Comparison of photosynthetic fluorescence characteristics of several submerged plants in Honghu Lake, China

Submerged plants are the pioneer species of eutrophic water remediation, and they are important for maintaining the health of aquatic ecosystem, while light is the main limiting factor for the growth of submerged plants. In this study, we measured the maximal quantum yields of photosystemII(Fv/Fm) and rapid light curves (RLCs) of five dominant submerged macrophytes in situ by using pulse-amplitude modulated fluorometer (Diving-PAM). Results revealed that P. crispus L. and M. verticillatum L. had the highest Fv/Fm value, all species’ Fv/Fm are less than 0.8. In addition, the variation trends of Fv'/Fm' and Fv/Fm were same. All species showed statistically significant differences in α, while P. crispus L. and M. verticillatum L. showed the highest α value in the five species. And the variation trends of rETRm and Ek were basically the same. It indicated that P. crispus L. and M. verticillatum L., both of which had high photosynthetic efficiency, had excellent ability to withstand hard light. Compared five species, P. crispus L. and M. verticillatum L. had resistance capacity to hard light as well as faster photosynthetic rate, and V. natans (Lour.) Hara had higher resistance capacity to low light. Thus, when submerged plants are used for water restoration, V. natans (Lour.) Hara could be regarded as a pioneer species in eutrophication water restoration. P. crispus L. and M. verticillatum L. will have better effects when used in shallow water areas.


Introduction
During the past several decades, most lakes around the world have been suffering from eutrophication (Smith and Schindler, 2009;Yang et al., 2017).The causes of lake eutrophication are various: Under the combined interference of natural and man-made factors, a large amount of nutrients are imported into the lake, resulting in an increase in carbon, nitrogen, phosphorus and other source elements in the water body, which leads to an increase in phytoplankton biomass and a decrease in water transparency, gradually transforming it from a nutrient-poor lake to a nutrient-rich lake (Qin et al., 2013;Bal et al., 2017;Liao et al., 2017).Lake eutrophication is the most serious water quality problem in the world, which directly causes the frequent occurrence of toxic algal blooms, the death of fish, the decline of biodiversity, the degradation of lake ecological structure and function and other related ecological problems (David et al., 2008;Wang and Wang, 2009).

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Notulae Botanicae Horti Cluj-Napoca Agrobotanici 2 Submerged plants are important structural shapers and functional maintainers of lake ecosystems. Using submerged plants to repair eutrophic lakes has the characteristics of low cost, low carbon and environmental protection, and is a truly sustainable management method .However, Light is one of the major factors limiting the growth of submerged macrophytes (Barko and Smart, 1981)and plays a decisive role in the growth and spatial distribution of submerged plants (Bornette and Puijalon, 2011).The decrease of water transparency caused by eutrophication directly leads to the deterioration of underwater light environment, which reduces the photosynthetic capacity of submerged plants, and then leads to the disappearance of a large number of submerged plants (Gudrun and Sara, 2011;Zhao et al., 2018). Our aim is to find a pioneering species with strong photosynthetic capacity in low-light environments with low water transparency, so as to provide a theoretical basis for eutrophication management of shallow lakes.
V. natans (Lour.) Hara, H. verticillata, M. verticillatum L., P. crispus L. and P. wrightii Morong are dominant species of submerged plant communities in shallow lakes (water depth is around 150cm) in the middle and lower reaches of the Yangtze River with high pollution tolerance and high pollution absorption. In recent years, the studies on these submerged plants mainly focus on the absorption of nitrogen, phosphorus and heavy metals (Yang et al., 2015;Zhang et al., 2015;Xie et al., 2016), mostly by laboratory simulation experiments.
Chlorophyll fluorescence dynamics technology is a rapid, simple, accurate and non-damaging measurement, which is an important means of detecting and analyzing the relationship between plant photosynthesis and the environment (Zhang, 1999;Küster and Altenburger, 2007;Liu et al., 2010). At present, fluorescence technology has been widely used in the physiological and ecological research of terrestrial higher plants, as well as large seagrass and coral, but less applied in the physiological and ecological research of aquatic higher plants in fresh water (Lamote and Dunton, 2006;Belshe et al., 2007;Hallik et al., 2012;Wang et al., 2015;Zhong et al., 2018;Liu et al., 2019). Existing studies on the fluorescence characteristics of higher aquatic plants in fresh water mostly focus on the comparative analysis of ecological factors on aquatic plant stress under experimental control conditions. However, studies on the comparison of photosynthetic fluorescence parameters of aquatic plants in natural habitats have rarely been reported (Hussain et al., 2019).
In this study, we measured main leaf fluorescence kinetics parameters and fast light response curve of five kinds of submerged plant by using in-situ detecting method, and compared their PSⅡ main fluorescence parameters characteristics in natural water, aiming at providing a theoretical basis for remediation of lake eutrophication by submerged plants.

Site and conditions
Honghu Lake (N29°42′～29°58′, E113°13′～113°29′) is the largest lake in Hubei province, China, lying on the north shore of the Yangtze River middle reaches, which plays a significant role in water supply, flood storage and drainage, pollution self-purification, agricultural and fishery production in the Jianghan Plain of the middle and lower reaches of the Yangtze River (Liu et al., 2004) (Li, 1997).

