Responses of growth and photosynthetic fluorescent characteristics in Ottelia acuminata to a water-depth gradient

ABSTRACT To assess the response of Ottelia acuminata to a water-depth gradient, we investigated the plant growth and leaf photosynthesis by setting three water depths (0.5, 1.0, and 1.5 m) in situ in Yilong Lake, Yunnan Province, China. The results showed that the growth and photosynthetic fluorescent characteristics of O. acuminata exhibited different responses to the water-depth gradient. The plant height, fresh weight, root length, and leaf number of O. acuminata, varied significantly with changes in the water depth. With regard to the photosynthetic fluorescent characteristics of leaves, the maximum quantum yield half-saturation light intensity and fluorescence parameter of photosystem II markedly improved with increasing water depth. The increase of photosynthetically active radiation resulted in a decreased photochemical quenching coefficient (qP). In contrast, the nonphotochemical quenching coefficient was relatively high in the leaves of O. acuminata in shallow water under high photosynthetically active radiation. The chlorophyll content of the leaves varied significantly with changes in the water depth. Higher chlorophyll a, chlorophyll b, and carotenoid contents were detected in the leaves of O. acuminata at the water depth of 1.5 m. The results of the growth and photosynthetic fluorescent characteristics of O. acuminata indicate a better protection mechanism against high light in the leaves of O. acuminata in shallow water and a higher photosynthetic efficiency, as well as a greater photosynthetic potential, in the leaves of O. acuminata in deep water.


Introduction
The effect of water depth on submerged plants comprehensively reflects multiple influencing factors (e.g. light, temperature, hydraulic pressure, and nutrient salts). Among these, light is the most important environmental factor that plays a decisive role in the growth and spatial distribution of submerged plants (Bornette and Puijalon 2011). Therefore, the effect of water depth on submerged plants can be accurately determined by monitoring plant growth and photosynthetic fluorescent characteristics. In response to the adverse conditions brought about by changes in water depth, submerged plants usually possess the ability to cope with an adverse environmental impact through self-regulation. This is mainly reflected in two aspects: first, the plants deal with the adverse environmental impact by altering their morphological appearance, including leaf length, leaf width, leaf number, and root length; second, the plants deal with the adverse environmental impact through physiological changes, such as activation of the antioxidant system (Chen et al. 2013) and changes in enzyme levels, chlorophyll CONTACT Liqing Wang lqwang@shou.edu.cn

Methods
In April 2015, O. acuminata seedlings with consistent growth were selected from the Yilong Lake demonstration base for the restoration of submerged plants. The seedlings had a plant height of 27 § 1.5 cm, a leaf number of 7 § 0.5, a biomass of 9.8 § 1.6 g, and a total root length of 110 § 6 cm. The demonstration base was a closed lake body enclosed by an earthen dam along the east side of Yilong Lake. The base had an area of 36,350 m 2 , an average water depth of 1.5 m (maximal 2.0 m), and a transparency of 1.55 m.

Experimental design
Three experimental sites were set in the western lake area of Yilong Lake (average water depth = 1.5 m, Secchi depth = 1.2 m). At each site, four bamboo stakes were vertically inserted into the bottom of the lake. Both ends of another four bamboo stakes were fixed to the vertical bamboo stakes, and the horizontal bamboo stakes, which were parallel to the water surface, were used to hang plastic baskets. The selected O. acuminata seedlings were grown in nutritional bowls (h = 10 cm, d = 8 cm), with one plant per bowl. The nutritional bowls were then placed in the plastic baskets (length = 60 cm, width = 48 cm, height = 12 cm), with 45 bowls per basket. The baskets were hung horizontally from the stakes, which were leveled by pulling a nylon rope. At each site, the plastic baskets were hung one of three depths (0.5, 1.0, or 1.5 m) with 24 numbered baskets per depth. Each experimental zone was enclosed using 5 £ 5 mm breeding nets to prevent herbivorous aquatic animals from impacting the experimental results.
The experiment began on 10 April 2015 and lasted 60 days. At the end of the experiment, the O. acuminata seedlings were taken from the 120 nutritional bowls held at the different water depths at the three experimental sites (40 bowls were selected at random at each site) to measure the growth and photosynthetic fluorescence parameters.
During the experiment, parameters, including water temperature (T), total nitrogen (TN) and total phosphorus (TP) concentrations, and chlorophyll a (Chl a) content, as well as light conditions at the three water depths, were monitored in the experimental zones at noon every 10 days. The results were then averaged, and the following mean parameter values were obtained for 0.5, 1.0, and 1.5 m, respectively (Table 1).

