Abstract
We investigated sugar (solute) accumulation in watermelon [Citrullus lanatus (Thunb.) Matsum. et Nakai] fruits at the immature stage. Watermelon plants were grown hydroponically in a nutrient solution with an electric conductivity (EC) of 1.2 S⋅m−1 (EC 1.2 regime); then, fruits were harvested 21 days after anthesis. The flesh of each fruit was divided into seven different parts to measure the sugar concentration and water status. The results indicated that the sugar concentration was higher in the center of the fruit flesh than in the other parts, such as around the pericarp. Moreover, the lowest osmotic potential was observed in the center of the fruit flesh, indicating solute accumulation. Concurrently, when the transport of photosynthates in the fruit was investigated using the 13CO2 isotope, the active solute accumulation in the center of the fruit flesh was observed, supporting the observed sugar accumulation in this part. Consequently, this active solute accumulation and distribution occurred in the center of the watermelon fruit, as demonstrated by the data of osmotic pressure and sugar concentration and supported by the observed active photosynthate accumulation. Additionally, we investigated these measurements by increasing the nutrient solution concentration 14 days after anthesis. As a result, fruit growth was slightly inhibited using the EC 3.0 regime, and 13C translocation was also inhibited in the fruit, especially in its center. Even though the sugar concentration and osmotic pressure of the fruit flesh were not clearly affected by high nutrient solution concentrations, the cell turgor of the central flesh of the fruit grown using the EC 2.0 and 3.0 regimes was lower than that of the fruit grown using the EC 1.2 regime. Treatments with higher nutrient concentrations might have negative effects on immature watermelon fruits.
Many fruits and vegetables accumulate sugars and other solutes during their growth. Watermelons, one of the attractive sweet crops, accumulate sugars in their fruits during their growth (Brown and Summers 1987; Yativ et al. 2010). It has been reported that the sugar concentration is high near the center of watermelon fruits (Fukuoka et al. 2008; Ikeda et al. 2011). In our previous study, we demonstrated that the presence of seeds was not a trigger for the accumulation of sugar in the center of the fruit (Kawamura et al. 2018). However, we did not investigate the mechanism of sugar accumulation in detail, especially the detailed distribution and deviation of sugars in the fruit. In our present study, we analyzed the sugar concentration in seven different parts of the fruit flesh to investigate the localization of sugars in the fruit. Moreover, we performed an isotope analysis to examine the translocation of photosynthates into the fruit; then, we analyzed the relationships among the sugar concentration, water status (osmotic pressure), and 13C ratio.
An isotope analysis is frequently performed to investigate the transport of photosynthates in plants (Finazzo et al. 1994; Okano et al. 1983; Yakushiji et al. 1998). In some studies, the translocation of photosynthates in Cucurbitaceae plants was investigated by an isotope analysis (Barzegar et al. 2013; Lima et al. 2020; Watanabe 2004). In this study, we applied the 13CO2 isotope to leaves of watermelon plants during fruit growth and determined the ratio of photosynthates translocated to different parts of the fruit flesh in which the sugar concentration and water status were measured and analyzed. Then, we determined the relationship between sugar accumulation and isotope translocation in the fruits. The fruits were harvested 21 d after anthesis. The cultivar Hitorijime–BonBon was used because it can be harvested 40 d after anthesis. Because the photosynthate transport may be inhibited in mature fruits, we investigated immature fruits.
One of the objectives of this study was to investigate the center of a watermelon fruit, which is considered the largest sink, using three different methods, namely, high-performance liquid chromatography (HPLC) to determine the sugar concentration, isopiestic thermocouple psychrometer to determine the water status (water potential and osmotic potential = osmotic pressure and turgor), and 13CO2 isotope analysis with a gas analyzer. Consequently, we demonstrated that the active photosynthate accumulation occurred in the center of the fruit, which was associated with the osmotic pressure difference that induced sugar accumulation.
Additionally, we determined the effects of different nutrient solution concentrations on fruit quality. Applying water stress is a well-known technique used to induce sugar accumulation in fruits of Satsuma mandarin (Yakushiji et al. 1996, 1998) and tomato (Nahar et al. 2011; Sanchez-Rodriguez et al. 2012). In watermelons, such a technique increases the amino acid (citrulline) concentration in leaves (Kawasaki et al. 2000). However, the water stress generated by the high nutrient solution concentration in hydroponics has not been frequently investigated; therefore, related measurements were also conducted at high liquid fertilizer concentrations and the results were analyzed.
