM-3 mM (6.7 ± 0.24 N) tomatoes in the first 6 days. From day 30, the firmness of GM-3 mM (3.9 ± 0.23 N) tomatoes maintained higher values than the rest of the treatments, especially WT-C (2.9 ± 0.17 N) tomatoes at day 30 (Figure 4).
LOX activity
WT-C tomatoes showed a maximum peak of LOX activity with 1433 ± 57.1 U/mg protein on day 12, whereas WT-3 mM tomatoes recorded the maximum activity of 1254 ± 38.72 U/mg protein on day 18. Conversely, both GM-C tomatoes with an activity of 1122 ± 32.83 U/mg protein and GM-3 mM tomatoes with 935 ± 36.37 U/mg protein showed the highest peak until day 24; the latter having significantly lower LOX activity (p < 0.05) among all treatments (Figure 5).
Electrolyte leakage
Electrolyte leakage gradually increased during ripening in storage. Transgenic fruits manifested a better response measured in lower electrolyte leakage than wild-type fruits. GM-3 mM tomatoes showed a significantly lower electrolyte leakage rate of 62.48 ± 3.30 % (p < 0.05) with respect to WT-C tomatoes with 92 ± 3.39 % at day 30 of storage. In turn, GM-C tomatoes recorded an electrolyte leakage of 69 ± 3.80 % and for WT-3 mM tomatoes it was 77 ± 3.77 %, finding no significant differences (Figure 6).
Total phenolics content
Regarding the total phenolics content in both, transgenic and wild-type fruits, this value increased during the entire storage time. By means of a two-way ANOVA, it was determined that time had a significant effect on phenolics content, as well as the different factors (fruits:WT or GM and oxalic acid concentration: 0, 3 and 10 mM) over time (p < 0.05).
Specifically in GM-3 mM tomatoes, it was significantly higher (p < 0.05) with a value of 11.76 ± 0.13 mg GAE/100 g FW, compared to WT-C tomatoes with 9.85 ± 0.14 mg GAE/100 g FW, to WT-3 mM tomatoes with 10.5 ± 0.18 mg GAE/100 g FW, to GM-C tomatoes with 11.0 ± 0.18 mg GAE/100 g FW at day 30 of storage. (Figure 7).
Discussion
The data show a positive effect during the ripening of GM fruits and the application of OA. By analyzing the mechanism of action of each of the factors (oxalic acid and silencing of TomloxB gene) we could argue that there was an additive effect, but also a synergistic effect. It is an additive effect, since both promote a different mechanism by which senescence in the fruit is delayed. Silencing of the TomloxB gene delays the metabolic and biochemical processes that lead to senescence. Furthermore, OA promotes the accumulation of antioxidant molecules and enzymes (ascorbate peroxidase, catalase, glutation reductase and others) which help to neutralize free radicals generated during the decompartmentalization of the cell membrane, maintaining textural quality and lowering softening during storage, and therefore contributes to the delay in the processes of oxidative deterioration in this membrane, preserving its integrity for a longer time and thus contribute to the delay of senescence (Hasan et al., 2023; Ali et al., 2021; Wu et al., 2011; Wang et al., 2009).
On the other hand, it could also be assumed that there was a synergistic effect in the fruits modified with the silencing of the TomloxB gene and the application of OA. There is a positive regulation between the TomloxB enzyme and ethylene, by decreasing the activity of the enzyme, attributed to the effect of silencing; ethylene production decreased due to a reduction in the activity of the enzyme ACC Oxidase (1-aminocyclopropane-1-carboxylic acid oxidase) at the Break stage of ripening (Velazquez-López et al., 2019), an enzyme required for the last step of ethylene production from methionine. In addition to this, OA also affects ethylene production by decreasing the activity of a key enzyme in the production pathway of this hormone from methionine, ACC synthase (1-aminocyclopropane-1-carboxylic acid synthase), and since ethylene is the key hormone in ripening, this process is also affected (Wu et al., 2011; Wang et al., 2009).
The treatments (WT and GM) with application of OA at a concentration of 3 mM showed the best results compared to their respective controls. Weight loss was lower, due to a mesocarp wall strengthening and a softening slowdown, caused by the formation of oxalate-pectin complex as a result of the application of OA (Martínez-Esplá et al., 2019). In this experiment, a smaller weight loss of 8.5 % was obtained in GM fruits, with 3 mM at day 30. Here the synergistic effect is shown, between the afore mentioned mechanisms of action of OA and the silencing of TomLoxB gene, which decreased the production and expression of the same enzyme, preventing the fatty acids that constitute the membrane from being oxidized by the enzyme (Velázquez-López et al., 2020; León-García et al., 2017). This also contributed to maintaining lightness (L*) longer in GM-C (18 %) and GM-3mM (23 %) tomatoes at day 15 and GM-C (10 %) and GM-3mM (12 %) at day 30, compared to WT-C control fruits (36 L*, day 15; 34 L*, day 30). The same trend was observed for color, measured as Hue angle, where a gradual decrease was recorded for GM-C, GM-3 mM and WT-3 mM tomatoes. Therefore, we can infer that the slow decrease in Hue angle may be attributed to a reduction in ethylene production, which promotes carotenoid biosynthesis (Sun et al., 2020). In this case, the decrease in ethylene production is a result of the synergy between OA and TomLoxB gene silencing.
