原著論文
Factors Explaining Variations in Soluble Solids Content of Apples during Ripening and Storage
2024 年 93 巻 2 号 p. 135-142
詳細
2024 年 93 巻 2 号 p. 135-142
Sweetness is one of the most important drivers of consumer preference in apples (Malus × domestica Borkh.). The increase in sugar during the ripening period of fruit is mainly brought about by the hydrolysis of starch accumulated before the ripening period. However, sugars are also continuously translocated into the fruit during the ripening period, as seen in watercored fruits. The objective of this study was to estimate the contribution of translocated sugars that accumulated in the apoplast to the increase in soluble solids content (SSC) of fruit during ripening. The amount of apoplastic solution (AS) tended to be high in the fruit of trees on vigorous rootstocks, such as ‘JM2’ and ‘Marubakaido’. On the other hand, fruit with more AS had lower SSC. Therefore, although AS increased during ripening, the contribution of AS to the increase in SSC was small. After fruit matured and during storage, dehydration increased the SSC of the fruit. On the other hand, the SSC decreased simultaneously due to a reduction in organic acids and any soluble solids, which was expressed as a decrease in titratable acidity (TA). Under standard refrigerated conditions, the increase in SSC due to dehydration and the decrease in SSC due to respiration were likely to be quantitatively comparable. The contribution of translocated sugars to the increase in SSC during ripening was small, suggesting that managing trees and fruit to increase starch accumulation before fruit ripening is crucial for the production of fruits with high sugar content.
Sweetness is one of the most important drivers of consumer preference in apples (Malus × domestica Borkh.) (Bonany et al., 2013; Endrizzi et al., 2015; Musacchi and Serra, 2018). Sweetness is a sensory trait that is evaluated by a trained panel, and soluble solids content (SSC)—expressed as °Brix—is commonly used to estimate sweetness (Aprea et al., 2017). Although SSC incorporates not only sugars, but also organic acids and inorganic salts (Kingston, 1992), sugars are the SSC major component, and they increase as fruit ripens. The increase in sugar content is brought about by the hydrolysis of starch accumulated during fruit development (Doerflinger et al., 2015), and a high correlation was observed between SSC at harvest and starch content two months before harvest (Iwanami et al., 2023).
On the other hand, there have been some reports that pre-harvest leaf picking to improve fruit coloration reduced the SSC of fruit (Matsumoto et al., 2017; Oba et al., 1996; Suzuki et al., 1987). The mechanism of this phenomenon has been interpreted to be that the translocation of sugars to the fruit was restricted due to the picking of healthy photosynthetic organs around the fruit. This suggests that direct sugar translocation from leaves to fruit before harvest also has a significant effect on the SSC of the fruit at harvest.
Some apple cultivars exhibit watercore in the late stage of maturity. Watercore is a phenomenon in which sorbitol, a main translocated sugar alcohol, accumulates in the apoplast of flesh tissues adjacent to the vascular core (Gao et al., 2005). The area of watercore widens as maturity progresses, indicating that sugars are continuously translocated into the fruit, even during the mature period. Therefore, it is expected that the SSC is higher in fruit with a high watercore score.
The objective of this study was to estimate the contribution of translocated sugars that accumulated in the apoplast to the increase in the SSC of fruit during ripening. Using a multiple regression model, we searched for factors that could explain the variation in the SSC of individual fruits and estimated the contribution of these factors to the SSC.
‘Fuji’ (Malus × domestica Borkh.), known as a cultivar in which watercore frequently occurs, was used to develop a regression model for explaining the tree- and fruit-dependent variation in the SSC of fruit during ripening and storage. The trees were grafted on four rootstocks (‘JM1’: dwarfing rootstock; ‘JM7’: dwarfing to semi-dwarfing rootstock; ‘JM2’: semi-dwarfing to semi-invigorating rootstock; ‘Marubakaido’: semi-invigorating rootstock, Iwanami et al., 2009) in 2010 (‘JM2’ and ‘Marubakaido’) and 2011 (‘JM1’ and ‘JM7’) and planted in the orchards (39°3'N, 141°3'E, 190 m altitude, 1,266 mm annual average precipitation) of the Division of Fruit Tree Production Research, Institute of Fruit Tree and Tea Science, NARO, Morioka, Japan. Trees were spaced at 1 m within-row and 4 m between-row intervals in ‘JM1’ and at 2 m within-row and 4 m between-row intervals in ‘JM7’ and trained as slender spindles, and at 8 m square intervals in ‘JM2’ and ‘Marubakaido’ and trained as open centers. All trees were managed identically in accordance with standard orchard practices. No leaves were picked to promote fruit skin coloration. To assess the degree of crop load, the number of total fruits harvested and trunk cross-sectional area (TCA) were recorded every year for all trees. The TCA was calculated by measuring the trunk circumference in the winter of the previous year at 20 cm above the grafting site, and the crop load (CL) was expressed as number of fruits per cm2 TCA.
