Comparison of the Ability to Control Water Loss in the Detached Leaves of Wedelia trilobata, Wedelia chinensis, and Their Hybrid

In the process of biological invasion, hybridization between invasive species and native species is very common, which may lead to the formation of hybrids with a stronger adaptability. The hybrid of Wedelia trilobata (an alien invasive species) and Wedelia chinensis (an indigenous congener) has been found in South China. In our previous study, we found that the hybrid showed heterosis under cadmium stress. However, the results of this experiment demonstrated that the leaves of the hybrid had no heterosis in controlling water loss. The results showed that the water loss rate of W. trilobata was the slowest, that of W. chinensis was the fastest, and that of the hybrid was in the middle. Compared with W. chinensis and the hybrid, W. trilobata accumulated more abscisic acid (ABA) in leaves to control water loss. After the leaves were detached, W. chinensis leaves suffered the most serious damage, the lowest maximum photochemical efficiency, the most serious membrane lipid peroxidation, and the largest accumulation of malondialdehyde and reactive oxygen species. Compared with W. chinensis and its hybrid, the leaves of W. trilobata could accumulate more antioxidant enzymes and antioxidants, and the total antioxidant capacity was the strongest. The results demonstrate that the ability of the hybrid to reduce water loss was lower than that of W. trilobata, but higher than that of W. chinensis. They showed that the drought resistance of the hybrid may be higher than that of W. chinensis, and it might threaten the survival of W. chinensis.


Introduction
In the process of plant growth, organisms are subjected to various adverse environmental stresses, among which drought is one of the most common abiotic stresses. The effects of drought stress on plants are mainly reflected in the effects on cell activity and organ and tissue function [1,2]. A decrease in the water content will result in the inhibition of photosynthesis, the slow growth of plants, and influence of the biomass or yield [3]. After drought stress, the metabolic process of cells is blocked, which leads to the accumulation of H 2 O 2 , 1 O 2 , and O 2 • − in cells [4]. The main component of the cell membrane is the phospholipid bilayer. A large number of reactive oxygen species will damage the properties of the cell membrane. The results of membrane lipid peroxidation are mainly an increase in the malondialdehyde (MDA) content and an increase in the cell membrane permeability [5]. Due to the destruction of the cell membrane and the increase in the permeability, the cell membrane will lose the function of selective passage, resulting in uncontrolled material passing in and out of the cell, resulting in the disorder of the cell metabolic function.

Changes of Water Loss in Detached Leaves
The leaf phenotype showed that the leaves of W. chinensis, W. trilobata, and their hybrid exhibited wilting with the prolongation of the leaf in vitro time, among which the W. chinensis displayed the most obvious performance ( Figure 1A,B), and the leaf edge angle had curled at 3 h ( Figure 1A). Figure 1C shows the changes of leaf water loss for the three plants. With the extension of the in vitro time, the water loss rate of leaves of W. chinensis was the highest, and that of W. trilobata was the lowest. The water loss rate of hybrid leaves was between those of its parents.

Changes of Water Loss in Detached Leaves
The leaf phenotype showed that the leaves of W. chinensis, W. trilobata, and their hybrid exhibited wilting with the prolongation of the leaf in vitro time, among which the W. chinensis displayed the most obvious performance ( Figure 1A,B), and the leaf edge angle had curled at 3 h ( Figure 1A). Figure 1C shows the changes of leaf water loss for the three plants. With the extension of the in vitro time, the water loss rate of leaves of W. chinensis was the highest, and that of W. trilobata was the lowest. The water loss rate of hybrid leaves was between those of its parents.

Abscisic Acid Content and Related Gene Expression in Detached Leaves
The ABA content of detached leaves increased significantly. The ABA content of W. trilobata was significantly higher than that of W. chinensis and its hybrid. The content of ABA increased by 43%, 207%, and 51% in W. chinensis, W. trilobata, and their hybrid, respectively (Figure 2A). ABA1 and NCED are the main regulatory genes in the ABA synthesis pathway. The relative expression levels of ABA1 and NCED in detached leaves were significantly up-regulated, and their trend was consistent ( Figure 2B,C). The results showed that the relative expression of ABA1 and NCED in the leaves of W. trilobata was significantly higher than that of hybrids, and that of hybrids was significantly higher than that of W. chinensis. PIP is a key gene in the synthesis of aquaporin, and the relative expression of the PIP gene in detached leaves also increased significantly ( Figure 2D). Moreover, the trend was consistent with that of ABA1 and NCED.