GM fruits had higher firmness than WT fruits, and the GM-3 mM treatment had the highest firmness retention. The application of OA decreased the fruit softening process by the formation of an oxalate-pectin complex that strengthens the mesocarp wall, conferring greater firmness to the fruit (Martínez-Esplá et al., 2019). Kant et al. (2013) showed that tomatoes treated with 3 mM OA recorded an improvement in tomato firmness for up to 21 d compared to the control that lasted 15 d, attributing this fact to a low solubilization of pectin, which in turn improved water holding capacity and decreased cell wall-degrading enzymes activity. While the effect of OA on WT tomato has shown to have an impact on cell wall degrading enzymes, for GM tomato the antisense insertion of the TomLoxB gene confers an additional effect, which bestows integrity to the membrane by decreasing the expression of lipoxygenase. This maintains intact the fatty acids of the membrane longer, thus preserving its integrity (León-García et al., 2017).
The 3 mM OA concentration (WT-3 mM and GM 3 mM) had a delaying effect on LOX activity and therefore the fruits showed a delay in senescence. Ding et al. (2007) also showed low LOX activity in mangoes treated with OA for up to 30 days of storage, attributing this effect to the ability of OA to inhibit peroxide accumulation. Therefore, the decrease in LOX activity in tomato is attributed to the action of OA, which induced resistance to the peroxidation of fatty acids present in the membrane and, together with the low expression of lipoxygenase in the tomato modified by the insertion of the TomLoxB gene in antisense, decreased the accumulation of hydroperoxides responsible for causing damage to the membrane and thus preserve its integrity, thereby extending its shelf life.
It is reported that OA can increase the activity of antioxidant enzymes such as catalase, ascorbate peroxidase, superoxide dismutase, among others. Application of 3 mM OA in tomato was effective in reducing electrolyte leakage in WT (61 %) and GM (52 %) fruits on day 21, compared to their WT-C (79 %) and GM-C (59 %) controls. At the same concentration, Kant et al. (2013) reported values of 45.66 % at 21 d of storage, while for the control fruit was 71.66 %. Similar results were obtained by Li et al. (2016), who observed that electrolyte leakage in tomatoes treated with OA was significantly smaller by 20 % compared to the control fruits, after been stored between 20 and 32 d. It is reported that OA can increase the activity of antioxidant enzymes, which act on free radicals released during the ripening and softening process of the fruit, relaxing membrane permeability. Electrolyte loss reflects membrane permeability and, indirectly, the effect of oxalic acid application (Nyanjage et al., 1999; Zheng et al., 2007c; Razzavi et al., 2017) which together with the silencing the TomLoxB gene, decreases the expression of lipoxygenase and thus the decompartmentalization and loss of tissue structure in tomato, providing a synergistic effect to extend the postharvest life of the fruit.
OA promotes the accumulation of total phenolic compounds, which favors the protective action against reactive oxygen species (ROS) involved in membrane destabilization. Its positive effect has been proven in peaches (Razavi et al., 2017) and mangoes (Razzaq et al., 2015), where a significant trend of increased total phenolics content was observed from day 7 to day 21 of storage compared to untreated fruit. It has also been reported that with reduced ethylene levels, there is an increase in phenolic content in tomatoes (Dominguez et al., 2016). In this experiment, the 3 mM concentration of OA in GM fruit was the treatment that showed the highest results of total phenolics over time (p < 0.05).
The effects of antisenescence and antistress might be associated with ethylene signaling regulated by OA. It has been demonstrated that OA delayed the ethylene climacteric peak and exhibited 2-fold lower rates of ethylene production (Wu et al., 2011). Wang et al. (2009) reported that reduced ethylene production in OA-treated fruit might be ascribed to the reduced ACC synthase activity. By virtue of the regulation of OA on ethylene, via ACC synthase, and knowing that ethylene is an indispensable and key hormone during the ripening process, it can be assumed that the 10 mM concentration of OA strongly blocked ACCS enzyme production and thus ethylene synthesis, and because of the above, the ripening processes were affected.
In turn, the 3 mM concentration was adequate to partially inhibit the activity of the ACC synthase enzyme, still allowing the production of the hormone, which is necessary to trigger those metabolic and biochemical processes involved in ripening.
Conclusions
Of the two concentrations of OA applied to WT and GM tomatoes, the 3 mM OA solution, was the one that showed more favorable effects on the physiology of GM tomatoes, producing greater lightness retention, delay in the appearance of red color, greater firmness and total phenolics content, lower weight loss, lipoxygenase activity and electrolyte leakage. All of these resulted in an extension of shelf life of 30 d. In turn, the untreated GM tomatoes showed an appropriate appearance up to day 24 of storage. However, increasing the concentration to 10 mM showed adverse effects on the physiology of both GM and WT tomatoes, that resulted in irregularities in the development of treated fruit such as premature shriveling and uneven coloration, with a shelf life of only 18 d, while the WT control tomatoes had a postharvest shelf life of 15 d.
