www.nature.com/scientificreports OPEN Leaf morphology and chlorophyll fuorescence characteristics of mulberry seedlings under waterlogging stress Liangyi Rao1,2*, Siyuan Li1,2 & Xue Cui1,2 Because of its high fooding tolerance, in recent years, mulberry has become a tree species that is used in plant restoration in impact zones in reservoir areas. Therefore, 3-year-old potted forage mulberry seedlings were used to investigate the mechanism of mulberry adaptation to fooding stress. An indoor simulated fooding method was adopted to study the morphology of mulberry seedling leaves and the changes in leaf chlorophyll fuorescence parameters and fuorescence imaging under diferent fooding times and depths. The results showed that the leaves of mulberry seedlings treated with shallow submergence remained healthy during the fooding period, while the leaves of mulberry seedlings treated with half submergence and full submergence showed diferent degrees of waterlogging symptoms in the middle and late fooding periods and formed adventitious roots at the base of the stem. Most of the chlorophyll fuorescence parameters decreased at the beginning of fooding, but the steady-state degree of closure of PS II reaction centres (1-qP_Lss) increased signifcantly. In the later stage of fooding, the fuorescence parameters showed relatively stable trends. Based on these results, we conclude that mulberry has high fooding tolerance due to a combination of morphological and physiological responses. Mulberry (Morus alba L.) is a deciduous tree or shrub in the mulberry family, that has a well-developed root system and has been widely cultivated worldwide for a long time 1. Several studies have reported that mulberry trees have good adaptability to waterlogging stress, and those planted in the water-level-fuctuating zone (WLFZ) of the Tree Gorges Reservoir area (TGRA) at elevations ranging from 170 to 175 m survived and grew well afer several winter foods2. Te germination rate of mulberry seedlings at an elevation above 170 m in the WLFZ reached 62.6% afer 90 days of submerged fooding. Although mulberry seedlings at elevations below 165 m experienced very long periods of fooding (214 days), most mulberry seedlings from that area could still germinate normally due to their developed root systems3. Other studies have also pointed out that mulberry trees exhibit high drought tolerance by increasing their root absorptive area, reducing stomatal conductance and enhancing their capacity for water retention 4. In addi- tion, it was found that mulberry trees could slow surface runof, protect soil resources and isolate the heavy metal pollutants in the TGRA5 . Terefore, mulberry trees are considered to be candidate species for vegetation restoration in the WLFZ of the TGRA. Photosynthesis in plants is highly sensitive to biotic and abiotic stresses 6, so studying the photosynthetic char- acteristics of plants is an efective way to reveal the mechanisms of their adaptation to diferent environments 7. Te dynamic changes in chlorophyll fuorescence in plants can characterize the local changes in the function of the photosynthetic apparatus8. Chlorophyll fuorescence imaging technology has been recognized as a non- destructive and non-invasive technique for detecting the relationship between photosynthesis and the plant environment9,10. In recent years, much research has been carried out on the efects of adversity stress on plant fuorescence characteristics. For example, the efects of waterlogging stress on the photosynthetic characteristics of Pterocarya stenoptera C. DC., Salix integra CV. ‘Hakuro nishki’, Hemarthria altissima (Poir.) Stapf et C. E. Hubb., Phragmites australis (Cav.) Trin. ex Steud. and Distylium chinense (Fr.) Diels11–15; the efects of salt stress on the photosynthetic characteristics of Morus alba L., Populus tomentosa Carr., Celtis sinensis Pers. and Ulmus pumila L. seedlings 16–18, and the efects of drought stress on the photosynthetic characteristics of rootstocks of 1College of Soil and Water Conservation, Beijing Forestry University, Beijing 100083, China. 