Materials and Methods
Plant materials.
Watermelon [Citrullus lanatus (Thunb.) Matsum. et Nakai ‘Hitorijime–BonBon’; Hagihara Farm Co., Ltd., Nara, Japan] seedlings that were grafted to the rootstocks of bottle gourd [Lagenaria siceraria (Mol.) Stanol ‘Kachidoki 2gou’; Hagihara Farm Co., Ltd.] were used in this study. The seedlings were supplied by Hagihara Farm in May 2020, and transplanted at a distance of 50 cm in three hydroponic culture beds (258 × 110 × 15 cm; 5 plants × 2 lines were grown in each bed). A nutrient solution (Otsuka Chemical Co., Ltd., Osaka, Japan) with electric conductivity (EC) of 1.2 S⋅m−1 and a pH of 5.8 to 6.2 was maintained at a depth of 7 cm using an EC meter (Atago Co., Ltd., Tokyo, Japan) and a pH meter (9652–10D/9652–20D; Horiba Advanced Techno Co., Ltd., Kyoto, Japan). The beds were maintained in a glasshouse that was ventilated whenever the air temperature exceeded 25 °C. Plants were grown vertically (Watanabe et al. 2001). One vine per plant was trained upward, and all other vines were removed. When it had produced more than 20 nodes, one fruit was retained. Female flowers were pollinated conventionally by hand. The fruits were supported with a ball net (Molten Co., Hiroshima, Japan) on bars placed at a height of 230 cm. Fruits were harvested 21 d after pollination, and the fruit flesh was divided into six or seven different parts. We defined the parts as the upper pericarp, upper outer flesh, upper flesh, central flesh, lower flesh, lower outer flesh, and lower pericarp for all the experiments in this study (Fig. 1). For the 13C analysis, the central part was divided into the upper mid flesh and lower mid flesh.
Sampling positions in watermelon fruit for water status and sugar concentration analyses. For the 13C analysis, the central part was divided into upper mid flesh and lower mid flesh.
Citation: HortScience 58, 5; 10.21273/HORTSCI17026-22
Additionally, plants were grown under different nutrient solution concentrations at an EC of 2.0 or 3.0 S⋅m−1. Nutrient solutions under these conditions were modified from 14 d after anthesis. As the control, we maintained the same culture condition of the EC at 1.2 S⋅m−1. The measurement and analysis methods used for the control were similar.
Sugar concentration.
For analyses of the sugar concentration and composition of the fruit flesh, we used an HPLC system (EZChrom Elite; Hitachi High-Technologies Co., Tokyo, Japan) equipped with a refractive index detector (5450; Hitachi High-Technologies Co., Tokyo, Japan). We analyzed each sugar reagent and created calibration curbs; then, we determined the concentrations of each sugar in the tissue sample. The cubes (1 × 1 × 1 cm) from each part (Fig. 1) were ground with a mortar and pestle. The ground flesh was placed in a 1.5-mL microtube (Azwan Co., Ltd., Osaka, Japan) and diluted 10-fold with distilled water. After dilution, the resulting juice was centrifuged (188,845×g, 15 min, 5 °C; CF15RXII model; Hitachi Koki Co., Ltd., Tokyo, Japan) and filtered using a syringe with a filter with a 0.45-μm pore size and 13-mm diameter (Watman; Global Life Sciences Technologies Japan K.K., Tokyo, Japan). Sugars were separated using an analytical HPLC system fitted with a column (5-mm TSKgel Amide-80 column; Tosoh Co., Tokyo, Japan) kept at 70 °C. The mobile phase was composed of acetonitrile and distilled water (80:20), and the flow rate was 1.0 mL⋅min−1.
Water status.
We measured the water status of the flesh with an isopiestic psychrometer (Model–3; Isopiestic Psychrometry Ltd., Lewes, DE, USA) (Boyer 1995b). The thermocouple chambers were coated with petrolatum and loaded with tissue samples (size 1 × 1 × 0.5 cm) immediately after sampling. After the water potentials were measured, the psychrometer chambers were sealed by parafilm and frozen at −80 °C; then, they were kept at room temperature for 1 hour to thaw. The osmotic potentials of the tissue samples were measured using the psychrometer again (Boyer 1995b). Turgor was calculated as the water potential minus the osmotic potential.