Abscisic Acid Content and Related Gene Expression in Detached Leaves
The ABA content of detached leaves increased significantly. The ABA content of W. trilobata was significantly higher than that of W. chinensis and its hybrid. The content of ABA increased by 43%, 207%, and 51% in W. chinensis, W. trilobata, and their hybrid, respectively (Figure 2A). ABA1 and NCED are the main regulatory genes in the ABA synthesis pathway. The relative expression levels of ABA1 and NCED in detached leaves were significantly up-regulated, and their trend was consistent ( Figure 2B,C). The results showed that the relative expression of ABA1 and NCED in the leaves of W. trilobata was significantly higher than that of hybrids, and that of hybrids was significantly higher than that of W. chinensis. PIP is a key gene in the synthesis of aquaporin, and the relative expression of the PIP gene in detached leaves also increased significantly ( Figure 2D). Moreover, the trend was consistent with that of ABA1 and NCED. . The data are shown as means ± standard errors (SE), and the study included five biological replicates. Different letters (a, b, c, d) on the bar represent significant differences at p < 0.05 (Duncan's test).

Changes of Chl Fluorescence and the Localization of Reactive Oxygen Species in Detached Leaves
In detached leaves, the maximal photochemical efficiency (Fv/Fm) of W. chinensis leaves was the lowest, and that of W. trilobata was the highest ( Figure 3A,B). Proline, as an osmoregulation substance, can regulate the osmotic pressure of cells, and the proline content of detached leaves increased ( Figure 3C). MDA is a product of membrane lipid peroxidation, which can reflect the degree of cell membrane damage. The content of MDA in leaves increased significantly, among which W. chinensis had the highest content, increasing by 194%, while W. trilobata had the lowest content, with an increase of 32%. The content of the hybrid was between that of its parents, increasing by 108% ( Figure 3D). It was found that the accumulation of ROS in detached leaves increased, and the accumulation of ROS in leaves of W. trilobata was the lowest ( Figure 3E,F). . The data are shown as means ± standard errors (SE), and the study included five biological replicates. Different letters (a, b, c, d) on the bar represent significant differences at p < 0.05 (Duncan's test).

Changes of Chl Fluorescence and the Localization of Reactive Oxygen Species in Detached Leaves
In detached leaves, the maximal photochemical efficiency (Fv/Fm) of W. chinensis leaves was the lowest, and that of W. trilobata was the highest ( Figure 3A,B). Proline, as an osmoregulation substance, can regulate the osmotic pressure of cells, and the proline content of detached leaves increased ( Figure 3C). MDA is a product of membrane lipid peroxidation, which can reflect the degree of cell membrane damage. The content of MDA in leaves increased significantly, among which W. chinensis had the highest content, increasing by 194%, while W. trilobata had the lowest content, with an increase of 32%. The content of the hybrid was between that of its parents, increasing by 108% ( Figure 3D). It was found that the accumulation of ROS in detached leaves increased, and the accumulation of ROS in leaves of W. trilobata was the lowest ( Figure 3E,F).

