Physiological Responses and Tolerance Evaluation of Five Poplar Varieties to Waterlogging

Waterlogging resistance of five poplar varieties, ‘Danhongyang’ (DHY), ‘Juba-261’ (JB-261), ‘Zongqiansanhao’ (ZQ-3), ‘Zhonglin-2025’ (ZL-2025), and ‘Nanlin-895’ (NL-895), was evaluated under the simulated waterlogging conditions. Data on changes in leaf color and morphology as well as in biochemical indices, such as chlorophyll, malonaldehyde, soluble protein, soluble sugar content, superoxide dismutase (SOD), peroxidases (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione peroxidase (GSH-PX) activities, relevant to submergence stress, were analyzed. The principal component analysis of the data identified the waterlogging resistance coefficient of the indices, which showed that waterflooding brought about different degrees of damage in the five poplar varieties, with DHY having the lowest waterlogging index. The leaf pigment content of the poplar was remarkably decreased by waterlogging, whereas malondialdehyde (MDA) and proline contents were enhanced, but in different extents among the poplar varieties. Nearly all other poplar varieties showed a tendency of decline in JB-261, ZQ-3, ZL-2025, NL-895, except for SOD activity in DHY, which increased under submergence stress. Poplar varieties had varying degree of changes in POD activity, and APX activity tended to increase upon waterlogging. GR also displayed increasing tendency in JB-261, ZL-2025 and NL-895, except for in ZQ-3, which declined under waterlogging stress. GSH-PX except for ZQ-3 displayed no significant change, which showed a tendency of decline in DHY, JB-261, ZL-2025, and NL895. Principal component analysis allowed us to reduce16 indices to four independent indices. The subordinate function analysis identified that the DHY variety had the highest waterlogging tolerance, whereas the NL-895 variety had the lowest waterlogging tolerance among tested varieties.


Introduction
Waterlogging is the second major agricultural disaster in China, accounting for approximately 24% of natural disasters (Wang et al., 2015). Excessive soil moisture disrupts the water balance of plants and has a remarkable impact on plant morphology and metabolism, thereby restricting plant growth (Thomas et al., 2003;Ye et al., 2003). Submergence deprives plants of oxygen and inhibits aerobic respiration, resulting in the accumulation of toxic substances such as ethanol and lactic acid. Thus, absorption of minerals was inhibited, changed in the hormonal level of plants and disordered metabolic (Visser et al., 1994；Geigenberger, 2003Kaelke and Dawson, 2003). In the mung bean under submergence stress, the activities of superoxide dismutase (SOD), peroxidase, glutathione reductase, and ascorbate peroxidase (APX) are elevated at the beginning of flooding to eliminate free radicals accumulating in the cells (Ahmed et al., 2002). Upon flood treatment, the primary root of Camptotheca acuminata Statistical analysis Calculation of waterlogging resistance coefficient (Zhu et al., 2017) was as follows. Waterlogging tolerant coefficient = [Process measurement / Control test value] × 100% (1). gradually rots; root activity declines, lactate dehydrogenase activity increases, and the O2 -, H2O2, and malonaldehyde contents of the leaf gradually increase. Submergence stress induces increase of the antioxidant enzyme activity in various plant cells in varying degrees, explaining the different waterlogging tolerance of plants to some extent (Keles and Öncel, 2002).
The Jianghan Plain in China is located in the middle and lower reaches of the Yangtze River. It has large areas of low wetlands and mudflats. The presence of vast wetland and aquatic plant species provide an opportunity for the development and study of waterlogging-resistant tree species. Because annual precipitation in Jianghan Plain has been increasing year by year recently, the demand for waterlogged poplar varieties has also been increasing. In spite of the great number of research and fruitful achievements on directive breeding, genetics, and seed breeding, systematic research on the waterlogging resistance of poplar, especially on its physiological and biochemical aspects, is still limited. As a result, the selection of waterlogging-resistant poplar varieties suitable for Jianghan Plain is still in the initial stage. Therefore, the present experiment was conducted to illustrate the response mechanism of different poplar varieties against submergence stress so as to establish a waterlogging evaluation system of poplar in Jianghan Plain.

