Responses of Swamp Cypress ( Taxodium distichum ) and Chinese Willow ( Salix matsudana ) Roots to Periodic Submergence in Mega-Reservoir: Changes in Organic Acid Concentration

: Organic acids are critical as secondary metabolites for plant adaption in a stressful situation. Oxalic acid, tartaric acid, and malic acid can improve plant tolerance under waterlogged conditions. Two prominent woody species ( Taxodium distichum -Swamp cypress and Salix matsudana -Chinese willow) have been experiencing long-term winter submergence and summer drought in the Three Gorges Reservoir. The objectives of the present study were to explore the responses of the roots of two woody species during ﬂooding as reﬂected by root tissue concentrations of organic acids. Potted sample plants were randomly divided into three treatment groups: control, moderate submergence, and deep submergence. The concentrations of oxalic acid, tartaric acid, and malic acid in the main root and lateral roots of the two species were determined at four stages. The results showed that T. distichum and S. matsudana adapted well to the water regimes of the reservoir, with a survival rate of 100% during the experiment period. After experiencing a cycle of submergence and emergence, the height and base diameter of the two species showed increasing trends. Changes in base diameter showed insigniﬁcant differences between submergence treatments, and only height was signiﬁcant under deep submergence. The concentrations of three organic acids in the roots of two species were inﬂuenced by winter submergence. After emergence in spring, two species could adjust their organic acid metabolisms to the normal level. Among three organic acids, tartaric acid showed the most sensitive response to water submergence, which deserved more studies in the future. The exotic species, T. distichum, had a more stable metabolism of organic acids to winter ﬂooding. However, the native species, S. matsudana, responded more actively to long-term winter ﬂooding. Both species can be considered in vegetation restoration, but it needs more observations for planting around 165 m above sea level, where winter submergence is more than 200 days.


Introduction
Reservoirs have played an essential role in human development throughout the world for thousands of years by providing water and controlling flooding, as well as generating power and electricity [1,2]. The Three Gorges Dam Reservoir is one of the largest reservoirs globally, located in the Yangtze River upper reaches in China [3]. Annually, the water level in the reservoir fluctuates by 30 m from 145 m to 175 m above sea level (ASL), the to dynamic water stress caused by submergence in winter and drought in summer. This research can recognize the physiological and ecological adaptation mechanisms of T. distichum and S. matsudana as reforestation species in the hydro-fluctuation zone of Three Gorges Reservoir. We hypothesized that: (1) Both species have strong waterlogging tolerance, but high-intensity waterlogging would inhibit plant growth to some degree. (2) Oxalic acid, tartaric acid, and malic acid in the two suitable plant roots will show increasing responses to a certain degree when suffering from the dynamic variation of soil moisture in the water-fluctuation zone. (3) The growth and adaptability of two woody plants are related to their characteristics of organic acid concentrations. The findings from the water rhythms in situ will enrich researcher knowledge about the adaptive tree species and benefit the reforestation practices in the Three Gorges Reservoir region.

Study Region
The experimental Plot (107 • 32 -108 • 14 E, 30 • 03 -30 • 35 N) is located along the Ruxi River, Shibaozhai, Gonghe Village, Zhong County, Chongqing Municipality, China. Ruxi River is a tributary of the Yangtze River, with a subtropical, southeastern monsoonal mountain climate. The region experiences an annual accumulative temperature of 5787 • C for days ≥10 • C, an annual average temperature of 18.2 • C, and highest and lowest temperatures of 40 • C and 0 • C, respectively. The highest temperatures mainly occur from July to September. There is a frost-free period of 341 days, total solar radiation of 83.7 × 4.18 kJ/cm −2 , and an annual sunshine ratio of 29%. The mean annual precipitation is 1200 mm, and most rainfalls are in May and June. Plants suffer drought due to high air temperature and relatively fewer rainfalls in July and August [43,44].
The plants are experiencing annual cycles of periodic submergence and drought growing in the hydro-fluctuation zone of the Three Gorges Dam (Figure 1). Water level gradually rises in the fluctuation zone from September to October. It attains the highest altitude (175 m ASL) from November through to January, then gradually decreases, reaching its lowest level at 145 m ASL from June to September. the Three Gorges Reservoir, and both are considered candidates for the incorporation of reforestation. The objectives of this study were to explore the responses of the roots of two woody species during flooding conditions. The concentration of organic acids can reflect the reactions to dynamic water stress caused by submergence in winter and drought in summer. This research can recognize the physiological and ecological adaptation mechanisms of T. distichum and S. matsudana as reforestation species in the hydro-fluctuation zone of Three Gorges Reservoir. We hypothesized that: (1) Both species have strong waterlogging tolerance, but high-intensity waterlogging would inhibit plant growth to some degree.
(2) Oxalic acid, tartaric acid, and malic acid in the two suitable plant roots will show increasing responses to a certain degree when suffering from the dynamic variation of soil moisture in the water-fluctuation zone.
(3) The growth and adaptability of two woody plants are related to their characteristics of organic acid concentrations. The findings from the water rhythms in situ will enrich researcher knowledge about the adaptive tree species and benefit the reforestation practices in the Three Gorges Reservoir region.