2Engineering Research Center of Forestry Ecological Engineering of Ministry of Education, Beijing 100083, China. *email: raoliangyi@ bjfu.edu.cn Scientifc Reports | (2021) 11:13379 | https://doi.org/10.1038/s41598-021-92782-z 1 Vol.:(0123456789) www.nature.com/scientificreports/ SS SS HS HS 9 5 FS FS 8 4 7 t t n n a a l l p p 6 r r e e p p s s 3 e e v v 5 a a e e l l f f o o r r 4 e e b b 2 m m u u n n 3 e e h h T T 2 1 1 0 0 0510 15 20 0510 15 20 Flooding time/d Flooding time/d (a) (b) SS SS HS HS 11 8 FS FS 10 7 9 s t t 6 o n 8 a o l r p s r u e 7 o i p 5 t i s t e n v e a 6 v e d l a 4 f f o 5 o r r e e b b m 3 m u 4 u n n e e h 3 h T T 2 2 1 1 0 0 0510 15 20 0510 15 20 Flooding time/d Flooding time/d (c) (d) Figure 1. Efect of submergence stress on leaf morphology in Morus alba: (a) Te number of curled or wilted leaves per plant; (b) Te number of brown spots or rotten leaves per plant; (c) Te number of fallen leaves per plant; (d) Te number of adventitious roots. Tis fgure was drawn using Origin Pro 2021 v. 9.8.0.200. Vitis vinifera Linn. and leaves of Fragaria × ananassa Duch.19,20 have been studied. However, studies on the chlo- rophyll fuorescence characteristics of mulberry seedlings under waterlogging stress have not been reported. Te main objectives of this study were to quantify the leaf morphology and chlorophyll fuorescence characteristics of 3-year-old mulberry seedlings under diferent depths of waterlogging stress and to reveal their photosynthetic physiological and ecological responses under diferent levels of waterlogging stress. Results Efects of waterlogging stress on leaf morphology in mulberry seedlings. Figure 1 shows the change in the leaf morphology of mulberry seedlings under diferent submergence depths. Te results showed that the seedlings under both SS and HS could grow well, and there were 3 slightly wilted leaves on average under FS. Tere were 3 wilted leaves and 2 defoliated leaves on average in the HS group afer 10 days of fooding, 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 afer 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 Scientifc Reports | (2021) 11:13379 | https://doi.org/10.1038/s41598-021-92782-z 2 Vol:.(1234567890) www.nature.com/scientificreports/ SS 600 HS FS aA aA aA bA 400 cdA cA bB dA bB bB cB eA cC cA Fo cC dC eC 200 fB 0 0510 15 20 Flooding time/d (a) SS 1000 HS FS aA aA aA 800 bA 600 bB cA bA cA cA Fm cC cB cB cB 400 dC dA dA eC fB 200 0 0510 15 20 Flooding time/d (b) Figure 2. Efect of submergence stress on Fo and Fm in Morus alba. Diferent uppercase letters indicate that the means of the diferent groups at the same time are signifcantly diferent (p < 0.05), and diferent lowercase letters indicate that the means of the same group at diferent times are signifcantly diferent (p < 0.05). Tis fgure was drawn using Origin Pro 2021 v. 9.8.0.200. seedling, and no adventitious roots developed. Te 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. Efects of waterlogging stress on initial fuorescence (Fo), and maximum fuorescence (Fm) under dark adaptation in mulberry leaves. Te initial fuorescence value (Fo) and the maximum fuo- rescence value (Fm) of mulberry seedlings signifcantly 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 signifcantly diferent (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 signifcant decrease of 9.5% (p < 0.05) on the 20th day of water- logging. Te Fo under HS and FS signifcantly increased, by 12.56% and 25.62%, on the 8th day of waterlogging (p < 0.05), respectively, and the Fo under FS showed a signifcant downward trend at 12–20 days of fooding. Fig- Scientifc Reports | (2021) 11:13379 | https://doi.org/10.1038/s41598-021-92782-z 3 Vol.:(0123456789) www.nature.com/scientificreports/ ure 2b shows that Fm under SS, HS, and FS decreased signifcantly, by 40.54%, 37.67% and 51.6%, respectively, afer 4 days of fooding (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. Te diferences under SS, HS and FS were signifcant (p < 0.05). Te Fm under SS and HS showed a relatively stable trend in the late stage of fooding, while the Fm under FS showed a signifcant decreasing trend (p < 0.05). In addition, the Fo and Fm of mulberry seed- lings under HS were signifcantly higher than those in SS and FS from days 4 to 16 of fooding.