Methods
Taking the dense growth of submerged plants in Honghu ravine as the sampling area, five common submerged plants: V. natans (Lour.) Hara, H. verticillata, M. verticillatum L., P. crispus L. and P. wrightii Morong (all these submerged plants were identified by Flora of China (Wu, 2007)) were selected as the research objects, and their leaf green fluorescence parameters were determined in situ by Diving-PAM. We selected 3 samples from each plant in the lake area with a water depth of 150cm randomly, and 1-2 fully developed healthy leaves were selected for each plant.
Fv/Fm is PSⅡ maximum photochemical quantum yield, they reflect the PSⅡ intrinsic light energy conversion efficiency,which can be described by the equation: where Fo is the minimal fluorescence (testing light is 0.15 μmol·m -2 ·s -1 ); Fm represents the maximal fluorescence (saturation pulse light is 4000 μmol·m-2·s-1).
PSⅡ photochemical effective quantum yield of photochemical reaction (Fv'/Fm') can be described by the equation: Fv'/Fm'= (Fm'-Fo')/Fm' where Fo' and Fm' represent the minimal and the maximal fluorescence under the condition of actinic light adaptation, respectively.
Then, actinic light was turned on at the intensity of 33, 104, 219, 342, 515, 708, 1042 and 1407μmol·m -2 ·s -1 , respectively. The fluorescence before turning on the saturation pulse was recorded as Ft, and the fluorescence measured after turning on the saturation pulse light was recorded as Fm', then we got variable fluorescence (△F):△F=Fm'-Ft ,the effective quantum yield (Y) can be calculated: Relative electron transport rate (rETR) was described by the equation (Ralph et al., 1998): rETR=Y×PAR×0.5×0.84 The least square method was used for the fast light curve fitting, and the equation we used is proposed by Platt (Platt et al., 1980). and Ralph (Ralph and Gademann, 2005). The equation is as follows: rETR= rETRm (1-e -α.PAR/rETRm ) e -β.PAR/rETRm where, rETRm is the maximum potential relative electron transfer rate in the absence of light inhibition, α is the initial slope of the rETR-PAR curve, reflecting the ability of plants to use light energy, β is the photoinhibition parameter. Thus, the half-saturated light intensity( Ek ) is Ek=rETRm/α.

Statistical analysis
Microsoft Excel 2018 and IBM SPSS Statistics 22.0 were used for statistical analysis of all data, all diagrams were drawn by Origin 2018 and one-way ANOVA was used to compare the differences between different data groups. 4 crispus L. and P. wrightii Morong. Moreover, the Fv ' /Fm ' of P. crispus L. is the highest, P. wrightii Morong is the second, and V. natans (Lour.) Hara is the lowest. However, the differences of Fv ' /Fm ' between H. verticillata and M. verticillatum L. are not significant, and their Fv ' /Fm ' are lower than P. crispus L. and higher than P. wrightii Morong, significantly. 110.35, P. crispus L. 106.15, and P. wrightii Morong 51.99, among them, the Ek differences between P. crispus L. and M. verticillatum L were not significant, however, they reached a significant level with other 3 plants 5 respectively (P<0.05). And these five submerged plants showed high light tolerance, P. crispus L. and M. verticillatum L. were the strongest, H. verticillata is in the middle level, V. natans (Lour.) Hara is next, P. wrightii Morong is the weakest.

Discussion
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).
Fv/Fm, 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 (Cao et al., 2012;Cheng et al., 2014). Fv/Fm changes little under non-stress conditions and measures approximately 0.83 in most higher plants; the value is significantly reduced in plants subjected to environmental stress. In the present study, Fv/Fm of 5 plants are less than 0.8, showing that the 5 kinds of submerged plants may be intimidated, meanwhile, outside stress may do some damage to the PSⅡ reaction center of submerged plants (Jian et al., 2016). While the variation trends of Fv' /Fm' and Fv/Fm were same. At present, there have been reports on the effects of UV-B radiation, polycyclic aromatic hydrocarbon pollution and the attachment of reticulosa on Fv/Fm of aquatic plants (Marwood et al., 2001;Li et al., 2009) and the effects of other factors on Fv/Fm is seldom reported. Combined with the water quality in the sampling area, the most likely reason for the decrease of Fv/Fm value of submerged plants in the sampling area is the attached filamentous algae (Song et al., 2009). As for the tolerance of Fv/Fm of different plants to stress factors, further studies are needed. α, 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, different species have different α values, P. crispus L. and M. verticillatum L. are relatively high in the five species. While the variation trends of rETRm and Ek were basically the same. P. crispus L. and M. verticillatum L., both of which have high photosynthetic efficiency, have excellent ability to withstand hard light. α of V. natans (Lour.) Hara is the lowest, it suggests that V. natans (Lour.) Hara has a strong ability to withstand weak light.
Previous report showed that with only 5% natural light intensity, V. natans (Lour.) Hara cell membrane permeability still had no obvious change, so it had a strong growth advantage in the bottom of natural water, which may be a pioneer species in the restoration of eutrophic water (Xiao et al., 2006).