Study Region
The experimental Plot (107°32-108°14° E, 30°03°-30°35° N) is located along the Ruxi River, Shibaozhai, Gonghe Village, Zhong County, Chongqing Municipality, China. Ruxi River is a tributary of the Yangtze River, with a subtropical, southeastern monsoonal mountain climate. The region experiences an annual accumulative temperature of 5787 °C for days ≥10 °C, an annual average temperature of 18.2 °C, and highest and lowest temperatures of 40 °C and 0 °C, respectively. The highest temperatures mainly occur from July to September. There is a frost-free period of 341 days, total solar radiation of 83.7×4.18 kJ/cm −2 , and an annual sunshine ratio of 29%. The mean annual precipitation is 1200 mm, and most rainfalls are in May and June. Plants suffer drought due to high air temperature and relatively fewer rainfalls in July and August [43,44].
The plants are experiencing annual cycles of periodic submergence and drought growing in the hydro-fluctuation zone of the Three Gorges Dam (Figure 1). Water level gradually rises in the fluctuation zone from September to October. It attains the highest altitude (175 m ASL) from November through to January, then gradually decreases, reaching its lowest level at 145 m ASL from June to September. Oct. Jul. Jan. Apr.

Experiment Design
Two-year-old potted seedlings of T. distichum and S. matsudana were cultivated in a greenhouse in Shibaozhai, Zhong County, Chongqing, China, close to the study area, with

Sample Analysis
Before collecting each sample, plant height and base diameter of two species were measured using a height measuring rod and vernier calipers, respectively.
Primary roots were separated from the laterals root of each plant when collecting samples and immediately transported to the laboratory in an icebox. They were placed in an oven at 110 • C for 15 min after washing with deionized water, then adjusted to 80 • C to dry until constant weight [45,46], and finally ground into a powder then sifted through a 1 mm sieve.
A 0.1 g sample of root powder was placed in a 10 mL centrifuge tube with 5 mL of ultrapure water and ultrasonicated for 1 h, then cooled to room temperature. After centrifugation at 8000 rpm for 10 min, the supernatant was filtered through a 0.45 µm syringe filter (Millipore, Danvers, MA, USA) to determine the concentrations of oxalic acid, tartaric acid, and malic acid [47].
A high-performance liquid chromatography method was used to determine the organic acid concentration in dried roots by the Sepax Sapphire C18 column (4.6 mm × 250 mm, 5 µm). The mobile phase aqueous and organic components were 95% 20 mmol·L −1 KH 2 PO 4 buffer (pH 2.5, adjusted with phosphoric acid) and 5% methanol. The HPLC instrument was coupled to an Agilent 1100 diode array multi-wavelength detector. The mobile phase flow rate was 0.9 mL·min −1 , the detection wavelength was 210 nm, the column temperature was 30 • C, and the injection volume was 20 µL [47,48]. Organic acids in main and lateral roots were measured in milligrams of organic acid per gram of the mains root and lateral roots dry matter (mg.g −1 , DW).

Statistical Analysis
SPSS 22.0 was used for statistical analyses. Paired sample t-tests were used to analyze the differences of growth between different experimental periods for the same treatment ( Table 2). Tukey's tests were applied to determine the significance of differences between different submerging treatment groups (Table 2). Repeated measures ANOVA was conducted to assess the effects of submergence, experimental period, and their interaction on the concentrations of organic acids for T. distichum and S. matsudana (Table 3). Repeated measurement analysis of variance and Bonferroni method were carried out to analyze the differences of organic acids among different experimental periods for the same plant [49] ( Figures 2 and 3). One-way ANOVA and Tukey's tests were carried out to analyze the differences of organic acids among different treatment groups for the same plant ( Figure 4). The p-value for statistical tests was 0.05.

