3.1. Changes of skin-pulp adherence of different varieties of grapes in the process of fruit development
In the process of fruit development, the skin-flesh adhesion of Flame Seedless, Qiuhongbao, Wanheibao, Jinghongbao, Hutai No.8, Crimson Seedless and Black Balado showed a decreasing trend (Fig. 1). Different varieties of grapes skin-flesh adhesion force varied significantly. The peel-flesh adhesions of Flame Seedless, Thompson Seedless and Wuhecuibao was the lowest among the 12 varieties. Black Balado had the highest peel-flesh adhesion, which was 3.03, 2.67, and 2.46 times higher than that of Flame Seedless at the same period, respectively. The decrease in skin-flesh adhesion ranged from 6.4–52.4% for different varieties of grapes, and the easy peeling trait may be formed with the process of fruit development.
3.2. Changes in the morphology of pericarp cells of different varieties of grapes in the process of fruit development
In the process of fruit development (Fig. 2a), the volume of pericarp cells of Thompson Seedless, Summer Black, Qiuhongbao, Wanheibao, Hutai No.8, Lihongbao, Zaoheibao, and Crimson Seedlees gradually increased, and the pericarp cells became loosely arranged with complete morphology and dense arrangement (Fig. 2b); the thickness of the pericarp of Summer Black, Hutai No.8, Lihongbao, Zaoheibao, and Crimson Seedless gradually decreased from the period of expansion to the ripening stage. During the expansion stage, the arrangement of the pericarp cells of Flame Seedless, Thompson White, Qiuhongbao, Jinghongbao and Black Balado was loose, and in the process of fruit development, the combination between neighboring cells became loose, and the degree of pulp cell cleavage increased. At the same time, the flattened and neatly arranged pericarp cells of Wanheibao, Hutai No.8, Lihongbao, Zaoheibao, and Crimson Seedless may be related to their more robust cell walls.
3.3. Changes in cell wall polysaccharide content during fruit development of different grape varieties
During the process of fruit development, the total dry matter content of cell walls of 12 varieties of grapes showed a decreasing trend (Fig. 3a and b). Among them, the decline of total dry matter content of cell wall of each variety in the pulp was obvious, and its decline (30.3%~64.8%) was higher than that of total dry matter content of cell wall in the skin (23.9%~51.4%). The decreases in the pericarp from the expansion stage to the color change stage were higher (5.5%~49.5%) than those from the color change stage to the ripening stage (7.2%~47.8%). During the developmental process of decreasing peel-flesh adhesion, the content of cellulose, hemicellulose, protopectin, chelator-soluble pectin, and water-soluble pectin in the peel and flesh of the fruit decreased continuously. Among them, the content of protopectin and chelator-soluble pectin decreased most rapidly. For example, the flame-free protopectin content in the pericarp decreased by 97.1% from 2067 µg·g− 1 FW at the expansion stage to 60 µg·g− 1 FW at the ripening stage, and by 93.9% in the pulp. Among the varieties the chelator-soluble pectin content in the peel decreased by 87.8–97.7%, and the chelator-soluble pectin content in the pulp decreased by 73.7–94.6%; the decreases in water-soluble pectin content were higher in the peel (84.3–92.5%) than in the pulp (43.1–87.8%).
3.4. Changes in the activities of enzymes related to cell wall polysaccharide degradation during the development of grape berries of different varieties
During the process of fruit development, the activities of xylanase, xyloglucan endosyltransferase, β-mannanase, polygalacturonase, pectin cleaving enzyme, pectin methylesterase, β-galactosidase, α-L-arabinofuranosidase of different varieties in the peel and pulp showed an increasing trend (Fig. 4a and b). Among them, cellulase and β-glucosidase activities showed a smoother change, showing a tendency to increase first and then decrease. Xylanase, xyloglucan endosyltransferase and polygalacturonase activities showed higher increases. From the expansion stage to the ripening stage, xylanase activity increased 0.37–2.55 times in the pericarp and 0.01–1.84 times in the pulp; xyloglucan endosyltransferase activity increased 0.38–2.37 times in the pericarp and 0.42–2.33 times in the pulp; and polygalacturonase activity increased 0.21–2.85 times in the pericarp and 0.58–2.85 times in the pulp. activity was elevated 0.58 to 2.43 times. The activity of pectin lyase enzymes increased rapidly in the range of 16%-43% before the color change stage and increased slowly in the range of 1%-11% after the color change stage. Pectin methylesterase and β-galactosidase showed a higher increase in activity in both the expansion to color change and color change to ripening stages. In the pericarp, pectin methylesterase and β-galactosidase increased by 0.69–1.07 and 0.21–0.55 times, respectively, and in the pulp by 0.29–1.53 and 0.05–1.02 times, respectively. Compared to the rise in the peel (1–341%), α-L-arabinofuranosidase had a significant increase in the pulp (85–365%). The above increase in polysaccharide-degrading enzyme activities may have promoted cell wall polysaccharide decomposition, which had an effect on the formation of easy peeling traits in grapes.