Sampling fruit for SSC measurementIn 2017, fruits were harvested three times at one-week intervals during the matured period (first to third week of November). Three fruits were harvested from each of 10 trees (30 fruits in total) for each sampling of ‘JM1’ and ‘JM7’ trees; 10 fruits from each of three trees (30 fruits in total) were harvested from trees on ‘JM2’ and ‘Marubakaido’.
In 2018, fruits were harvested five times at one-week intervals during the ripening period (first to fifth week of October) and three times at one-week intervals during the matured period (first to third week of November). Four fruits were harvested from each of three trees (12 fruits in total) for each sampling of trees on all four rootstocks. Moreover, 16 fruits per tree harvested during the second week of November were stored in a chamber controlled at 2±1°C and 90±5% RH (air storage) until four months after harvest, and four fruits per tree were sampled from the chamber on a monthly basis.
In 2019, fruits were harvested once in the second week of October as the ripening period and once in the second week of November as the matured period. Furthermore, fruits harvested in the second week of November were stored in the above-mentioned chamber until the second week of March, and they were taken out every month for measurements. The number of fruits for each sampling was the same as in the 2018 test.
Measurement of traits related to SSCApoplastic solution (AS) was measured by a centrifuge method according to Iwanami et al. (2017). Cylindrical fruit specimens of 24.0 mm in length and 20.5 mm in diameter were excised from the outer part of the equatorial region of each fruit with a cork borer and a pair of parallel blades and placed in a centrifugal filter unit (Vivaspin 20; Sartorius AG, Göttingen, Germany). The unit was centrifuged at 1,500 × g for 60 min. Juice extracted by centrifugation was calculated as (Wi − Wf)/Wi × 100 (%), where Wi and Wf = the weight of the cylinder before and after centrifugation, respectively.
The water content (WC) of flesh tissue per volume (cm3) was measured by drying two additional cylinders excised from the same region of the fruit at 90°C for 24 h. The degree of watercore was evaluated by giving a score of 0 to 4 from no to severe occurrence of watercore, and the starch index (SI) was evaluated by applying an iodine solution to a slice of the equatorial part of the fruit with a thickness of 5 mm and assigning an index of 0 (0%) to 5 (100%), depending on the degree of staining.
The remaining fruit from which the cylinders and slice were excised were used to measure the titratable acidity (TA) and SSC. Crude juice was extracted with a juicer. Following filtration, 5 mL of the juice was titrated with an automatic titrator (AUT-701; TOA-DKK, Tokyo, Japan) to pH 8.0 with 0.1 N NaOH. Titration results were calculated as the malic acid (g) per 100 mL of sample juice. The SSC of the juice was measured using a digital refractometer (PR-100; Atago, Tokyo, Japan).
Statistical analysisWe developed a multiple regression model for predicting SSC using the CL of each tree and the AS, WC, SI and TA of each fruit, as variables. Coefficients of the variables in the model were estimated as follows:
| [1] |
where SSCi,j,k,l is the SSC of the lth fruit sampled on the kth date from the jth tree in the ith year; CLi,j is the crop load of the jth tree in the ith year; ASi,j,k,l, WCi,j,k,l, SIi,j,k,l and TAi,j,k,l are the apoplastic solution, the water content, the starch index and the titratable acidity, respectively, of the lth fruit sampled on the kth date from the jth tree in the ith year; and β1–β11 are an intercept of the regression model and coefficients of each variable.
The coefficients of Eq. [1] were estimated using R software (R Core Team) version: 4.2.2. To choose the best set of predictor variables, a stepwise method was applied, in which the decision to include or exclude the variables in the model was made based on the Akaike information criterion (AIC). The AIC can lead to a suitable regression model by comparing the number of parameters with the magnitude of the error in the model. The accuracy of the model was described by the bias between the predicted and measured values, the correlation coefficient (r) and the root mean square error (RMSE).