Parameter determination
The number of leaves was counted. Fresh weight was determined after the plants had been washed with distilled water and dried with absorbent paper. Plant height was measured with a 1 mm precision ruler. The total root length was determined using a root scanner (WinRhizo, Regent Instrument Inc., Ville de Qu ebec, Quebec, Canada).
The chlorophyll content of the leaves was determined by spectrophotometry colorimetry after extraction with 80% acetone (Institute of Plant Physiology and Ecology 1999).
Determination of the photosynthetic fluorescence parameters (Schreiber et al. 1997) began with the measurements made at 7-9 am (9 June 2015) using a Diving-PAM underwater fluorometer and WinControl data acquisition software (Germany). The measurement site of the leaf was first subjected to shading treatment for 20 min using a blade clamp. The blade clamp was then opened, and the measuring light was turned on to obtain the initial fluorescence (F 0 ). Subsequently, the saturation pulse was turned on [saturation pulse intensity = 4000 mmol/ (m 2 ¢s) for 0.8 s] to measure the maximum fluorescence (F m ). There are 120 individuals sampled in the experiment and three experimenters at the same time, and each of which handles 40 samples. The acquisition of experimental data is completed in the shortest time, and the experimental error is reduced as much as possible. Each plant measurement was repeated twice, and the results were used to calculate the variable fluorescence (F v ), the maximum quantum yield (F v /F m ), and the fluorescence parameter (F v /F 0 ). Calculations were made using the following formulae: Determination of the rapid light response curve began with measurements made at 9-11 a.m. Actinic light was turned on at the light intensity of 0, 60,152,286,431,631,840,1098, and 1263 mmol/ (m 2 ¢s), and the actinic light radiation at each intensity lasted 10 s. After each photosynthetically active radiation (PAR), the fluorescence before turning on the saturation pulse was recorded as F t , and the fluorescence measured after turning on the saturation pulse light was recorded as F m 0 . The results were used to calculate the effective quantum yield (Yield): The photochemical quenching coefficient (qP) was calculated as and the nonphotochemical quenching coefficient (qN) was calculated as The relative electron transport rate (rETR) was derived from the Yield and the PAR: rETR ¼ Yield:PAR Â 0:5 Â 0:84: The rapid light response curve was fitted by the least squares method, and the light response curve of the mean rETR was drawn according to the following: where P is the electron transfer rate; P m is the maximum electron transport rate (ETR max ); a is the initial slope of photosynthetic curve, which reflects photosynthetic utilization efficiency; b is the photosynthetic suppression parameter; E k is the half-saturation light intensity, indicating plant tolerance to high light; and E m is the maximum saturation light intensity.

Data analysis
Statistical analysis and graph plotting were performed using Excel 2007 (Microsoft Corp., Redmond, WA, USA), SPSS 19.0 (IBM SPSS, Somers, NY, USA), and Origin 8.0. All data were tested for normality and homogeneity before analyses. The effects of water depths on the growth and photosynthetic fluorescent characteristics of O. acuminata were evaluated by oneway ANOVA and means were compared by Duncan's multiple range test, with the depths as dependent variables, with plant height, total length, leaf number, fresh weight, F 0 , F m and so on as fixed factors.

Responses of growth to water depth in O. acuminata
The growth indicators of O. acuminata showed positive responses to water depth. All plant height, total root length, leaf number, and fresh weight varied with changes in water depth. The plant height was significantly higher at 0.5 and 1.0 m than at 1.5 m (P < 0.05), but no significant difference was found between the former two groups (P > 0.05) ( Figure 1). The mean plant height at 1.0 m reached 68.7 cm, which was the highest group; the lowest plant height appeared at 1.5 m, only 57.2 cm. The roots exhibited a growth trend consistent with that in plant height. The total root length was highest for the 1.0 m group (521.6 cm) and it was lowest for the 1.5 m group (196.5 cm). There were significant differences in total root length between the three groups (P < 0.05) ( Figure 1). The growth trend of leaf number differed from those of leaf length and root length. At various water depths, the leaf number ranked 0.5 m > 1.0 m > 1.5 m, and significant differences were detected between groups (P < 0.05) ( Figure 1). The highest leaf number for the 0.5 m group was 27.6. Moreover, the three groups showed significant differences in fresh weight (P < 0.05). The fresh weight was highest for the 1.0 m group, with a mean of 31.2 g/plant; lowest for the 1.5 m group, with a mean of 18.3 g/ plant; and intermediate for the 0.5 m group, with a mean of 26.8 g/plant ( Figure 1).