3.5. Cluster analysis of peelability of different varieties of grapes based on the peel-flesh adhesion force during the ripening period
Cluster analysis based on the peel-flesh adhesion force during the ripening period of the fruit of 10 Eurasian species of grapes can be divided into 2 categories (Fig. 5). The first category is easy to peel varieties, such as Flame Seedless, Thompson Seedless, Wuhecuibao, Wanheibao, Zaoheibao, Jinghongbao, Lihongbao, Qiuhongbao, Crimson Seedless; the second category is not easy to peel varieties such as Black Balado.
3.6. Correlation analysis between peel-meat adhesion and cell wall polysaccharides in different varieties of grape berries
The measured peel-flesh adhesion force and 16 polysaccharide indicators were analyzed by Pearson correlation. There were highly significant positive correlations between peel-flesh adhesion and total cell wall dry matter, cellulose, hemicellulose, protopectin, chelator-soluble pectin, and water-soluble pectin in the pericarp, highly significant negative correlations with cellulase, xylanase, xyloglucan endosyltransferase, β-mannanase, pectin methylesterase, and β-galactosidase, and highly significant negative correlations with β-glucosidase, polygalacturonase, α-L-arabinofuranosidase, and insignificant correlation with pectin lyase enzyme (Table 1). Peel-flesh adhesion was positively correlated with cell wall polysaccharide content in the pericarp and pulp, and negatively correlated with cell wall polysaccharide degrading enzyme activities in the pericarp and pulp, suggesting that the low content of cell wall polysaccharides in the pericarp and pulp as well as the high activity of cell wall polysaccharide degrading enzymes may be closely related to the low level of peel-flesh adhesion, and may be one of the physiological bases in the process of formation of pericarp separability trait.
Table 1
Correlation between peelability and cell wall polysaccharide related indexes in different varieties of grape berries
Part | Peelability | Cell wall material | Cellulose | Hemicellulose | Protopectin | CDTA-soluble pectin | Water soluble pectin | Cellulase | β-glucosidase | Xylanase |
Skin | Adherence | 0.790** | 0.683** | 0.720** | 0.608** | 0.660** | 0.693** | -0.552** | -0.393* | -0.645** |
Pulp | Adherence | 0.769** | 0.713** | 0.670** | 0.722** | 0.667** | 0.775** | -0.449** | -0.479** | -0.560** |
Part | Peelability | Xyloglucan endotransglycosylase | β-mannanase | Polygalacturonase | Pectate lyase | Pectin methyl esterase | β-galactosidase | α-L-Arabinofuranosidase |
Skin | Adherence | -0.529** | -0.539** | -0.403* | -0.142 | -0.501** | -0.498** | -0.398* |
Pulp | Adherence | -0.516** | -0.524** | -0.363* | -0.296 | -0.493** | -0.498** | -0.490** |
**P<0.01, *P<0.05. |
Cellulase activity was positively correlated with β-glucosidase, xylanase, xyloglucan endoglycosyltransferase, polygalacturonase, pectin lyase, enzyme, pectin methylesterase, β-galactosidase, and α-L-arabinofuranosidase, and was not significantly correlated with β-mannanase (Fig. 6). It suggests that there may be a synergistic relationship between cellulase and the above polysaccharide degradation-related enzymes in the process of influencing the formation of easy-to-skin traits in grapes.