In 2017, SSC and related traits were measured during the matured period of fruit. The CL of the trees used was a standard value and the average values of the CL for each tree of the four rootstocks were approximately equal (CL = 2.2, 1.8, 2.1 and 2.1 in ‘JM1,’ ‘JM7,’ ‘JM2’ and ‘Marubakaido,’ respectively). The SSCs of fruit from trees on dwarfing rootstocks of ‘JM1’ and ‘JM7’ were higher than those on semi-invigorating rootstocks of ‘JM2’ and ‘Marubakaido’, especially in the third week of November (Fig. 1A). On the other hand, the watercore score was relatively higher in trees on ‘JM2’ and ‘Marubakaido’ than in trees on ‘JM1’ (Fig. 1B). Apoplastic solution (AS) and water content (WC) were also higher in trees on ‘JM2’ and ‘Marubakaido’ than in trees on ‘JM1’ and ‘JM7’ (Fig. 1C, D).
Changes in the soluble solids content (SSC), watercore score, apoplastic solution (AS), water content (WC), starch index (SI) and titratable acidity (TA) in fruit of ‘Fuji’ grafted on four different rootstocks (‘JM1,’ ‘JM7,’ ‘JM2’ and ‘Marubakaido’) from the first week of October to the third week of November and during storage in 2017 to 2019. Vertical bars indicate the standard error (n = 30 in 2017, n = 12 in 2018 and 2019).
In 2018, we started measuring the traits from the beginning of the ripening period. The CLs of the trees used were all higher than those in the previous year, especially the those of ‘Marubakaido’ (CL = 2.7, 2.1, 2.5 and 3.3 in ‘JM1,’ ‘JM7,’ ‘JM2’ and ‘Marubakaido,’ respectively). As the fruit ripened, the SI and TA decreased, and the SSC and watercore score increased (Fig. 1G, H, K, L). The AS and WC also increased (Fig. 1I, J). As in the previous year, the SSC of the ‘JM7’ tree was consistently higher than that of the other rootstock trees (Fig. 1G). One of the reasons for this was that the CL of the ‘JM7’ trees was lower than that of other rootstocks. The AS and WC of the ‘JM2’ and ‘Marubakaido’ trees also tended to be higher (Fig. 1I, J). During storage, traits other than the SSC decreased gradually, while the SSC remained almost unchanged (Fig. 1G–L).
In 2019, the CL was low for all trees used and there was no difference in the CL among the four rootstocks (CL = 1.5, 1.4, 1.4 and 1.5 in ‘JM1,’ ‘JM7,’ ‘JM2’ and ‘Marubakaido,’ respectively). There was no large difference in the SSC between the four rootstocks during the ripening period, but the SSC was higher in the fruit of the ‘JM7’ tree during storage (Fig. 1M). The AS and WC were generally lower than in the previous year (Fig. 1I, J, O, P). The AS and WC of ‘JM2’ trees were higher as compared to those of trees with the other rootstocks (Fig. 1O, P).
Comparing the AS in fruits with each watercore score without distinguishing between rootstocks and years, it was found that when the watercore score was greater than 2.0, fruits with higher watercore scores tended to have higher AS (Fig. 2).
Relationships between watercore scores and the apoplastic solution (AS) during the ripening and mature periods in ‘Fuji’ fruit from 2017 to 2019. Vertical bars indicate the standard error calculated by combining data for the four different rootstocks (n = 7–172).
To clarify factors related to changes in the SSC during ripening and storage and factors that made the differences in SSC between fruits, we created the multiple regression model shown in Equation 1 and searched for factors that could explain the differences in SSC based on the AIC. Since there was a correlation between the watercore score and the AS, the AS was used as a variable in the regression analysis. Prior to the analysis, we confirmed that no trait with a particularly strong correlation was found among each variable (Table 1). As a result of critically selecting variables based on the AIC, all variables—CL, AS, WC, SI and TA—were necessary to explain the difference in the SSC. Also, the size of the effect, indicated by the coefficient of each factor on the SSC, was different among the periods of ripening, maturation and storage (Table 2). The predicted values obtained by the regression model and the measured values of SSC were close (RMSE = 0.589), with almost no bias (0.021) (Fig. 3), indicating that the selection of variables and the estimation of coefficients of the variables were appropriate. Therefore, changes in the SSC during ripening and storage and differences in the SSC between fruits within a tree could be explained by the five variables, regardless of the type of rootstock and different growing year of the trees.