Plant materials and treatments
The experimental site was located in the botanical garden of Yangtze University in Jingzhou, Hubei, China (around N30.35, E112.14). The experimental materials were 1-year old poplar cuttings taken from five cultivars: 'Danhongyang' (DHY), 'Juba-261' (JB-261), 'Zhongqian-3' (QZ-3), 'Zhonglin-2025' (ZL-2025) and 'Nanlin-895' . Planting of the cuttings was started on March 20, 2017. The stem cuttings were planted in plastic flowerpots (10 cm in inner diameter and 25 cm in depth). The culture medium was vermiculite, coconut shell powder and pearl, uniformly mixed with the ratio of 5: 3: 2 and placed in the greenhouse for seedling cultivation, two cutting seedlings of each pot. During this period, the cultivation and management measures were undertaken to maintain consistency until the end of the flooding. The cutting seedlings that reached at uniform growth of about 80 cm height were selected for waterlogging treatment on July 17. The experiment was designed as a random group with 3 replications, and every cultivar was treated with 9 pots, three plants per pets. The flooding treatment was such that the water surface was about 10 cm above the soil surface, and water was replenished daily to keep the water level constant. Control plants were watered every evening to maintain the soil moisture content at 60-80% of the maximum field water capacity.
During this period of the experiment, the morphological characteristics were recorded every 10 days with photo record. After flooding for 65 days, three mature leaves in the middle section of the plants were harvested and immediately frozen in liquid nitrogen to store at -80 °C. The frozen materials were used to determine their physiological and biochemical indices.
Principal component analysis of waterlogging tolerant was performed on the waterlogging tolerant of individual photosynthetic indexes. The original single indicator was transformed into a new independent comprehensive indicator.
The membership function value of the composite index was obtained by the formula of Zhou et al. (2003). U(Xj) = (Xj-Xmin) / (Xmax-Xmin), j = 1, 2, ……，where, Xj represents the Jth comprehensive index, Xmin represents the Jth minimum comprehensive index value, Xmax represents the Jth maximum comprehensive index value. If a certain index is negatively correlated with waterlogging tolerance, the value of its waterlogging tolerance membership function can be calculated through the inverse membership function.
The specific formula is as follows: X(μ) =1-(X-Xmin) / (Xmax-Xmin). The comprehensive value of waterlogging tolerance of varieties was obtained by summing up the specific waterlogging subordinate values of each index. The higher the comprehensive value was, the stronger the waterlogging resistance.
The weight of each comprehensive index was calculated according to the contribution rate of the comprehensive index (Rui-Juan et al., 2013). Wj =Pj/∑(j=1)^nPj =1, 2, ……. Where, Wj value represents the importance of the Jth comprehensive index in all comprehensive indexes, and Pj is the contribution rate of the Jth comprehensive index of each variety.
Comprehensive assessment was determined as follows: Data were analyzed with one-way ANOVA using SPSS22.0 for Windows (SPSS Inc., Chicago, IL). The means were compared with Duncan's multiple range tests. P <0.05 was considered to be statistically significant.

Effect of waterlogging stress on the growth of poplar varieties
Biomass change is an important index of plant resistance. Once the five poplar varieties experienced submergence stress, their biomass change displayed remarkable differences among them (Table 1). During the submergence treatment for 65 d, the biomass of 'Danhongyang' (DHY) increased by 140% compared with that before submergence, indicating that DHY can grow well even under submergence condition. However, the biomass of the (JB-261, QZ-3, ZL-2025 and NL-895 varieties was increased by only 15 to 45% indicating that they were severely affected by waterlogging stress. On the basis of biomass change, the waterlogging tolerance of DHY was judged to be higher than those of the four other varieties.

Effect of waterlogging stress on the leaf morphology
The changes in the leaves and root system in the five poplar varieties after submergence for 65 d were as follows. Decaying and yellowing in the large area of the leaves, as well as margin rolling, leaf abscission, were observed in four poplar varieties, namely, ZO-3, JB-261, ZL-2025, and NL-895. Only a small area of yellowing is found in the leaf at the lowest level ( Fig. 1), and emerald is still relatively high as a whole, especially for ZL-2025, which has a waterlogging injury index as high as 0.69 (Table 2), thereby showing poor waterlogging resistance. Nevertheless, no large-scale yellowing and rolling were found in the DHY leaf, whose waterlogging injury index was only 0.28, thereby showing strong waterlogging resistance.
The stem bases of all the five poplar varieties underwent submergence treatment showed varying degree of adventitious root appearance.