In this study, the cell wall polysaccharide content in the pulp was lower than that in the peel. However, the absolute value of the difference between the correlation coefficient of cell wall polysaccharide content in the peel and peel-flesh adhesion and the correlation coefficient of the pulp was low. It suggests that the factors affecting the decrease in peel-flesh adhesion may be related to the influence of cell wall polysaccharide degrading enzymes and other influences that change the level of polysaccharide content. The level of cell wall polysaccharide content varied in different parts of the plant, but a more active feedback mechanism of cell wall polysaccharide content on the activity of cell wall polysaccharide-degrading enzymes may not be universal.
3.7. Based least squares regression analysis of peel-flesh adhesion and cell wall polysaccharides in different varieties of grape berries
In the correlation loading plot, the linear distance between “17” (total dry matter of pulp cell wall) and “A” (peel-flesh adhesion) was the lowest among the 32 indicators. This indicates that the decrease in the total dry matter content of pulp cell wall may be closely related to the decrease in peel-flesh adhesion (Fig. 7). Meanwhile, “4” (pericarp protopectin), “5” (pericarp chelator-soluble pectin), and “6” (pericarp water-soluble pectin) had the largest distances from A in the first and fourth quadrants, suggesting that changes in pericarp pectin content may have a lower influence on the changes in peel-flesh adhesion.
Based on the 32 cell wall polysaccharide-related indices measured, the peel-flesh adhesion of different varieties at different periods was scored by different principal component observations (Fig. 8). “L” (Black Balado-expanding stage) and “K” (Crimson Seedless-expanding stage) were located in the first quadrant of the axes and were farthest away from the origin, which indicated that the peel-flesh adhesion between Black Balado and Crimson Seedless were highest during the expanding stage, which is in line with the measured values. Taking the lowest observed value on the horizontal and vertical axes as the origin to establish a coordinate system, the observed values of peel-flesh adhesion “X” (Black Balado-veraison stage) was lower than those of “B” (Thompson Seedless-expanding stage), “F” (Wanheibao-expanding stage), “J” (Zaoheibao-expanding stage), “E” (Qiuhongbao-expanding stage), “G” (Jinghongbao-expanding stage), “H” (Hutai No.8-expanding stage), and “I” (Lihongbao- expanding stage), which indicates that the decrease in peel-flesh adhesion may be lower than the decrease in cell wall polysaccharide content. And the degree of change in cell wall polysaccharide content may not be the main factor affecting the differences in peel-flesh adhesion. The differences in peel-flesh adhesion between different varieties may be closely related to the differences in total cell wall dry matter or cell wall polysaccharide content at the expansion stage, which may be related to the fact that varieties with higher adhesion at the expansion stage have a more active polysaccharide synthesis mechanism as well as higher levels of polysaccharide synthesis-related enzyme activities.
The standardized regression coefficients of pectinase, pectin lyase, pectin methyl esterase, β-galactosidase, and α-L-arabinofuranosidase in the peel and pulp were positive, whereas their correlation coefficients with the peel-flesh adhesion force were negative (Fig. 9). It suggested that the levels of activity of polysaccharide-degrading enzymes involved in the hydrolysis of pectin, such as polygalacturonase, pectin lyase, pectin methyl esterase, β-galactosidase, and α-L-arabinofuranosidase may not be a major factor influencing the peel-flesh adhesion force. Since the linear distances between pulp cellulose, pulp cell wall total dry matter and peel-pulp adhesion were the smallest in the correlation loadings, the absolute values of the standardized regression coefficients for pulp cellulase (-0.018) were 37.5% of the absolute value of pulp xylanase (-0.048) and 20.2% of that of pulp β-mannanase (-0.089), respectively, It suggested that the degradation of hemicellulose, which is involved in cellulose cross-linking, may be one of the factors constituting the decrease in the total dry matter content of cellulose and cell wall.
3.8. Principal component analysis of cell wall polysaccharide related indexes of different varieties of grape berries
The principal component scores of different varieties at different periods were predicted using cell wall polysaccharide as a negative score index and cell wall polysaccharide degrading enzyme as a positive score index for peelability (Table 2). The scores of Qiuhongbao-ripening stage, Summer Black-ripening stage, Lihongbao-ripening stage, Hutai No.8-ripening stage and Jinghongbao-ripening stage were relatively high, and the observed values of the scores of Lihongbao-expanding stage, Hutai No.8-expanding stage, Jinghongbao-expanding stage, Qiuhongbao-expanding stage, Summer Black-expanding stage were low (Table 3). This suggests that the cell wall polysaccharide content and degradation enzyme activity of Hutai No.8, Lihongbao, Qiuhongbao, Jinghongbao and Summer Black changed more obviously during the development process, and the peelability may have changed significantly.