Correlations among the CL (crop load), AS (apoplastic solution), WC (water content), SI (starch index), TA (titratable acidity) and SSC (soluble solids content) in ‘Fuji’ grafted on different rootstocks.
Estimated coefficients of the regression model variables expressed as Eq. [1].
Relationships between measured and predicted soluble solids content (SSC) in fruit of ‘Fuji’ from 2017 to 2019. Estimated values were obtained from Eq. [1] using data from the four different rootstocks.
Assuming that the CL of a tree affects the SSC of all fruits on the tree equally, the lighter the CL, the higher the SSC will be (Fig. 4).
Contribution of crop load to the predicted soluble solids content (SSC) obtained from Eq. [1] and Table 2. Values of each variable other than crop load were as follows: AS = 0.05, WC = 0.75, SI = 1.0 and TA = 0.35.
The values of the AS, WC, SI and TA varied between fruits within a tree, and the effects of the four variables on the SSC differed during the ripening period, mature period and storage. The AS increased from approximately 0.03 to 0.06 g·g−1FW from October to November (Fig. 1I), but had little effect on the SSC (Fig. 5A). Similar to the AS, the WC increased during ripening (Fig. 1J), but had little effect on the SSC (Fig. 5B). During the mature period, the WC, however, negatively affected the SSC (Fig. 5F); fruits on trees with vigorous rootstocks (‘JM2’ and ‘Marubakaido’) tended to have higher WC (Figs. 1D and J), and the SSC of the fruits was correspondingly lower than that of the other rootstocks (Fig. 1A, G). Therefore, high WC causes low SSC. During storage, the WC gradually decreased (from 0.8 to 0.72) (Fig. 1J). The decrease in WC in that range increased the SSC by about 0.8 °Brix (from 13.6 to 14.4) (Fig. 5J). From these facts, a decrease in the WC, that is, the degree of dehydration, has a large effect on the SSC after fruit maturation.
Contributions of apoplastic solution (AS), water content (WC), starch index (SI) and titratable acidity (TA) to the predicted soluble solids content (SSC) during ripening periods (A, B, C and D), mature periods (E, F, G and H) and storage periods (I, J, K and L). The arrows in the figure indicate the direction of change in each trait during each period. The predicted values were obtained from Eq. [1] and Table 2. Dotted lines indicate the upper and lower 95% confidence limits for the predictions. Variables other than the ones shown in the plate were given fixed values as follows: AS = 0.05, WC = 0.75, SI = 3.0 and TA = 0.4 during the ripening period; AS = 0.05, WC = 0.75, SI = 1.0 and TA = 0.35 during the mature period; AS = 0.05, WC = 0.75, SI = 0 and TA = 0.25 during the storage period.
The SI decreased greatly during ripening (4 to 1.5) (Fig. 1K), which induced a large increase in the SSC (Fig. 5C). Since most of the starch had been hydrolyzed by the mature period, the effect of decreasing SI on increasing SSC was not observed during the mature and storage period (Fig. 5G, K).
The lower the TA, the lower the SSC at any stage (Fig. 5D, H, L). Since TA is one of the components of the SSC, and it continued to decrease at a constant rate from the first week of October through the second week of March (Fig. 1L), it could be said that the SSC decreased accordingly. However, the SSC decreased by nearly 1 °Brix (from 14.8 to 13.9 °Brix) for the 0.1 g/100 mL decrease in TA from 0.4 to 0.3 g/100 mL (Fig. 5H). This suggests that a decrease in organic acid does not directly lower the SSC value, but that a decrease in some soluble solids that occurs synchronously with the decrease in TA lowers the SSC value.
After the ripening period, at the beginning of November, and during storage, changes in the SSC were small (Fig. 1G, M). It could be said that the increase in the SSC associated with a decrease in the WC (Fig. 5F, J) and a decrease in the SSC associated with a decrease in the TA during these periods (Fig. 5H, L) occurred at the same time, and the changes in the SSC were small because the two were in balance.
Apoplastic solution (AS) is closely related to juiciness and freshness and is an important trait that determines fruit quality (Iwanami et al., 2017). In this study, since fruit with high watercore scores also had high AS values (Fig. 2), it can be assumed that fruits with high watercore scores are juicy and fresh. AS tended to be high in fruits of trees on vigorous rootstocks such as ‘JM2’ and ‘Marubakaido’ (Fig. 1C, I). It has been reported that the ability of roots to absorb and transport water increased as the degree of rootstock vigor increased (Iwanami et al., 2009, 2011). In this study, the WC also tended to be higher in trees on vigorous rootstocks compared to those on dwarfing rootstocks (Fig. 1D, J). Therefore, the high AS and the high WC may reflect the vigor of trees or the water uptake and transport capacity of the roots. The AS and WC of ‘Marubakaido’ trees in 2019, however, were lower than expected (Fig. 1O, P). This may be because the trees weakened due to the high CL in the previous year.