Responses of photosynthetic fluorescence parameters to water depth
With an increase in water depth, the F 0 and F m did not vary significantly in the leaves of O. acuminata (Duncan test, p = 0.058, n = 2) ( Figure 2). Nonetheless, both the F v /F m and F v /F 0 markedly increased with increasing water depth (Duncan test, p = 0.039, n = 2), i.e. 1.5 m > 1.0 m > 0.5 m Figure 1. Responses of growth characteristics to water depth in Ottelia acuminata.
( Figure 2). It is shown that with the increase of water depth, the ability of the light reaction center to use the weak light increases significantly. With a continuous increase in water depth, the qP was lower for the shallow water group than for the deep water group under the low PAR treatment, which suggests a higher light-use efficiency in the leaves of the O. acuminata in deep water under low light. With a continuous increase of PAR [0-282 mmol/ (m 2 ¢s)], a sharp decline was observed in the qP for the 0.5 m group, a slow decline for the 1.5 m group, and a moderate decline for the 1.0 m group (Figure 3). With a further increase of PAR [282-12,352 mmol/ (m 2 ¢s)], the qP decline slowed for the 0.5 m group, whereas it accelerated for the 1.5 m group.  The qN exhibited the opposite trend to the qP. With a continuous increase of PAR, the qN constantly increased in each treatment group. Different groups had similar qN values under low PAR. When the light intensity was continuously enhanced, the qN rapidly increased in the leaves of O. acuminata for the 0.5 m group; the increase was relatively slow for the other two groups, and the group difference was significant (Duncan test, p = 0.021, n = 2). The highest values were 0.87 for the 0.5 m group, 0.48 for the 1.5 m group, and 0.63 for the 1.0 m group (Figure 3). The above results indicate a higher self-protection ability of leaves in O. acuminata against high light in shallow water.
Among the different groups, the Yield showed the same trend of response to water depth in the leaves of O. acuminata. With an enhancement of PAR, the Yield declined exclusively. The difference occurred because the decrease was fastest for the 0.5 m group, intermediate for the 1.0 m group, and slowest for the 1.5 m group (Figure 3).

Responses of the light response curve to water depth
Water depth exhibited a significant effect on the light response curve in leaves of O. acuminata. a, which reflects photosynthetic efficiency, varied significantly in leaves of O. acuminata among the three water depths. The a value was lowest at 0.5 m, intermediate at 1.0 m, and highest at 1.5 m; the difference was significant among the groups (P < 0.05) (Figure 4). The ETR max constantly increased with increasing water depth, from 22.935 mmol photons/ (m 2 ¢s) at 0.5 m to 29.324 mmol photons/ (m 2 ¢s) at 1.5 m; the increase was 27.86%, reaching statistical significance (P < 0.05) (Figure 4). The E k exhibited the same trend as the ETR max . The E k value at 1.5 m improved by 44.12% compared with that at 0.5 m; the difference was significant (Duncan test, p = 0.023, n = 2) (Figure 4). The E m varied larger with changes in water depth, and significant difference was found between groups (Duncan test, p = 0.019, n = 2).

Responses of photosynthetic pigment contents in leaves of O. acuminata to water depth
Photosynthetic pigment contents showed positive responses to water depth in leaves of O. acuminata. The lowest Chl a, Chl b, carotenoid (Car), and Chl a + Chl b contents were found at 0.5 m, which showed significant differences compared with the other two groups (P < 0.05) ( Figure 5). The parameter values were intermediate at 1.0 m and highest at 1.5 m; however, no significant difference was found between the two groups (P > 0.05). Comparing the leaves of O. acuminata at 1.5 m with those at 0.5 m, we found that the Chl a, Chl b, Car, and Chl a + Chl b contents increased and the differences reached high significance (Duncan test, p = 0.006, n = 2), by 261%, 203%, 421%, 247%; little change occurred in Chl a/Chl b or Car/Chl a, and no significant difference was found between groups (Duncan test, p = 0.061, n = 2)) ( Figure 5). The results showed that the content of chlorophyll increased with the increase of water depth, but the proportion of different kinds of chlorophyll had not changed obviously.