Growth of T. distichum and S. matsudana Plants
The survival rate was 100% throughout the experiment for both species. The heights and base diameters of T. distichum and S. matsudana plants were increased after one year of growth (Table 2). Compared to the initial values at T0 (15 September 2015), the plant height of two species in the SS and MS groups and the base diameter of T. distichum in the SS group at T3 (15 September 2016) were significantly higher. Winter submergence signif-

Growth of T. distichum and S. matsudana Plants
The survival rate was 100% throughout the experiment for both species. The heights and base diameters of T. distichum and S. matsudana plants were increased after one year of growth (Table 2). Compared to the initial values at T0 (15 September 2015), the plant height of two species in the SS and MS groups and the base diameter of T. distichum in the SS group at T3 (15 September 2016) were significantly higher. Winter submergence significantly inhibited the growth height of plants in the DS groups. In contrast, the base diameter of T. distichum and S. matsudana plants showed fewer differences among different treatment groups. Table 3 shows the repeated measures of ANOVA results for organic acid concentrations in the roots of T. distichum following different water treatments, experimental periods, and their interaction. The experimental period had the most significant effect on the concentrations of organic acids in the root system. Different water treatments significantly affected tartaric acid in the main and lateral root and malic acid in the main root. In addition, tartaric acid and malic levels in the main root were significantly affected by treatment and experimental period interaction.

Organic Acid Concentrations in T. distichum Roots
The response of the oxalic acid, tartaric acid, and malic acid concentrations in T. distichum roots to soil moisture was shown in Figure 2. The concentrations of three organic acids in the main and lateral roots displayed similar responses to the changes in soil moisture in the fluctuating water zone. Most of them increased during the flooding stress period (T0-T1), then decreased during the recovery growth period (T1-T2), and were no significant changes under mild drought stress (T2-T3). The concentrations of three organic acids in roots were influenced by winter submergence. The concentrations of tartaric acid in lateral roots in the MS and DS groups and malic acid in lateral roots in the DS group were significantly decreased compared with that in SS group. After emergence in spring, three organic acid concentrations had no differences among the three water treatments.

Organic Acid Concentrations in S. matsudana Roots
Different flooding treatments in the water fluctuation zone significantly affected oxalic acid concentration in the lateral roots and the tartaric acid concentrations in the main and lateral roots of S. matsudana (Table 3). Except for malic acid in the main roots, oxalic acid and tartaric acid in both the main and lateral roots, and malic acid in the lateral roots of S. matsudana were significantly affected by different experimental periods. The interaction between treatment and experimental periods had little effect on organic acids in the roots of S. matsudana. Only the oxalic acid concentration in lateral roots was significantly affected.
The response of the oxalic acid, tartaric acid, and malic acid concentrations in S. matsudana roots to soil moisture was shown in Figure 3. The concentrations of oxalic acid in the main and lateral roots and tartaric acid in the lateral roots were increased during the flooding stress period (T0-T1), then decreased during the recovery growth period (T1-T2), and were no significant changes under mild drought stress (T2-T3). The concentrations of malic acid in the main and lateral roots and tartaric acid in the main roots increased during the flooding stress period (T0-T1) and the recovery growth period (T1-T2), and then decreased under mild drought stress (T2-T3). Under water submergence in winter, the concentration of oxalic acid in the lateral roots and tartaric acid in the main roots were significantly increased. All acid concentrations in each treatment showed no significant difference after emergence from winter flooding. Figure 4 showed the difference in mean concentrations of three organic acids in the whole experimental period between two species. The concentrations of organic acids in the roots of S. matsudana were higher than those of T. distichum generally. The effects of different treatments on the concentrations of oxalic acid in the main and lateral roots of T. distichum and S. matsudana were similar. Under flooding stress, there were no significant differences in oxalic acid concentrations in the main and lateral roots of T. distichum and S. matsudana among the three treatment groups. The tartaric acid concentrations in the main and lateral roots of T. distichum gradually decreased with increasing flooding intensity, but it was the opposite trend for S. matsudana. The malic acid concentrations in the main roots of S. matsudana in MS and DS groups were significantly higher than those in the SS group. No significant differences in the concentrations of malic acid in lateral roots of T. distichum and S. matsudana were showed. The malic acid concentrations in the main and lateral roots were more sensitive to water flooding in S. matsudana than T. distichum.

