Effects of waterlogging stress on leaf morphology in mulberry seedlings
Figure 1 shows the change in the leaf morphology of mulberry seedlings under different submergence depths. The results showed that the seedlings under both SS and HS could grow well, and there were 3 slightly wilted leaves on average under FS. There were 3 wilted leaves and 2 defoliated leaves on average in the HS group after 10 days of flooding, and a few adventitious roots began to appear at the base of the stem. In the SS group, there slight wilting and falling of mulberry leaves were observed on the 15th day after submergence, and there were 5 wilting leaves and a few adventitious roots per plant. In the SS group, there were 3 defoliated leaves and 2 wilted leaves per mulberry seedling, and no adventitious roots developed. The HS group showed an average of 7 adventitious roots per plant. Additionally, there were 8 wilted leaves, 10 defoliated leaves and 4 brown spots per plant under HS.
Effect of submergence stress on leaf morphology in Morus alba: (a) The number of curled or wilted leaves per plant; (b) The number of brown spots or rotten leaves per plant; (c) The number of fallen leaves per plant; (d) The number of adventitious roots. This figure was drawn using Origin Pro 2021 v. 9.8.0.200.
Effects of waterlogging stress on initial fluorescence (Fo), and maximum fluorescence (Fm) under dark adaptation in mulberry leaves
The initial fluorescence value (Fo) and the maximum fluorescence value (Fm) of mulberry seedlings significantly decreased over time. Figure 2a shows that the Fo values of mulberry seedlings under SS, HS, and FS decreased by 31.27%, 22.51%, and 42.45%, respectively, on day 4 and were significantly different (p < 0.05). On days 12 and 16, the Fo of mulberry seedlings under SS increased by 0.85% and 6.87%, respectively, and there was a significant decrease of 9.5% (p < 0.05) on the 20th day of waterlogging. The Fo under HS and FS significantly increased, by 12.56% and 25.62%, on the 8th day of waterlogging (p < 0.05), respectively, and the Fo under FS showed a significant downward trend at 12–20 days of flooding. Figure 2b shows that Fm under SS, HS, and FS decreased significantly, by 40.54%, 37.67% and 51.6%, respectively, after 4 days of flooding (p < 0.05). On day 7, the Fm of mulberry seedling leaves decreased by 12.65% under SS but increased by 28.08% and 40.27% under HS and FS, respectively. The differences under SS, HS and FS were significant (p < 0.05). The Fm under SS and HS showed a relatively stable trend in the late stage of flooding, while the Fm under FS showed a significant decreasing trend (p < 0.05). In addition, the Fo and Fm of mulberry seedlings under HS were significantly higher than those in SS and FS from days 4 to 16 of flooding.
Effect of submergence stress on Fo and Fm in Morus alba. Different uppercase letters indicate that the means of the different groups at the same time are significantly different (p < 0.05), and different lowercase letters indicate that the means of the same group at different times are significantly different (p < 0.05). This figure was drawn using Origin Pro 2021 v. 9.8.0.200.
Effects of waterlogging stress on the maximum photoperiod quantum yield (QY_max) of PS II and the potential activity of PS II with primary light energy (Fv/Fo) in mulberry leaves
The effects of waterlogging stress at different depths on the maximum photoperiod quantum yield (QY_max) and the potential activity of PS II (Fv/Fo) in mulberry leaves are shown in Fig. 3a,b. After 4 days of waterlogging, the QY_max of mulberry seedlings under SS, HS, and FS significantly decreased (p < 0.05), by 18.11%, 29.54%, and 22.1% respectively (Fig. 3a). Under SS, the QY_max of mulberry seedlings continued to decrease during the flooding period, decreasing by 11.82% and 31.39% on the 8th and 16th days, respectively (p < 0.05). The QY_max under HS and FS increased significantly, by 26.11% and 18.57%, on day 4 (p < 0.05) and decreased by 15.68%, 15.55% and 52.29% under HS on days 12, 16 and 20, respectively (p < 0.05). The decreases in QY_max under HS on days 12, 16 and 20 were significant (p < 0.05). Under FS, significant decreases in QY_max of 15.31% and 22.25% (p < 0.05) occurred on the 12th and 16th days, respectively, and a nonsignificant decrease of 7.54% was observed on the 20th day. Figure 3b shows that the Fv/Fo of mulberry seedlings under SS, HS, and FS decreased significantly, by 29.2%, 43.15% and 34.48%, respectively (p < 0.05). On days 8, 12 and 16 during the flooding period, the Fv/Fo of mulberry seedlings under SS decreased by 17.69%, 5.63%, and 16.76%, respectively; it decreased by 39.49% on the 20th day, which was a significant difference (p < 0.05). The Fv/Fo of mulberry seedlings under HS and FS significantly increased, by 43.01% and 32.24% respectively, on day 8. However, there was a significant downward trend from days 12–20 of flooding (p < 0.05).