13CO2 analysis.
Three leaves below the pollinated flower cluster and a 100-mL beaker were covered with a polyethylene bag. Then, 2 g of Ba213CO3 (SK–660PH; Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) in a beaker was mixed with 3.5 mL of lactic acid (Naito Shoten Co., Ltd., Aichi, Japan) generated in the bag by adding 13CO2 (1.5 mol⋅L−1), which was absorbed by the three lower leaves of the pollinated flower cluster. These three leaves are hereafter called treated leaves and stems. A total of 12 plants were analyzed by measuring 0.9 to 2.5 mg of each of the following parts: roots, lower parts of the stem and leaves, treated leaves, upper parts of the stem and leaves, and seven different parts of the fruit flesh already described. Each part of the plant was dried in an oven (MOV–212F; Sanyo Electric Co., Ltd., Osaka, Japan) at 80 °C for at least 48 h. Furthermore, the parts of the fruit samples were dried in a vacuum freeze-dryer (FDU–2200; Tokyo Rika Kikai Co., Ltd., Tokyo, Japan). Then, the dry weight of each sample was determined. Powdered samples were set in the 13CO2 analyzer (EX−130S; Japan Spectroscopy Co., Ltd., Tokyo, Japan) for analysis. The samples were completely burned in the CO2 analyzer. The resulting 12CO2 and 13CO2 were irradiated with infrared light. The amount of 13C contained in each sample was determined by using different absorption wavelengths in the infrared region. The amount of 13C assimilate distributed to each part of the plant was determined by substituting the measured values, dry matter weight, and dry matter weight of the samples quantified during analysis using the following equations; from these results, the ratio of the 13CO2 assimilate distributed to each part was calculated.
The 13C absorption of the whole plant (g) means the sum of the 13C absorptions of each organ and part. Data from experiments were subjected to a one-way analysis of variance, and the means were evaluated using Tukey’s honestly significant test (P < 0.05).
Results
Fruit growth.
The fruit weights and circumferences obtained during this experiment are shown in Fig. 2. When the plants were subjected to stress using a high nutrient solution concentration, the fruit size tended to decrease with the EC 3.0 regime, although the difference did not reach statistical significance (Fig. 2).
Fruit weights (A) and fruit circumferences (B) of watermelon at 21 d after anthesis with electrical conductivity (EC) 1.2 (blank bar), 2.0 (stripe bar), and 3.0 (solid bar) regimes. Error bars indicate the SE (n = 4). Lowercase letters indicate a significant difference at P < 0.05 by Tukey’s test. n.s. = not significant.
Citation: HortScience 58, 5; 10.21273/HORTSCI17026-22
Sugar concentration.
The sugar concentration (Fig. 3) was determined as the sum of the glucose, fructose, and sucrose concentrations in each part of the fruit flesh measured during the HPLC analysis (Tables 1 and 2). In the central flesh of the fruit grown with the EC 1.2 regime, the glucose, fructose, and sucrose concentrations were significantly higher than those in other parts of the fruit flesh (Table 1). As shown in Fig. 3, the highest sugar concentration was found in the center of the fruit. The sugar concentration gradually decreased toward the fruit skin (surface) (Fig. 3).
Sugar concentration (sum of the glucose, fructose, and sucrose concentrations) of each part of the watermelon flesh at different nutrient solution concentrations [electrical conductivity (EC) 1.2 (blank bar), 2.0 (stripe bar), and 3.0 (solid bar) regimes]. Error bars indicate the SE (n = 4). Lowercase letters indicate significant difference at P < 0.05 by Tukey’s test between the sampling positions of the control. n.s. under the X axis means not significant for each regime at different sampling positions. There are no data for the upper pericarp of the control.
Citation: HortScience 58, 5; 10.21273/HORTSCI17026-22
Sugar concentrations (glucose, fructose, and sucrose) of various parts of watermelon flesh at different nutrient solution concentrations [electrical conductivity (EC) 1.2, 2.0, and 3.0 regimes]. Values are means ± SE (n = 4). Values within lines followed by the same letter are not significantly different at P < 0.05 by Tukey’s test. There are no data for the upper pericarp of the control.