Effect of waterlogging stress on the content of leaf pigment, soluble sugar, and soluble protein of the poplar varieties. Fig. 2 shows that the contents of chlorophyll a, chlorophyll b, carotenoid and the total chlorophylls of five poplar varieties under waterlogging treatment were remarkably lower than those of the corresponding control group, albeit with different degrees among varieties. It is of note that decreases in contents of the total chlorophyll and carotene of JB-261 under submergence treatment was lower compared to control values, and the chlorophyll a, chlorophyll b, carotenoid and the total chlorophylls are all 60-80% of the control group.  However, ZL-2025 and NL-895 exhibited most severe decrease in chlorophyll a, chlorophyll b, carotenoid, and the total content of chlorophyll contents. The values were all lower by approximately 35% than that of the control group. The four pigment indices of 'Zongqiansanhao' (ZQ-3) under submergence stress remained at approximately 50% of the control group. These results showed that DHY and JB-26 under submergence condition could keep their leaf pigment content at a high level. Hence, the analysis confirmed the stronger waterlogging resistance of DHY and JB-26 than other poplar varieties. In addition, chlorophyll a / chlorophyll b (chl a/chl b) was no significant changes in DHY and ZQ-3 of control compared to waterlogging, suggesting that it was slight resistance under waterlogging stress. However, chl a/chl b appeared to be an increasing trend in the JB-261 and ZL-2025 of control compared to waterlogging and decreasing tendency in the NL-895 of control compared to waterlogging (Fig. 2E).
Submergence for 65 d caused no significant changes in soluble sugar content in the leaves of ZL-2025 and NL-895 ( Fig. 3A). At the same time, soluble sugar in the leaves of the other three varieties exhibited considerable increase, 25% for DHY, 70% for ZQ-3, and 65% for JB-261 compared to that of the control group.
Submergence treatment had different impacts on the soluble protein content among the poplar varieties. The three other poplar varieties all had remarkable increases in 661 ZQ-3, JB-261 and NL-895, compared with the control group, in which DHY and ZL-2025 had large increasing amplitudes. The soluble protein in the leaf in DHY and ZL 2025 increased by 80% and 94% compared with the control group, respectively. Fig. 4A shows that MDA content in the ZL-2025 leaf was 41% higher than that of the control group. In contrast, MDA content in DHY, JB-261 and NL-895 were lower than the control group by 26%, 28% and 24%. ZQ-3 did not show remarkable change in MDA content. The proline content increase was highest in the leaves of DHY up to 49%, and in JB-261, ZL-2025 and NL-895 were higher than the control group by 38%, 33% and 33%. (Fig. 4B). However, proline content of ZQ-3 had no significant difference compared with the control group.

Effect of submergence stress on antioxidant enzyme activity in the leaf of poplar varieties
Submergence stress can greatly affect SOD enzyme activity (Fig. 5A) in the leaves of poplar trees, but with different degrees of influence. Under submergence stress, T-SOD and CuZn SOD activities in the leaf of only the DHY variety were higher by 45 and 118% than those of the control group, respectively ( Fig. 5A and B).   (Yiu et al., 2009). The data well explains adaptability of DHY, JB-261, and NL-895 against the submergence stress. When subjected to submergence stress for 65 d, ascorbate peroxidase (APX) activity in the leaf of all the five poplar varieties was remarkably induced (Fig. 5D). DHY and JB-261 were 345 and 195% higher than that of the control group (P<0.05), respectively. The remaining three other poplar varieties, ZQ-3, ZL-2025, and NL-895, also showed significant yet lower than those of the other two ranging 103 to 68% increase. This outcome again reconfirmed the high water-logging tolerance of DHY and JB-261.
Submergence caused little change in GR activity for DHY and increase in the activity for JB-261, ZL-2025, and NL-895, ranging from 40 to 155% (Fig. 5E). Even though ZQ-3 showed decrease in GR activity, the extent was only 17% (Fig. 5E). Therefore, change in GR activity did not explain water-logging tolerance among the varieties. It is possible that preexisting high GR activity in DHY did not call for further induction of GR.
The effect of submergence on GSH-PX activity in the five poplar varieties again did not follow the pattern of water-logging stress (Fig. 5F). GSH-PX activities in four poplar varieties were significantly lowered upon waterlogging stress by 31 to 69%, except for ZQ-3 which showed no significant difference. comprehensive index can be estimated according to the size of the contribution rate. The comprehensive index value of each poplar was calculated according to the index coefficient of each comprehensive index (Table 5) and the waterlogging resistance coefficient of each single index (Tables 3 and 6). The weight of four comprehensive indices can be calculated according to the CI contribution rate of each principal component, namely, CI (1)-CI (4), as given above. The D value of the comprehensive waterlogging resistance of poplar varieties was calculated according to the comprehensive evaluation formula (Table 6), in which the maximum D value of DHY is 0.671. The minimum D value of NL-895 is 0.082. The waterlogging resistance of the five poplar varieties was ranked according to the D value: DHY>JB-261>ZQ-3>ZL2025>NL-895. The results showed that DHY poplar had the highest waterlogging tolerance, whereas NL-895 poplar had the lowest tolerance.