Changes of the Antioxidant Capacity in Detached Leaves
Plants can reduce oxidative stress in cells by synthesizing antioxidants. The contents of SOD, CAT, and POD were increased significantly in detached leaves. The contents of SOD, CAT, and POD in W. trilobata were the highest, and those of W. chinensis were the lowest ( Figure 4A-C). Total phenols and flavonoids were nonenzymatic antioxidants, and their contents also increased significantly in detached leaves ( Figure 4D,E). Furthermore, the trend was consistent with SOD, CAT, and POD. The total antioxidant capacity (TAC) reflected the antioxidant capacity of nonenzymatic

Changes of the Antioxidant Capacity in Detached Leaves
Plants can reduce oxidative stress in cells by synthesizing antioxidants. The contents of SOD, CAT, and POD were increased significantly in detached leaves. The contents of SOD, CAT, and POD in W. trilobata were the highest, and those of W. chinensis were the lowest ( Figure 4A-C). Total phenols and flavonoids were nonenzymatic antioxidants, and their contents also increased significantly in detached leaves ( Figure 4D,E). Furthermore, the trend was consistent with SOD, CAT, and POD. The total antioxidant capacity (TAC) reflected the antioxidant capacity of nonenzymatic antioxidants. The content of total antioxidant substances in the leaves of W. trilobata was significantly higher than that of the hybrid, and that of the hybrid was higher than that of W. chinensis ( Figure 4F).
Plants 2020, 9, x FOR PEER REVIEW 6 of 12 antioxidants. The content of total antioxidant substances in the leaves of W. trilobata was significantly higher than that of the hybrid, and that of the hybrid was higher than that of W. chinensis ( Figure 4F).

Discussion
The hybrid leaves showed no heterosis in controlling water loss. As the main organ of water loss in plants, most of the water is lost through leaves. Therefore, the ability of plant leaves to control water loss can reflect the drought resistance of plants. In this paper, the water loss rate of detached leaves of W. chinensis, W. trilobata, and their hybrid was compared. The results showed that the water loss rate of leaves of W. trilobata was the lowest, and that of leaves of W. chinensis was the highest. The water loss rate of hybrid leaves was between that of their parents, and it was closer to that of W. chinensis. The phenotype of detached leaves showed that W. chinensis leaves wilted first. Water loss in leaves mainly occurs through the stomata on the leaves, and the stomatal opening is positively correlated with the water loss of plants. Studies have shown that under drought stress, plants will reduce stomatal opening or close stomata to reduce water loss and improve drought resistance [24,25]. Many studies have shown that ABA plays a key role in stomatal regulation. The The data are shown as means ± standard errors (SE), and the study included five biological replicates. Different letters (a, b, c, d, e) on the bar represent significant differences at p < 0.05 (Duncan's test).