Sugar concentrations (glucose, fructose, and sucrose) of various parts of watermelon flesh at different nutrient solution concentrations [electrical conductivity (EC) 1.2, 2.0, and 3.0 regimes]. There are no data for the upper pericarp of the control.
When the plants were treated with highly concentrated nutrient solutions (EC 2.0 regime and EC 3.0 regime in (Fig. 3), the sugar concentration gradient of the fruit had the same tendency as that of the control (EC 1.2 regime). However, even though different conditions were used, the sugar concentration was not significantly different among all the parts of the fruit flesh (Fig. 3).
Water status.
When the water status for each watermelon flesh sample was measured, the lowest water potential was observed in the center of the fruit flesh (Fig. 4A, blank bars). The lowest osmotic potential was also similarly observed in the center (Fig. 4B, blank bars). Turgor tended to be high in the center of the fruit flesh (Fig. 4C, blank bars).
Water status of various parts of watermelon flesh at different nutrient solution concentrations [electrical conductivity (EC) 1.2 (blank bar), 2.0 (stripe bar), and 3.0 (solid bar) regimes]. (A) Water potential. (B) Osmotic potential. (C) Turgor. Error bars indicate the SE (n = 4). Lowercase letters indicate significant difference at P < 0.05 by Tukey’s test between the sampling positions of the control. n.s. under the X axis means not significant for each treatment at different sampling positions.
Citation: HortScience 58, 5; 10.21273/HORTSCI17026-22
Under stress (EC 2.0 and 3.0 regimes in (Fig. 4), the tendencies of the data were similar to those of the control (EC 1.2 regime; blank bars in Fig. 4). However, as the sugar concentration increased, the stress of the high nutrient solution concentration had no effect on the water status of watermelon fruits. Although the turgor was not significantly different between the regimes at each part of the flesh, it tended to increase in the center of the fruit under stress (Fig. 4C).
Isotope analysis.
When we applied Ba213CO2 to watermelon leaves and analyzed the content and ratio using a 13C analyzer, the 13C absorption was higher at the fruit rather than at other parts of the fruit (Fig. 5), and the highest rate of 13C isotope transport was observed in the fruit rather than in other parts of the plant (Fig. 6A). Within the fruit, the highest 13C isotope sharing ratio was observed near the center of the flesh (upper mid flesh and lower mid flesh in Fig. 6B). When we applied a high hydroponic culture solution concentration to the plants, the ratio of photosynthates within the fruit showed a similar behavior to that of the control (Fig. 6B). The upper outer flesh of the fruit grown with the EC 1.2 regime had a significantly higher sharing ratio than that of the fruit grown with the EC 2.0 and 3.0 regimes (Fig. 6B). Although the difference was not statistically significant, the 13C isotope sharing ratio was higher for the fruit grown with the EC 1.2 regime than for that grown with other regimes (Fig. 6B). When stress was induced by applying a high nutrient solution concentration, the 13C absorption in the fruit grown with the EC 3.0 regime was the lower than those in the fruits grown with the EC 1.2 and 2.0 regimes (Fig. 5). The 13C sharing ratio of the fruit grown with the EC 1.2 regime was higher than those of the fruits grown with the EC 2.0 and 3.0 regimes, but lower than those of the lower leaves (Fig. 6A).
13C absorption of watermelon plants (treated leaves and stem, upper leaves and stem, and lower leaves and stem mean the leaves with the Ba213CO3 application, including the internode stem, upper part of the plant including stems, and lower part of the plant including stems, respectively) at different nutrient solution concentrations [electrical conductivity (EC) 1.2 (blank bar), 2.0 (stripe bar), and 3.0 (solid bar) regimes]. Error bars indicate the SE (n = 4). Lowercase letters indicate significant difference at P < 0.05 by Tukey’s test between the sampling positions of the control. n.s. under the X axis means not significant for each treatment at different sampling positions.