Discussion
Water depth is a major environmental factor that influences plant growth and distribution. Submerged plants have the ability to self-regulate and adapt to the environment; they can adjust the distribution of resources in the plant body for adapting to the environment, which is often manifested as changes in the growth and physiological parameters (Li et al. 2008). Photosynthetic fluorescence parameters in leaves closely relate to photosynthesis in plants and accurately reflect the actual situation of plant photosynthesis in certain circumstances (Guo and Tan 2015). Thus, the growth and photosynthetic fluorescent characteristics of a plant can accurately reflect plant physiology and growth performance at different water depths. In the current study, the results show that water depth has a significant effect on the growth and photosynthetic fluorescent characteristics of O. acuminata. Havens (2003) believed that the water depth is tightly linked to a reduction in light intensity. At a certain water depth, when light intensity has not reached the compensation point for plant growth, submerged plants will change their leaf features and physiological adaptability in response to the changing environment; whereas, in shallow waters, submerged plants often receive high light far above the light compensation point, resulting in high light suppression (Blanch et al. 1998). In the present study, the growth characteristics of O. acuminata showed a series of positive responses to water depth. Specifically, due to high light suppression, the plant height at 0.5 m was lower than that at 1.0 m; whereas, at 1.5 m under low light intensity, the growth of O. acuminata was subjected to low light suppression, and the highest leaf length was recorded at the 1.0 m water depth. Under high light suppression, O. acuminata plants in shallow water showed reduced plant height and increased leaf number to adapt to the high light environment; in deep water, O. acuminata plants showed increased plant height and reduced leaf number to achieve the purpose of photosynthesis. The experimental results indicate that in O. acuminata, the response mechanisms of growth characteristics to water depth are similar to those in V. natans .
Changes in F 0 are associated with the initial electronic excitation density of photosystem II (PSII) antenna pigments and chlorophyll content; F m represents the fluorescence yield when the antenna pigments are completely closed (Barbara et al. 1987). Our results showed that neither F o nor F m varied significantly in response to water depth (P > 0.05). This suggests that in an environment with changing water depth, no significant differences occur in the electron density of the PSII reaction center or the fluorescence yield on completely closed antenna pigments in the leaves of O. acuminata. When the depth of water is between 0.5 and 1.5 m, and other situations where lake depths are greater need to be discussed in future studies.
F v /F m , the maximum quantum yield of PSII, reflects the potential light energy conversion efficiency of the PSII reaction center, and this efficiency is independent of species (Cheng et al. 2014). F v /F o reflects the potential activity of PSII. In this study, we observed a significant reduction in the F v /F o at 0.5 m, which indicates a significant increase in the potential activity of the PSII reaction center with increasing water depth (P < 0.05).
After being harvested by antenna pigments, light energy is consumed mainly by three competitive pathways: photochemical electron transfer, chlorophyll fluorescence emission, and heat dissipation. Only a small portion of energy is consumed by chlorophyll fluorescence emission, while the majority is consumed by photochemical electron transfer and heat dissipation. qP represents the light energy adsorbed by the antenna pigments of PSII that is used for photochemical electron transfer; this parameter reflects the openness of the PSII reaction center and the number of electrons participating in CO 2 fixation. qN reflects the light energy adsorbed by antenna pigments that cannot be used for electron transfer but consumed in the form of heat. When the antenna pigments of the PSII reaction center adsorb excessive light energy that cannot be dissipated timely, it will cause damage to the photosynthetic structure. Therefore, nonphotochemical quenching is a self-protection mechanism for plant tissue (Van and Snel 1990). The level of qN indicates the self-protection ability of plants against excessive light energy. In the present study, under low PAR, the qP was higher in leaves of O. acuminata at 1.5 m; an increase of PAR resulted in a rapid decrease in the qP in deep water and a slow decrease in shallow water. This indicates that when the PAR was low, the PSII reaction center exhibited higher 'openness' and CO 2 fixation ability in leaves of O. acuminata in deep water; under high PAR, the trend was the opposite. In light conditions, the qN data in shallow water remained higher than in deep water, i.e. 0.5 m > 1.0 m > 1.5 m, suggesting a better light protection mechanism in the leaves of O. acuminata in shallow water. Our observation is in agreement with the result of Yang et al. regarding the response of photosynthetic fluorescent characteristics in leaves of V. natans to water depth. Together, these findings prove the common response mechanisms of photosynthetic reaction in O. acuminata and V. natans. In shallow waters, the leaves of O. acuminata possess higher heat dissipation ability and thereby protect the structure of the PSII reaction center against the damage of excessive high-energy electrons. In deep waters, however, the leaves of O. acuminata have limited nonphotochemical quenching capacity under high PAR conditions and thus cannot maintain their structural stability through effective heat dissipation, resulting in decreased photosynthetic ability and subsequent growth inhibition. In contrast, the leaves of O. acuminata have better light utilization efficiency under low PAR conditions in deep waters. This is an adjustment in the plant physiological structure by O. acuminata based on the living conditions, as well as part of the adaptability of plants.