Table 2
Load values and contribution rates of cell wall polysaccharide related indicators
Indicator | Principal Component 1 | Principal Component 2 | Principal Component 3 | Principal Component 4 |
Peel cell wall material | -0.931 | 0.195 | -0.150 | 0.108 |
Pulp cell wall material | -0.850 | 0.186 | -0.140 | 0.345 |
Peel cellulose | -0.905 | 0.172 | -0.051 | 0.091 |
Pulp cellulose | -0.880 | 0.182 | -0.013 | 0.333 |
peel hemicellulose | -0.888 | 0.286 | -0.016 | 0.187 |
pulp hemicellulose | -0.917 | 0.149 | 0.125 | 0.180 |
peel protopectin | -0.933 | 0.134 | 0.160 | 0.049 |
pulp protopectin | -0.904 | 0.076 | -0.126 | 0.235 |
peel CDTA-soluble pectin | -0.930 | 0.198 | 0.132 | 0.044 |
pulp CDTA-soluble pectin | -0.936 | 0.216 | 0.067 | 0.158 |
peel water-soluble pectin | -0.913 | 0.152 | 0.126 | 0.152 |
pulp water-soluble pectin | -0.887 | 0.152 | -0.111 | 0.272 |
peel cellulase | 0.736 | -0.517 | 0.062 | 0.160 |
pulp cellulase | 0.666 | -0.434 | 0.190 | 0.381 |
peel β-glucosidase | 0.561 | -0.522 | 0.124 | 0.215 |
pulp β-glucosidase | 0.515 | -0.520 | 0.288 | 0.266 |
peel xylanase | 0.865 | 0.207 | 0.310 | 0.103 |
pulp xylanase | 0.747 | -0.117 | 0.356 | 0.340 |
Peel xyloglucan endoglycosyltransferase | 0.828 | 0.384 | 0.238 | 0.116 |
pulp xyloglucan endoglycosyltransferase | 0.815 | 0.377 | 0.261 | 0.194 |
peel β-mannanase | 0.647 | 0.458 | 0.541 | -0.083 |
pulp β-mannanase | 0.574 | 0.565 | 0.544 | -0.095 |
peel polygalacturonase | 0.879 | 0.113 | -0.019 | 0.217 |
pulp polygalacturonase | 0.890 | 0.069 | -0.172 | 0.290 |
peel pectate lyase | 0.740 | 0.146 | -0.502 | 0.293 |
pulp pectate lyase | 0.837 | 0.066 | -0.468 | 0.085 |
peel pectin methyl esterase | 0.925 | 0.305 | -0.144 | 0.021 |
pulp pectin methyl esterase | 0.922 | 0.253 | -0.207 | 0.013 |
peel β-galactosidase | 0.913 | 0.227 | -0.235 | 0.032 |
pulp β-galactosidase | 0.885 | 0.413 | -0.092 | 0.034 |
peel-α-L-arabinofuranosidase | 0.722 | 0.217 | -0.299 | -0.021 |
pulp-α-L-arabinofuranosidase | 0.812 | 0.279 | -0.253 | -0.004 |
eigenvalue | 22.151 | 2.816 | 2.110 | 1.207 |
variance explained rate (%) | 69.222 | 8.801 | 6.276 | 3.773 |
Cumulative contribution (%) | 69.222 | 78.023 | 84.299 | 88.072 |
Table 3
Comprehensive score and ranking of peelability in different varieties at different stages
Sample | 1st Principal Component Score | 2nd Principal Component Score | 3rd Principal Component Score | 4th Principal Component Score | Combined Score | Rank |
Flame Seedless-ripening stage | -626.88 | 220.24 | 134.72 | 166.78 | -453.954 | 1 |
Thompson Seedless-ripening stage | -907.37 | 270.25 | 119.20 | 212.79 | -668.556 | 2 |
Wuhecuibao-ripening stage | -975.43 | 283.41 | 136.22 | 234.76 | -718.576 | 3 |
Qiuhongbao-ripening stage | -1154.53 | 313.31 | 180.34 | 227.18 | -853.539 | 4 |