Citation: HortScience 58, 5; 10.21273/HORTSCI17026-22
The 13C sharing ratio (A) of watermelon plants (treated leaves and stem, upper leaves and stem, and lower leaves and stem mean the leaves with the Ba213CO3 application, including internode stem, upper part of plant including stems, and lower part of the plant including stems, respectively) and various parts of watermelon fruit (B) at different nutrient solution concentrations [electrical conductivity (EC) 1.2 (blank bar), 2.0 (stripe bar), and 3.0 (solid bar) regimes]. Error bars indicate the SE (n = 4). Lowercase letters indicate significant difference at P < 0.05 by Tukey’s test between the sampling positions of the control. n.s. under the X axis means not significant for each treatment at different sampling positions. The asterisk under the X axis in B indicates significant difference at P < 0.05 by Tukey’s test.
Citation: HortScience 58, 5; 10.21273/HORTSCI17026-22
Discussion
There have been many reports of the investigation of sugar accumulation in fruits (Cheng et al. 2018; Yamaki 2010; Zhang et al. 2018); however, most of them did not focus on the detailed deviation and translocation within a fruit. In this study, the fruits at 21 d after anthesis (which are not mature yet) were used, but a sugar concentration gradient could already be observed (Fig. 3).
Soil culture was used during most earlier studies using applied cultivation technology. Because it can be difficult to control the soil water status and fertilizer condition, the plants were grown hydroponically to maintain their water status during this study. A water potential difference is required to move solutes in fruits (Boyer 1995a). Osmotic pressure is controlled by sugars, glycerol, amino acids, sugar alcohols, and various low-molecular-weight metabolites (Boyer 1995b). Both the water and osmotic potentials were lowest in the center of the fruit rather than in the other parts (Fig. 4). A low osmotic potential means that the solute accumulated as sugars in watermelon fruits (Figs. 3 and 4). This solute accumulation in the center of the fruit was demonstrated by two different methods (Figs. 3 and 4) during this study.
The 13C isotope analysis has been performed to investigate the translocation and sharing of photosynthates to fruits (Finazzo et al. 1994; Okano et al. 1983; Yakushiji et al. 1998). For watermelons, there is limited research of the use of isotopes during experiments. Watanabe (2004) demonstrated that 13C translocated mostly to the fruit rather than to the other parts of the plants, which is in agreement with the results of our experiment (Figs. 5 and 6A). Furthermore, we found that the photosynthates are actively translocated to the center of the watermelon fruit (Fig. 6B). Yakushiji et al. (1998) demonstrated that sugar accumulation, as supported by the data of the isotope analysis, is induced by water deficit stress in Satsuma mandarin fruits. The sugar concentration and isotope ratio were clearly correlated in our study (Figs. 3 and 6B), with translocation of the 13C isotope to the center of the fruit (Fig. 6B).
Even though the plants grown with the EC 3.0 regime tended to have lower 13C absorption of the whole plant, there were no significant differences between treatments with the three EC regimes (Fig. 5). Photosynthetic activity was presumed to have little effect on EC regimes up to 3.0 during this experiment. Fruit enlargement was also slightly suppressed with the EC 3.0 regime, but there were no significant differences between treatments in this study (Fig. 2). Under such conditions, the fruit showed not only the highest 13C absorption but also the highest sharing ratio in the whole plant (Figs. 5 and 6A). As reported by Yakushiji et al. (1998), the watermelon fruit is considered the largest sink for photosynthetic assimilates.