Discussion
The hybrid leaves showed no heterosis in controlling water loss. As the main organ of water loss in plants, most of the water is lost through leaves. Therefore, the ability of plant leaves to control water loss can reflect the drought resistance of plants. In this paper, the water loss rate of detached leaves of W. chinensis, W. trilobata, and their hybrid was compared. The results showed that the water loss rate of leaves of W. trilobata was the lowest, and that of leaves of W. chinensis was the highest. The water loss rate of hybrid leaves was between that of their parents, and it was closer to that of W. chinensis. The phenotype of detached leaves showed that W. chinensis leaves wilted first. Water loss in leaves mainly occurs through the stomata on the leaves, and the stomatal opening is positively correlated with the water loss of plants. Studies have shown that under drought stress, plants will reduce stomatal opening or close stomata to reduce water loss and improve drought resistance [24,25]. Many studies have shown that ABA plays a key role in stomatal regulation. The exogenous application of ABA or endogenous increase in ABA expression can cause stomata to close, thus reducing water loss [26,27]. The results showed that the ABA content of detached leaves increased significantly, and that of W. trilobata was significantly higher than that of W. chinensis and hybrids, which was consistent with the results of water loss. In Arabidopsis thaliana, it was found that the overexpression of ABA synthesis-related genes could increase the ABA content in leaves, reduce the water loss of detached leaves, and improve the drought resistance of plants [26]. ABA1 and NCED are the main regulatory genes in the ABA synthesis pathway, and their expression levels are positively correlated with the ABA content. In Arabidopsis, drought can increase the expression of NCED and the content of ABA. Compared with wild type specimens, the ABA content in ABA-deficient mutants decreased, and drought resistance decreased under drought stress [28,29]. The results of this study showed that the expression of ABA1 and NCED in detached leaves increased significantly. The expression level of ABA1 and NCED in W. trilobata was the highest, and that in W. chinensis was the lowest. PIP is a key gene in the synthesis of aquaporin. The increase in PIP expression can increase the content of aquaporin. Aquaporin is the most important aquaporin on the cell membrane, and can affect the water transport and regulation between cells. Figure 2 shows that the PIP expression increased significantly, which was consistent with ABA1 and NCED expression. Studies have shown that increasing the content of aquaporin in leaves can improve the drought resistance of plants [30].
Fv/Fm is one of the most important parameters of chlorophyll fluorescence in plant leaves, and its value will not change significantly under natural conditions. However, it will decrease under stress (high temperature, low temperature, drought, etc.) [31,32]. The results of this study showed that the Fv/Fm of leaves decreased significantly, indicating that the detached leaves were damaged. Among them, W. chinensis leaves exhibited the lowest values, while those of W. trilobata were the highest. It was suggested that the leaves of W. trilobata were the least damaged, while the leaves of W. chinensis were the most seriously damaged. Drought stress is often accompanied by oxidative stress, and the accumulation of ROS in plants increases significantly. It was found that drought could increase the accumulation of ROS in rice leaves [25]. The results demonstrated that the accumulation of ROS in detached leaves increased. A large amount of ROS can cause toxicity to plant cells, which can destroy biological macromolecules, attack cell membranes, and accelerate leaf damage [33]. In this experiment, the accumulation of ROS in the leaves of W. chinensis was the highest, and that in the leaves of W. trilobata was the lowest, indicating that the leaves of W. chinensis were under the most serious oxidative stress. MDA is a product of membrane lipid peroxidation and can indicate the damage of stress to plants. The content of MDA and the change of plasma membrane permeability are important indicators reflecting the degree of membrane lipid peroxidation and plasma membrane damage. In the study of Zoysia japonica, it was found that with the extension of the drought time, the content of MDA in leaves increased gradually [34]. The results revealed that the content of MDA in detached leaves increased significantly, indicating that the detached leaves were damaged. Among them, the content of MDA in leaves of W. chinensis was the highest, and that of W. trilobata was the lowest, indicating that the leaves of W. chinensis were the most seriously damaged.
In the long-term evolution of plants, the mechanism of stress adaptation is restricted by heredity. On the one hand, plants scavenge reactive oxygen radicals by enhancing the activities of antioxidant enzymes (SOD, CAT, and POD), in order to maintain the stability and integrity of the cell membrane [35]. The results showed that the activities of SOD, CAT, and POD in detached leaves of W. chinensis, W. trilobata, and their hybrid increased significantly. Among them, the enzyme activity in the leaves of W. trilobata was the strongest, the enzyme activity in the leaves of W. chinensis was the weakest, and the enzyme activity in the leaves of the hybrid was between that of its parents. A previous study showed that increasing the activities of SOD, CAT, and POD could significantly reduce the accumulation of ROS in plant leaves [23]. On the other hand, plants can also accumulate nonenzymatic antioxidants to improve the antioxidant capacity and scavenge ROS. These nonenzymatic antioxidants include vitamins, phenols, and flavonoids [36]. Studies have shown that increasing the contents of total phenols and flavonoids in plant leaves can reduce the accumulation of reactive oxygen species under stress [23]. The results of this study showed that the contents of total phenols and flavonoids in detached leaves of W. chinensis, W. trilobata, and their hybrid increased significantly, and the trend was consistent with enzyme activity.

Plant Materials
W. trilobata is one of the invasive species in South China, W. chinensis is a native species in South China, and their hybrid has been found in the field. W. trilobata, W. chinensis, and their hybrid were propagated asexually and planted in the experimental field of the biological garden of South China Normal University, Guangzhou, China. The annual average temperature and rainfall are 22.8 • C and 1736 mm, respectively, belonging to the subtropical humid monsoon climate [37]. Mature leaves at the fourth leaf position were selected as research materials. Five replicates were performed.