Effect of submergence stress on QY_max and Fv/Fo in Morus alba. Different uppercase letters indicate that the means of the different groups at the same time are significantly different (p < 0.05), and different lowercase letters indicate that the means of the same group at different times are significantly different (p < 0.05). This figure was drawn using Origin Pro 2021 v. 9.8.0.200.
Effects of waterlogging stress on the closure degree (1-qP_Lss) and the rate of steady-state fluorescence decay (Rfd_Lss) of the PS II reaction centre in mulberry leaves
The effects of waterlogging stress on the closure degree (1-qP_Lss) and the rate of steady-state fluorescence attenuation (Rfd_Lss) of the PS II reaction centre in mulberry leaves are shown in Fig. 4a,b. The 1-qP_Lss of mulberry seedling leaves increased sharply after 4 days of waterlogging stress, by 13.25%, 45.33%, 69.42% under SS, HS and FS respectively (p < 0.05), compared with that before waterlogging (Fig. 4a). The increases in 1-qP_Lss under SS, HS, and FS on day 4 were significant. The 1-qP_Lss in all three groups of 8–16 days of flooding showed a relatively stable trend, and the 1-qP_Lss under SS after 20 days of flooding decreased significantly by 10.84%, compared with that on previous days. In addition, the leaf 1-qP_Lss values under HS and FS were higher than that under SS during the whole flooding period, and there were significant differences between the two groups at 4, 8, 16 and 20 days of flooding (p < 0.05). The Rfd_Lss of SS, HS, and FS decreased by 65.76%, 61.6%, and 84.89% respectively after 4 days of flooding (p < 0.05), and the values were significantly different (Fig. 4b). The Rfd_Lss values under SS and HS showed a gentle downward trend after 8–20 days of flooding. The Rfd_Lss under FS significantly increased, by 78.31%, after 12 days of flooding and decreased by 45.59% after 16 days of flooding. In addition, the Rfd_Lss values under SS and HS were significantly higher than that under FS (p < 0.05) after 4, 8 and 16 days of flooding.
Effect of submergence stress on 1-qP–Lss and Rfd–Lss values in Morus alba. Different uppercase letters indicate that the means of the different groups at the same time are significantly different (p < 0.05), and different lowercase letters indicate that the means of the same group at different times are significantly different (p < 0.05). This figure was drawn using Origin Pro 2021 v. 9.8.0.200.
Effects of waterlogging stress on Morus alba chlorophyll fluorescence imaging
The fluorescence imaging results for QY_max of mulberry seedlings under different submergence depths are shown in Fig. 5. The results showed that the leaves of mulberry seedlings under the three flooding depth treatments exhibited strong fluorescence values before flooding and that the fluorescence intensity of mulberry seedlings under the flooding depth treatments decreased as the flooding period continued. Among them, the decrease in fluorescence values of mulberry seedlings was the slowest under SS, and there was a significant decrease at 20 days after flooding under SS. The fluorescence values of mulberry seedlings under HS and FS decreased significantly on the 4th day of flooding, and the fluorescence values of FS were significantly lower than those of SS and HS on the 20th day of flooding. In addition, the fluorescence intensity of QY_max at the tip of the mulberry seedling leaves decreased first; then, the fluorescence value at the edge of the blade began to decrease, and the decrease finally extended to the entire leaf blade.
Fliorescence imaging showing the effect of submergence stress on QY_max values in Morus alba. The figure was drawn using Photoshop CS6 V13.0.
Correlations between various chlorophyll fluorescence indexes of mulberry leaves under waterlogging stress
Table 1 shows that the various chlorophyll fluorescence indicators were highly correlated. Fm had a very significant positive correlation with Fo, QY_max, Fv/Fo, and Rfd_Lss and a very significant negative correlation with 1-qP_Lss. Fo had a very significant positive correlation with QY_max, Fv/Fo, and Rfd_Lss and a very significant negative correlation with 1-qP_Lss. QY_max has a very significant positive correlation with Fv/Fo and Rfd_Lss and a significant negative correlation with 1-qP_Lss. 1-qP_Lss had a very significant negative correlation with Rfd_Lss.
Source: Ecology - nature.com