Because of the effect of the nutrient solution concentration, the 13C sharing ratio in the fruit with the EC 1.2 regime was apparently higher than those with the EC 2.0 and 3.0 regimes (Fig. 6A). However, with these regimes, the 13C sharing ratio in the lower leaves and stem was higher than that in the control, depending on the nutrient solution concentration (Fig. 6A). This indicates that the photosynthates of high EC regimes remained in the leaves, and their translocation was inhibited in the fruits. Although we did not measure the photosynthetic rate of leaves, it may be reduced with an EC 2.0 regime or higher in watermelon plants. Based on these results, it is reasonable to consider that a high nutrient solution concentration in hydroponic culture inhibits the translocation of photosynthates in immature watermelon fruits. In this study, it was confirmed that the fruit growth and weight tended to be suppressed with the EC 3.0 regime rather than with the EC 1.2 and 2.0 regimes (Fig. 2), suggesting that the EC 3.0 regime negatively affected the fruit growth and sugar accumulation in the hydroponically cultured watermelon. Although the EC 2.0 regime did not affect fruit growth (Fig. 2), it affected the translocation of photosynthates (Fig. 6A), suggesting that the nutrient solution concentration affects fruit growth at EC higher than 2.0 S⋅m−1. Because we used immature fruits (20 d after anthesis) in this experiment, watermelon fruits may respond differently to nutrient stress when they are in the mature stage. Therefore, we should further investigate their response at various growth stages. Even though the translocation in fruits was inhibited by the high nutrient solution concentration (Fig. 6A), the sugar concentration did not differ among the treatments in each part in the fruit (Fig. 4). One of the possible reasons for this was the concentration effect in fruit cell sap. In particular, the fruit growth with the EC 3.0 regime was inhibited compared with that with the EC 1.2 regime (Fig. 2), suggesting that the translocation of photosynthates was inhibited (Fig. 6A). Based on this result, the sugar concentration with the EC 3.0 regime might become similar to that with the EC 1.2 regime (Fig. 3). For Satsuma mandarin fruits, it has been reported that fruit growth was inhibited by drought (water deficit) stress, but the sugar content was increased (Yakushiji et al. 1998). Even though the type of stress applied to Satsuma mandarin fruits is different than that applied to watermelons (i.e., the cultivation management in this study did not involve stress application to watermelon plants), it did not inhibit fruit growth significantly (Fig. 2), and sugar concentrations were not different significantly (Fig. 3).
The vascular bundle morphology of a watermelon fruit was described in our previous report (Yoshii et al. 2013) and in another report (Kanahama and Saito 1987). The vascular bundle in a watermelon fruit runs from the peduncle to the bottom of the fruit, along with the skin, and then toward the center of the fruit. The same applies to the sieve tubes running in parallel with an ordinary vascular bundle. Based on these findings, photosynthates are considered to be transported from the peduncle to the center of the fruit, along with the skin. The highest sugar concentration was observed in the center of the fruit (Fig. 3), which was correlated with the highest 13C sharing ratio in the center of the fruit (upper mid flesh and lower mid flesh) (Fig. 6B).
The sugar concentration in the center of the flesh was higher than that around the fruit (Fig. 3). Similarly, the 13C sharing ratio was significantly higher in the center of the fruit (upper mid flesh and lower mid flesh) than around the fruit (Fig. 6B), indicating that the center of the flesh is the main sugar sink of the fruit. Except for the lower mid flesh with the EC 1.2 regime, 13C sharing ratios with the EC 1.2 regime were higher than those with the EC 2.0 and 3.0 regimes, and the ratio tended to decrease depending on the nutrient solution concentration (Fig. 6B). In the lower mid flesh, the 13C sharing ratios with the EC 2.0 and 3.0 regimes were slightly higher than that with the EC 1.2 regime (Fig. 6B) because the vascular bundle in a watermelon fruit runs from the surface of the fruit (i.e., the bottom of the fruit to the center part of the fruit). With the EC 2.0 and 3.0 regimes, compared with the EC 1.2 regime, the translocation and accumulation of photosynthates in the upper mid flesh might be inhibited by the high nutrient solution concentration. In the upper outer flesh, upper flesh, lower flesh, and lower outer flesh, the 13C sharing ratios with the EC 1.2 regime were all higher than those with the EC 2.0 and 3.0 regimes; however, there was no significant difference in the lower mid flesh (Fig. 6B). This suggests that the lower mid flesh had a higher sink activity than the other parts of the watermelon fruit, even under the stress of the high nutrient solution concentration.
Because one fruit grew from one plant in this experiment, fruits were subjected to various stress conditions. Some reports indicated that sugar accumulation in Cucurbitaceae fruits is affected by stress (Kirnak and Dogan 2009; Lima et al. 2020). However, the experimental design of those studies differed from that used in this study. During most of the experiments, stress was applied using a salt (NaCl) solution or limited water irrigation (draught) under soil culture condition (Kirnak and Dogan 2009; Lima et al. 2020; Proietti et al. 2008). During this experiment, plants were cultured hydroponically and the culture solution was enriched by increasing the ratio of fertilizers. Further research of the relationship between the growth conditions of a watermelon fruit and stresses in hydroponic culture should be performed.
In conclusion, active solute accumulation occurred, particularly in the center of the watermelon fruit, as confirmed by measuring the water status and analyzing the sugar concentration in the fruits and by 13CO2 isotope application and analysis.
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