Summer Black-ripening stage | -1311.84 | 347.48 | 102.10 | 333.30 | -974.793 | 5 |
Lihongbao-ripening stage | -1411.70 | 346.58 | 164.95 | 293.90 | -1050.58 | 6 |
Hutai No.8-ripening stage | -1411.60 | 353.48 | 133.74 | 312.55 | -1051.23 | 7 |
Jinghongbao-ripening stage | -1422.74 | 356.67 | 127.81 | 331.48 | -1059.29 | 8 |
Flame Seedless-veraison stage | -1641.52 | 353.79 | 203.52 | 283.05 | -1228.21 | 9 |
Zaoheibao-ripening stage | -1654.72 | 387.27 | 114.72 | 378.52 | -1237.47 | 10 |
Wanheibao-ripening stage | -1754.59 | 424.46 | 204.91 | 347.42 | -1307.16 | 11 |
Wuhecuibao-veraison stage | -1891.12 | 386.31 | 211.01 | 324.30 | -1418.83 | 12 |
Summber Black-veraison stage | -2407.47 | 467.03 | 147.87 | 472.30 | -1814.77 | 13 |
Crimson Seedless-ripening stage | -2481.59 | 546.31 | 142.40 | 525.02 | -1863.23 | 14 |
Thompson Seedless-veraison stage | -2501.59 | 466.19 | 176.16 | 452.65 | -1887.65 | 15 |
Black Balado-ripening stage | -2898.88 | 615.57 | 134.35 | 624.82 | -2180.58 | 16 |
Jinghongbao-veraison stage | -3718.05 | 696.23 | 234.09 | 643.14 | -2808.47 | 17 |
Qiuhongbao-veraison stage | -3847.00 | 715.54 | 297.33 | 613.62 | -2904.66 | 18 |
Zaoheibao-veraison stage | -4185.63 | 779.89 | 292.50 | 710.05 | -3160.59 | 19 |
Hutai No.8-veraison stage | -4476.15 | 799.40 | 188.01 | 823.84 | -3389.56 | 20 |
Lihongbao-veraison stage | -4500.13 | 819.42 | 174.23 | 824.44 | -3407.36 | 21 |
Wanheibao-veraison stage | -5596.48 | 1051.37 | 412.34 | 899.79 | -4225.69 | 22 |
Crimson Seedless-veraison stage | -6009.33 | 1131.62 | 468.44 | 871.86 | -4539.35 | 23 |
Wuhecuibao-expanding stage | -7899.32 | 1456.23 | 900.92 | 1012.76 | -5955.54 | 24 |
Flame Seedless-expanding stage | -8088.56 | 1473.13 | 880.36 | 1041.36 | -6102.83 | 25 |
Black Balado-veraison stage | -8882.63 | 1607.53 | 744.70 | 1388.00 | -6708.33 | 26 |
Thompson Seedless-expanding stage | -11326.45 | 2089.33 | 1066.20 | 1509.87 | -8552.84 | 27 |
Summer Black-expanding stage | -14167.68 | 2598.92 | 1529.32 | 1697.98 | -10694 | 28 |
Wanheibao-expanding stage | -19231.09 | 3522.42 | 2188.25 | 2079.96 | -14518.1 | 29 |
Qiuhongbao-expanding stage | -20186.66 | 3815.81 | 2272.77 | 2212.44 | -15228.1 | 30 |
Zaoheibao-expanding stage | -20641.20 | 3884.54 | 2390.34 | 2243.01 | -15568.8 | 31 |
Jinghongbao-expanding stage | -21897.35 | 4019.64 | 2218.77 | 2495.16 | -16544 | 32 |
Hutai No.8-expanding stage | -22284.69 | 4081.51 | 2369.34 | 2501.53 | -16831.3 | 33 |
Crimson Seedless-expanding stage | -23143.37 | 4206.41 | 2394.78 | 2766.07 | -17480.6 | 34 |
Lihongbao-expanding stage | -23229.92 | 4231.29 | 2151.15 | 2758.28 | -17563.8 | 35 |
Black Balado-expanding stage | -27843.75 | 5106.01 | 2632.35 | 3463.49 | -21038.2 | 36 |