Comprehensive analysis and evaluation of poplar waterlogging resistance
The principal component analysis was performed based on the related waterlogging-resistance coefficients of morphological and biological indices of the five poplar varieties (Table 5).
It can be seen from the correlation coefficient matrix ( Table 3) that all physiological indicators have positive and negative correlations to different extent, so that the information they provided was overlapped. The contribution rate of the first four comprehensive indicators CI (1)-CI (4) was 0.474, 0.307, 0.134, and 0. 084. If the sum of the four indicators reaches 1.0, the contribution of all indicators is fully integrated. This contribution transferred the 16 original single indices to four new and relatively independent comprehensive indices, which completely represent the information content of the 16 indices. At the same time, the relative importance of each 664    outstanding waterlogging resistance among the five poplar varieties. MDA is the final product of membrane lipid peroxidation. The protective ability of the tissues is weakened when MDA accumulation is high. Generally, the degree of membrane lipid peroxidation in the poplar varieties with high flood-resistance is lower than the varieties with low flood-resistance (Loreti et al., 2010). In the present study, the MDA content of the tested poplar varieties, with exception of ZL-2025, were decreased compared with that of the control group. Especially, DHY, JB-261N, and NL-895 showed significant decrease. This finding indicates that these three poplar varieties had an ability to prevent membrane lipid peroxidation and thus explains high resistance of the varieties against waterlogging. Proline accumulation in the plant leaf is usually caused by osmotic stress ). In the present experiment, the observed significant increase in proline content of DHY under waterlogging condition could account for the high resistance of DHY against water stress.
To resist the toxicity of reactive oxygen species under submergence stress, plants also have developed complex antioxidant defense systems. Primary defense employs nonenzyme substances, such as ascorbic acid, glutathione, and vitamin E and further defense is achieved by enzymes such as active-oxygen-processed enzymes (SOD and POD), APX, GR, glutathione peroxidase (GSH-PX), as well as the antioxidant regeneration enzymes. Combined force of these systems can alleviate the damage of reactive oxygen species to plants under submergence stress (Thirunavukkarasu et al., 2009). Kumutha et al. (2009) showed that the antioxidant enzyme activity of the waterlogging-resistant genotype of cajanus increased continuously when exposed to the submergence condition, whereas the antioxidant enzyme activity of the sensitive genotype decreased after submergence for 2 d. High SOD activity is known to be important for plants to resist submergence stress (Tan et al., 2009). In the present experiment, T-SOD and CuZn SOD activities in the DHY variety under submergence stress increased significantly, thereby showing strong resistance toward oxidative stress. The POD activities of DHY, JB-261, and NL895 were remarkably higher than that of the control group (Fig. 5C). The high POD activities of the varieties can explain considerable waterlogging resistance of these poplar cultivars, as Zhu (2017) had demonstrated with peony. Although the APX activities of all the tested poplar varieties were considerably higher than that of the control group (Fig. 5D), DHY exhibited the highest fold increase (about 300% increase compared to control) among the varieties. This again indicates high resistance of DHY against waterlogging.
GSH-PX, together with SOD, peroxide (POD), and catalase (CAT), constitutes the major cellular antioxidant enzyme system (Blokhina et al., 2003;Islam and Macdonald, 2004). GSH-PX mainly removes the lipid and hydrogen peroxides, and thus protects the macromolecules of the biological membrane from being destroyed by Oxidation. Ascorbic acid-glutathione cycle, where GR plays an important role in maintaining intracellular ASA and GSH levels, is an important means to detoxify reactive oxygen in plants (Shu et al., 2011). The present experimental result showed that the GR activities in the Discussion Submergence stress inhibits plant growth from many aspects. However, under submergence condition, plants with waterlogging tolerance can resist the damages brought by environmental changes by transforming their Physiology signs and morphologies (Bailey-Serres and Voesene, 2008). Submergence stress changes chlorophyll content and Composition and destroys photosynthetic mechanism and performance by slowing down the growth of leaves and roots, thus inhibiting the growth and biomass accumulation of plants (Voesenek et al., 2013;Kissmann et al., 2014). The degree of growth inhibition varied with poplar clones. Under submergence stress, the biomass of DHY could still maintain growth, albeit in a lower rate compared with that without submergence, showing strong waterlogging resistance. However, JB-261 was the most affected, with its roots severely inhibited by waterlogged stress, and showed poor waterlogging resistance.