Water Loss Assay
The detached leaves were incubated at room temperature and weighed at 60 min intervals. The water loss (%) was calculated by dividing the difference between the initial fresh weight and the postincubation fresh weight by the initial fresh weight.

Hormone Determination
In this study, 0.1 g leaves were weighed and ground with 0.5 mL PBS (50 mM; pH 7.3) on ice and then left at 4 • C for 2 h. The grinding solution was centrifuged at 4 • C for 20 min at 5000× g, and the supernatant was the hormone extract. The contents of the hormone were determined by a plant ELISA Kit (Zike, Shenzhen, China).

Determination of Enzyme Activity
In this investigation, 0.2 g leaves were homogenized in 1.5 mL phosphate buffer (50 mM) containing 2% (w/v) polyvinylpyrrolidone, 0.1% (v/v) Triton-X-100, and 100 mM ethylenediaminetetraacetic acid disodium salt (EDTA-Na 2 ). The grinding fluid was centrifuged at 13,000× g for 15 min at 4 • C, and the resulting supernatant was retrieved as an enzyme extract.
The activity of POD was evaluated according to the method described by Chance and Maehly [40], with moderate adjustments. In total, 0.1 mL of the enzyme extract was mixed with 1.875 mL phosphate buffer (50 mM; pH 7.0), 1 mL H 2 O 2 (30 mM), and 0.025 mL 2-methoxyphenol. The absorbance was recorded nine times (20 s each time) at 470 nm (UV-2450, Shimadzu, Kyoto, Japan). The activity of POD (1 unit) was estimated as the quantity of enzyme needed for a 0.01-unit increase in OD within 1 min.
The activity of SOD was evaluated according to the method published by Giannopolitis and Ries [41], with moderate adjustments. In total, 0.1 mL of the enzyme extract was mixed with 1.7 mL phosphate buffer (50 mM; pH 7.8), 0.3 mL methionine (130 mM), 0.3 mL NBT (0.75 mM), 0.3 mL EDTA-Na 2 (0.1 mM), and 0.3 mL riboflavin (0.02 mM). The reaction was conducted at a photosynthetic photon flux density of 4500 Lux for 15 min. Subsequently, the absorbance was determined immediately at 560 nm (UV-2450, Shimadzu, Kyoto, Japan) after the reaction was terminated in the dark. The amount of enzyme that resulted in 50% suppression of the photochemical reduction in NBT was defined as the unit of enzyme activity.
The activity of CAT was evaluated according to the method described by Chance and Maehly [40], with moderate adjustments. A total of 0.1 mL of the enzyme extract was mixed with 2.9 mL H 2 O 2 (30 mM). After 15 s of incubation, the absorbance was recorded nine times (20 s each time) at 240 nm (UV-2450, Shimadzu, Kyoto, Japan). The activity of CAT (1 unit) was estimated as the quantity of enzyme needed for a 0.01-unit increase in the optical density (OD) within 1 min.

Determination of Total Phenols and Flavonoids
The total phenols were determined by the Folin-Ciocalteu method, as described by Ainsworth and Gillespie [42]. In the procedure, 0.05 g leaves were cut into pieces and placed in a centrifuge tube containing 1.5 mL 95% methanol and extracted at 4 • C for 24 h. Then, 1 mL 10% Folin-Ciocalteu and 2 mL 0.7 M Na 2 CO 3 were mixed and added to a 0.5 mL sample. The absorbance was measured at 765 nm (UV-2450, Shimadzu, Kyoto, Japan), and the concentration was calculated according to the standard curve of gallic acid.
The flavonoids were determined according to the method described by Heimler et al. [43]. In this procedure, 0.05 g leaves were cut into pieces and placed in a centrifuge tube containing 1.5 mL 95% methanol and extracted at 4 • C for 24 h. The 0.15 mL sample was mixed with 1.85 mL deionized water, 0.2 mL 5% NaNO 2 , 0.3 mL 10% AlCl 3 (freshly prepared), and 1 mL 1 M NaOH. The absorbance was measured at 510 nm (UV-2450, Shimadzu, Kyoto, Japan), and the concentration was calculated according to the standard curve of catechin.