Discussion
At present, adaptive woody plants such as T. distichum and S. matsudana were screened by simulated flooding tests and in situ planting analysis [23]. This study has shown that both T. distichum and S. matsudana adapt well to soil regimes in the water fluctuation zone of Three Gorges Reservoir. The seedlings of two species survived and grown well after winter submergence, although the flooding more than 200 days at 165 m ASL still inhibited the growth height of plants to a certain degree ( Table 2). The plants planted around 165 m ASL should be paying more attention to their growth and survival after years of periodical winter flooding. The growth of T. distichum and S. matsudana at 170 m ASL recovered significantly after the emergence and showed no significant differences with plants at 175 m ASL.
Flooding is often accompanied by secondary stresses such as low temperature, low light, and low dissolved oxygen. The photosynthesis of plants is limited under flooding. Studies have shown that the survival of plants under flooding stress and the restoration of growth after flooding are closely related to the regulation of certain metabolic pathways [18]. Organic acids are secondary metabolites related to adaptation that play important roles in defenses against adverse conditions. The responses of oxalic acid, tartaric acid, and malic acid in roots of T. distichum and S. matsudana were different to winter flooding in the hydro-fluctuation zone. However, two species could adjust their organic acid metabolisms to the normal level after emergence in spring.
Under winter flooding stress, relative to the control group, the tartaric acid concentrations in the main and lateral roots and the malic acid concentration in the main roots of T. distichum in the DS group decreased significantly. However, the concentrations of tartaric acid in the main and lateral roots of S. matsudana in the DS group increased significantly, and the malic acid concentration in the main roots of the flooded group was increased significantly, while the other organic acids were not altered significantly ( Figure 4). Thus, tartaric acid and malic acid responses in S. matsudana roots were more active than those in T. distichum roots.
Oxalic acid is one of the most important secondary metabolites in plants under adverse stress conditions, it can stimulate peroxidase activity and improves the antioxidant capacity [33]. Herein, the concentrations of oxalic acid in the roots of T. distichum and S. matsudana were greatly affected by the experimental period but not significantly affected by different flooding treatments. The concentration of oxalic acid in the main roots of T. distichum was significantly increased at the initial stage of water withdrawal (T1), and gradually decreased during the normal growth period; no significant changes showed under mild drought stress (Figure 2). The concentrations of oxalic acid in the main and lateral roots of S. matsudana increased gradually under water flooding stress, and remained high during the initial stages of plant reoxygenation, then decreased during the normal growth period, until the drought period (Figure 4), consistent with the simulation results [40]. Metabolic imbalance of active oxygen in flooding and the early stage of reoxygenation can result in severe oxidative stress. Thus, increased oxalic acid concentrations in the main roots of T. distichum and the main and lateral roots of S. matsudana may be associated with increased antioxidant capacity. During the recovery stage, reactive oxygen species production and elimination gradually achieve a balance; the oxalic acid concentration of roots may provide energy for plant growth through catabolic pathways, resulting in a decrease in oxalic acid concentration during recovery growth after emergence in spring. Throughout the whole experiment, the concentration of oxalic acid in the main roots of T. distichum was more sensitive than that in the lateral roots, while the concentration of oxalic acid in the lateral roots of S. matsudana was more sensitive than that in the main roots, but the roots of both plants responded positively to flooding in the water fluctuation zone. Relevant studies have pointed out that lateral root plays a more important role in responding to abiotic stress for plants [50], and adaptive plants can make full use of lateral root metabolism regulation to enhance the ability of water flooding tolerance [23].
The concentrations of tartaric acid in the main and lateral roots of S. matsudana was ordered by DS group > MS group > SS group. Some studies have previously reported that organic acids not only function as important intermediates in the energy balance and flow in plants but also participate in plant adaption to abiotic stress [51,52]. The DS group maintained a higher root tartaric acid concentration than the MS group. Therefore, we speculated that tartaric acid in the S. matsudana root system could respond positively to flooding stress in the water fluctuation zone [39]. The concentration of tartaric acid in S. matsudana differed between main and lateral roots, indicating different response strategies. Studies have shown that the change of organic acid concentration is closely related to the adaptation mechanism to environmental stress [52].In this study, changes in tartaric acid concentration in S. matsudana lateral roots over time were similar to those of oxalic acid in roots. The concentrations of oxalic acid and tartaric acid in roots are controlled by synthesis, decomposition, transport, and secretion [53], indicating that tartaric acid may perform similar physiological functions to oxalic acid upon changes in soil moisture in the water fluctuation zone. The synthesis of plant oxalic acid is mainly through the photorespiration glycolic acid pathway [54]. However, there is limited research work available on tartaric acid concentration mechanism under water-stressed conditions, and it is very important to be carried out further studies in this field.

Conclusions
The exotic species T. distichum and native species S. matsudana are two adaptive woody plants for reforestation in the hydro-fluctuation zone of the Three Gorges Reservoir. The two species can grow very well between 170 m to 175 m ASL in the hydro-fluctuation zone of Three Gorges Reservoir. It needs more observations for plants planted around 165 m ASL, where the duration of winter submergence is more than 200 days.
The concentrations of three organic acids in the roots of two species were influenced by winter submergence. After emergence in spring, two species could adjust their organic acid metabolisms to the normal level. Among three organic acids, tartaric acid was the most sensitive response to water submergence, which deserved more studies in the future. The exotic species, T. distichum, had more stable metabolism of organic acids to winter flooding. The native species, S. matsudana, responded more actively to long-term winter flooding. Two species showed the different organic acid regulation mechanisms facing water regimes, and both species can be applied in vegetation restoration.