Many studies showed that when plants suffer from submergence stress, chlorophyll a, chlorophyll b, and total chlorophyll contents in the leaf of the plants decrease. Ge et al. (2014) showed that water stress decreases not only chlorophyll (Zhou et al., 2017) but also carotenoid content. In the present study, chlorophyll and carotenoid content both displayed a decreasing tendency compared with the control group. The decreasing tendencies of chlorophyll and carotenoid in the leaves of DHY and JB-261 were lower compared with those of other poplar varieties, indicating DHY and JB-261 had stronger waterlogging tolerance than other poplar varieties.
Soil flooding exposes plant roots to hypoxia or anoxia, which severely limits availability of energy and sugar, and produces toxic substances such as ethyl alcohol and acetaldehyde (Sairam et al., 2008), severely affecting plant growth. High levels of soluble substances, such as soluble sugar and protein (Yin et al., 2009), accumulate in plants to ensure adjustment toward osmotic stress. Pociecha (2013) posited that plants could improve their waterlogging resistance and survival under submergence stress by accumulating additional carbohydrate stores. Kreuzwieser et al. (2015) assumed that some sensitive plants consume soluble sugar after being submerged for a while, whereas some waterlogging-resistant plants maintain a stable and adequate supply of carbohydrates in the process of flooding to regulate glycolysis, which is the key for woody plants to survive under hypoxic stress. In the present experiment, the soluble sugar content of DHY, ZQ-3, and JB-261, all showing high waterlogging resistance, still remained at a high level. However, other factors need to be combined to determine which variety has the strongest waterlogging tolerance. Soluble protein content in plants affects its growth development and aging process. Under waterlogging condition, the variety with strong waterlogging resistance has more soluble protein contents than the variety with weak waterlogging resistance (Lorenzo et al., 1995). Although soluble protein content of all the five poplar varieties were higher than that of the control group, DHY and ZL-2025 showed the highest fold increase in protein content (P<0.01) compared with that of the control group, suggesting that DHY and ZL-2025 had the most four other poplar varieties are either negatively (ZQ-3) or positively (JB-261, ZL-2025, and NL-895) affected by the submergence stress, except for DHY whose activity remained virtually unchanged by water stress (Fig. 5E). Submergence stress lowered GSH-PX activities in all the tested poplar varieties, but in different degrees. The activities in DHY and ZQ-3 were least affected, indicating higher waterlogging tolerance.
Waterlogging-resistant woody plants can adapt to flooding through morphological adaptation and physiological regulation (Shimamura et al., 2010;Argusa et al., 2015;Loreti et al., 2016). Therefore, waterlogging resistance is a compound trait, including morphological growth, physiology, biochemistry, and molecular expression (Sairam et al., 2008). A single index is not accurate enough to reflect the waterlogging resistance of plants. Thus, multiple indices should be used to evaluate the waterlogging resistance of plants . In this study, changes in morphology characteristics, growth status, and physiological and biochemical indices of plants were referred to assess the waterlogging tolerance of five poplar varieties in conjunction with principals component analysis. Multi-index method was used to measure the waterlogging tolerance of the five poplar varieties. In this study, the waterlogging tolerance of various poplar varieties was comprehensively evaluated by using the methods of principal component analysis of membership function as follows: DHY>JB-261>ZQ-3>ZL2025>NL-895. This result is basically consistent with the observed result of the morphology. Of the five poplar varieties, DHY had the highest waterlogging tolerance, whereas NL-895 the lowest tolerance. In the present study, the waterlogging resistant germplasm selection and waterlogging tolerance evaluation system for poplar varieties were established in terms of the characteristics of the response mechanism to waterlogging stress. The results provide a theoretical basis for exploring the mechanism of waterlogging resistance and selecting varieties with high waterlogging resistance, thereby establishing a reference for breeding poplar with waterlogging resistance. In order to further verify poplar varieties strong waterlogging resistance, further studies will be carried out, and transcriptome data will be analyzed in the follow-up to study the specific reasons for the expression of different waterlogging resistance genes in different varieties from the transcriptional level, so as to provide theoretical basis for their stress tolerance breeding.

Conclusions
Our results have been obtained by determining multiple physiological indexes which have closely related to the waterlogging resistance of poplar varieties. Principal component analysis of five poplar varieties was carried out; through the confirmation of morphology, injury index and physiology, we finally determined that DHY has the strongest waterlogging resistance of the five varieties. Therefore, DHY with strong waterlogging resistance in production can be selected and planted with areas with more rainfall. In areas where there was not much rain, JB-261, ZQ-3, ZL2025, and NL-895 varieties with moderate waterlogging resistance can be selected. 666