Fruits with high AS did not necessarily have a high SSC. Rather, the SSC of fruits with high AS tended to be low (Fig. 1A, C, G, I). Therefore, although the AS increased during ripening, the contribution of AS to the increase in the SSC was small (Fig. 5A). Tanaka et al. (2016) also mentioned that the SSC of watercored fruit was not necessarily high, based on some data in the literature comparing the sugar contents of watercored and non-watercored fruits. Iwanami et al. (2023) reported that there is a high correlation between the starch content two months before harvest and the SSC at harvest in ‘Fuji’. This indicates that the SSC of fruits at harvest is determined two months before harvest. In the present study, the increases in the SSC during the ripening period could largely be explained by the decreases in SI (Fig. 5C), which is consistent with the results of Iwanami et al. (2023).
After maturation and during storage, the SSC could be increased by decreasing the WC (Fig. 5F, J). °Brix, which is the unit of SSC, is a value that represents the sucrose content per 100 g of solution; therefore, the value increases as the water content of the solution decreases, even if the amount of soluble solids is constant. We calculated how much the SSC values would change when the amount of soluble solids was kept constant and only the water content in the solution changed. If fruit has a soluble solids content of 14% and a water content of 0.8 g·cm−3, the soluble solids content will be 15% if the water content is 0.74 g·cm−3. That is, a decrease in the water content of 0.06 g·cm−3 increases the SSC by 1 °Brix. A difference of 0.06 g·cm−3 in water content was within the range among fruits of different rootstocks observed in this study (Fig. 1D) and within the range of changes during fruit ripening and storage (Fig. 1J).
After maturation and during storage, changes in the SSC are small (Fig. 1A, G, M), although the concentration of soluble solids due to dehydration could increase the value of SSC (Fig. 5F, J). This is because the increase in the SSC is masked by the decrease in soluble solids that occurred in tandem with the decrease in the TA (Fig. 5H, L). Since sugars and malic acid are the main substrates for respiration in apples (Hulme and Rhodes, 1971), the soluble solids that decreased with the TA are considered to be sugars and it is logical that the SSC decreases during storage. Nevertheless, many publications have reported that SSC changed very little during refrigerated storage of at least 90 days after harvest (Fang et al., 2020; Kolniak-Ostek et al., 2014; Li et al., 2010; Shirzadeh and Kazemi, 2011). Therefore, under standard refrigeration conditions, the increase in the SSC due to dehydration and the decrease in soluble solids due to respiration were likely to be comparable.
The effect of the translocation of sugars during the ripening period on the SSC was small, suggesting that the reduction in the SSC observed in the pre-harvest leaf picking was induced not because of less translocated material, but for other reasons. Many studies have reported that pre-harvest leaf picking reduced the TA of fruit at harvest (Kume and Kudo, 1982; Oba et al., 1996; Suzuki et al., 1987). Iwanami et al. (2016) also observed that the TA decreased as the number of leaves picked increased and speculated that leaf picking allowed the fruit to receive more direct sunlight, which raised the fruit temperature and promoted the consumption of malic acid, a respiratory substrate. The present study showed that the SSC decreased with a decrease in the TA (Fig. 5D, H), suggesting that the reason for the decrease in SSC by leaf picking is not because the translocation of sugars to fruits is reduced, but because the consumption of sugars that are the same respiratory substrate as malic acid, is increased.
In conclusion, the increase in the SSC was mainly caused by the hydrolysis of starch into sugars during ripening, and the dehydration of fruit also increased the SSC after maturity and during storage. On the other hand, the SSC decreased at the same time due to a decrease in organic acids and soluble solids that was observed as a decrease in the TA. The contribution of translocated sugars to the increase in the SSC during the ripening and maturation periods was small, suggesting that managing trees and fruit to increase starch accumulation before fruit ripening is be important for the production of fruits with higher sugar content. Until now, little attention has been paid to tree management that increases starch accumulation in the fruit. We hope that further research on this point will lead to progress in improving fruit quality.