Determination of Total Antioxidant Capacity
The total antioxidant capacity (TAC) of leaves was evaluated by measuring the rate of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging [44]. In this procedure, 0.05 g leaves were cut into pieces and placed in a centrifuge tube containing 1.5 mL 95% methanol and extracted at 4 • C for 24 h. The 0.3 mL sample was mixed with 2.7 mL 120 µM DPPH. The absorbance was measured at 517 nm (UV-2450, Shimadzu, Kyoto, Japan), and the concentration was calculated according to the standard curve of DPPH.

Detection of Chl Fluorescence
Chl fluorescence was measured by the Chl fluorescence imaging system (Technologica, Essex, UK). The minimum fluorescence (F0) and the maximum fluorescence (Fm) of the leaves and stems were measured using a 6000 µmol m −2 s −1 saturating pulse. The maximum photochemical efficiency (Fv/Fm) was calculated as Fv/Fm = (Fm − F0)/Fm [45].

Determination of Proline
A 0.2 g sample was ground with 10 mL 80% ethanol, and the homogenate was then extracted with 0.01 g activated carbon in the dark for 1 h. The extract was filtered, 1 g zeolite was added, and the sample was shaken for 15 min and centrifuged for 5 min at 3000× g. Then, 2 mL supernatant, 2 mL acetic acid, and 2 mL ninhydrin solution (1.25 g of ninhydrin was dissolved in 30 mL acetic acid and 20 mL 6 M of phosphoric acid) were mixed in boiling water for 20 min. The absorbance of the solution was measured after cooling at 517 nm (UV-2450, Shimadzu, Kyoto, Japan), and the concentration was calculated according to the standard curve of proline.

Determination of Malondialdehyde
The MDA was measured according to the method published by Sun et al. [22]. A 0.2 g sample was homogenized with 2 mL 10% trichloroacetic acid. After centrifuging at 4000× g for 15 min at 4 • C, the supernatant (1 mL) was mixed with an equal volume of 0.67% 2-thiobarbituric acid and then boiled for 20 min. The absorbance of the solution was measured after cooling at 600, 532, and 450 nm using a UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan).

Tissue Localization of Reactive Oxygen Species
The tissue localization of H 2 O 2 was conducted according to the method described by Liu et al. [46]. Leaves were placed in 0.2 M phosphate buffer (pH 7.0) containing 1 mg mL −1 3,3 -diaminobenzidine (DAB) for 2 h at room temperature after brief vacuum infiltration three times (10 min each), and the leaves were then decolored in 95% ethanol.
The tissue localization of O 2 •− was conducted according to the method described by Liu et al. [46].
Leaves were placed in 0.2 M phosphate buffer (pH 6.4) containing 1 mg mL −1 nitroblue tetrazolium (NBT) and 10 mM NaN 3 for 4 h at room temperature after brief vacuum infiltration three times (10 min each), and the leaves were then decolored in 95% ethanol.

Statistical Analysis
SPSS Statistics 19.0 (IBM, New York, NY, USA) software was used to analyze the data. Data were subjected to one-way analysis of variance (ANOVA) with Duncan's multiple range test to compare means, and significance set at two-tailed p < 0.05. All analyses were conducted using SigmaPlot version 12.5 (SYSTAT Software, Richmond, CA, USA).

Conclusions
In summary, the ability of the hybrid of W. trilobata and W. chinensis to control water loss in leaves was weaker than that of the invasive species W. trilobata, but stronger than that of the native species W. chinensis. In the process of leaf water loss, the water loss rate of W. trilobata was the lowest, and that of W. chinensis was the highest. The antioxidant capacity of W. trilobata was the strongest, while that of W. chinensis was the weakest. W. chinensis suffered the most serious oxidative stress, while W. trilobata suffered the least. The results indicate that the drought resistance of the hybrid is not superior to that of its parents but is stronger than that of the native species. The survival of native species is threatened not only by invasive species but also by hybrids. Therefore, the emergence of hybrids may accelerate the extinction of native species.