Yield, which indicates the actual photosynthetic efficiency of PSII under light conditions, is the efficiency of a plant for absorbing and supplying photons to the PSII reaction center (Yu et al. 2015). In the current study, the Yield under low PAR [0-152 mmol/ (m 2 ¢s)] ranked 0.5 m > 1.0 m > 1.5 m. With an increase of PAR [PAR > 152 mmol/ (m 2 ¢s)], the Yield showed a rapid decrease at 1.5 m and a slow decrease at 0.5 m, indicating higher actual photosynthetic efficiency in leaves of O. acuminata in deep water under high PAR conditions. a, which reflects the level of the light-harvesting capacity of leaves, is associated with the light absorption coefficient of leaves and the light utilization efficiency of PSII. In this study, the a value was gradually increased with an increase of water depth, indicating higher light harvesting and light use capabilities of O. acuminata in deep water. rETR max and E k showed the same trends, both of which markedly increased with increasing water depth (P < 0.05); this suggests higher electron transfer efficiency and high light tolerance, as well as higher photosynthetic potential in the leaves of O. acuminata in deep water.
Chlorophyll is the major pigment for plant photosynthesis, and chlorophyll content reflects, to a certain extent, the photosynthetic capacity of plants (Zhang and Chen 2016). Chl a mainly plays a role in converting light into electrons, whereas Chl b is a major constituent of the light-harvesting pigments in plants; thus, the relative value of Chl a/Chl b reflects the size of the light-harvesting pigment system (Anderson and Aro 1994). Car is a photosynthetic pigment as well as an endogenous antioxidant whose presence is conducive to the protection and stabilization of the structure of lightharvesting complexes (He et al. 2001). In the present study, the chlorophyll contents were significantly lower in the leaves of O. acuminata at 0.5 m than at 1.0 and 1.5 m (P < 0.05), indicating that the significant increase of chlorophyll content was favorable for the effective absorption of light energy by O. acuminata in deep water under low light conditions. However, Chl a/Chl b did not significantly differ with increasing water depth (P > 0.05). This suggests that the light-harvesting capacity of the O. acuminata leaves did not change greatly at different water depths and that O. acuminata improved its photosynthetic capacity in low light conditions only through increasing the Chl a, chl b, and Car contents in the leaves. The Car content varied significantly with increasing water depth (P > 0.05), indicating that under high light irradiation, the structure of the light-harvesting complexes was more stable in the leaves of O. acuminata in deep water. The leaves exhibited a higher ability to harvest light under high light conditions, which is in agreement with the light-harvesting capacity indicated by a. The Car content is an intrinsic structural factor, and a is the experimental pattern shown due to differences in the Car content.
In summary, we found that the growth and photosynthetic fluorescent characteristics in leaves of O. acuminata demonstrated positive responses to water depth. The growth and photosynthetic fluorescence state of plants varied significantly with the various water depths. In shallow water, O. acuminata often lived under high light intensity, where it reduced the chlorophyll content in the plant body to attenuate the light harvest and thereby protected photosynthetic structures from damage; on the other hand, the plant maintained high heat dissipation in photosynthesis to protect photosynthetic structures (Zhou et al. 2011). In deep water, O. acuminata often lived in a low light environment, where it improved the chlorophyll content in the plant body and reduced heat dissipation to increase the photosynthetic efficiency; the plants commonly had relatively high ETR and E k as well as high photosynthetic efficiency, but their self-protection ability under high light was lower compared with the plants in shallow water. Thus, O. acuminata can adjust the growth and physiological parameters in the growth process based on the habitat conditions to better adapt to the environment and create a more conducive space for growth and development. Therefore, plants grown in different living environments possess particular physical structures adapted to the environment; when the environment changes, the plant has to re-adjust the physical structures. Intense changes in environmental variables may cause growth inhibition and even the death of plants. If the water level suddenly drops, O. acuminata plants living in deep water will be exposed to highlight and growth inhibition or death may occur due to a lack of self-protection mechanism under high light. If the water level is elevated, growth inhibition or death may occur in O. acuminata in shallow water due to relative low photosynthetic efficiency. This is one of the main reasons why a large amount of O. acuminata has gradually declined in plateau lakes in China.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Fengbin Zhao is a doctoral graduate student at the Shanghai Ocean University, China. He focus on the ecological restoration of submerged plants in the freshwater ecosystem.
Wei Zhang is a post-doctoral researcher at Shanghai Ocean University, China. He focus on the phytoplankton community ecology and ecological restoration in freshwater system.
Yanhong Liu is an independent ecological researcher and educator who is a university president in Yunnan, China.
Liqing Wang is an professor at Shanghai Ocean University as an ecologist in freshwater systems.