Physiological Responses of the Submerged Macrophyte Stuckenia pectinata to High Salinity and Irradiance Stress to Assess Eutrophication Management and Climatic Effects: An Integrative Approach

: Stuckenia pectinata , a submerged macrophyte of eutrophic to hyper-eutrophic fresh to brackish waters, faces management and climatic-forced increment of salinity and irradiance in Vistonis Lake (Greece) that may endanger its existence and the ecosystem functioning. A pre-acclimated clone under low irradiance and salinity conditions was treated to understand the effects of high salinity and irradiance on a suite of subcellular (chlorophyll a ﬂuorescence kinetics and JIP-test, and chlorophyll content) to organismal (relative growth rate—RGR) physiological parameters. The responses to high irradiance indicated the plant’s great photo-acclimation potential to regulate the number and size of the reaction centers and the photosynthetic electron transport chain by dissipation of the excess energy to heat. A statistically signiﬁcant interaction ( p < 0.01) of salinity and irradiance on Chl a , b content indicated acclimation potential through adjusting the Chl a , b contents. However, no signiﬁcant ( p > 0.05) difference was observed on Chl a/b ratio and the RGR, indicating the species’ potential to become acclimatized by reallocating resources to compensate for growth. Thus, the regulation of photosynthetic pigment content and photosystem II performance consisted of the primary growth strategy to present and future high salinity and irradiance stressful conditions due to eutrophication management and the ongoing climatic changes.


Introduction
Submerged macrophytes are aquatic plants of remarkable phenotypic plasticity [1] that grow across contrasting conditions, from pristine to degraded lakes, estuaries, and coastal lagoons [2,3]. They provide food and habitat for invertebrates, larvae and juvenile fishes [4]. They also play a crucial role in water quality improvement by stabilizing the sediments [2]. In addition, they take up excess nitrogen and phosphorus and excrete allelopathic substances [5], contributing to reductions in nuisance algal blooms [6]. Therefore, they are valuable components maintaining the functioning of ecosystems [7,8].
Stuckenia pectinata is a shade-adapted plant [18] with a relatively low irradiance optimum (50-60 µmol photons m −2 s −1 ) for photosynthesis [1,18]. Under highly turbid eutrophic conditions, it lengthens its bundles in the upper water layer without exposing salinity laboratory conditions was used to avoid seasonality or acute effects on species physiology. We applied a suite of physiological parameters representative of subcellular (Chl a fluorescence and Chl content) to organismal (growth) processes. The results will be valuable (a) in gaining an insight into on the photosynthetic and growth responses that allow the species to inhabit the brackish and clear water depths, (b) in contributing a management strategy to protect the freshwater habitats of the lake under the ongoing climatic changes.

Plant Material
A S. pectinata strain isolated from Vistonis Lake (Greece) was cultivated for two years in a PVC tank with tap water in the laboratory at 15-18 • C and 30 µmol photons m −2 s −1 , 14 h light per day. Twenty-four apical shoots of the plant were randomly chosen for the experiment and pre-acclimated for 14 days at increased salinity from 4 to 19 (1 salinity degree per day), in 2 L aquariums at 21-22 • C and 60 µmol photons m −2 s −1 , 14 h light per day. The eutrophic medium was renewed once a week, and it consisted of artificial aerated medium (60 µmol/L N-NO 3 − and 2 µmol/L P-PO 4 − ) produced by Münster sea salt (Meersalz) diluted in resin-filtered tap water (<15 µS cm −1 ).

Experimental Design and Treatments
A factorial experiment (n = 6) was carried out in culture chamber at 24-26 • C and lasted ten days in which combined effects of salinity (two levels: 9 and 19) and irradiance (two levels: 100 and 400 µmol photons m −2 s −1 , MI and HI, respectively) on the pre-acclimated S. pectinata were tested. There were four experimental conditions. C1: salinity 9 and irradiance 100 µmol photons m −2 s −1 , C2: salinity 19 and irradiance 100 µmol photons m −2 s −1 , C3: salinity 9 and irradiance 400 µmol photons m −2 s −1 , C4: salinity 19 and irradiance 400 µmol photons m −2 s −1 . Irradiance was provided for 14 h per day by LED Fyto-Panels (81 × 27 cm; Photon Systems Instruments, Drasov, Czech Republic). Two hundred milliliters of the medium was renewed every day inside small glass jars covered by transparent glass (2 mm) to avoid evaporation. All glass jars used for the experiment were placed on shakers to avoid medium stratification.

Chlorophyll-a Fluorescence Measurements
Chlorophyll a fluorescence measurements were carried out on experimental days 1, 5, and 8. The polyphasic Chl a fluorescence kinetics (OJIP) were measured using a continuous excitation plant efficiency analyzer (Handy PEA; Hansatech Instruments Ltd., Norfolk, UK). Before the measurement of each specimen, a dark adaptation of 15 min was conducted. Plant leaves were illuminated with continuous red light (wavelength in peak 650 nm) from three diodes of 3.000 µmol photons m −2 s −1 . The fast fluorescence rise kinetics was recorded from 10 µs to 1 s. The fluorescence intensity at 20 µs, 50 µs, 100 µs 300 µs, 2 ms, and 30 ms was measured, and the maximum fluorescence was extracted. Several basic parameters were calculated from the extracted data, and the basic parameters derived by the JIP-test models included nine biophysical parameters (Table 1).
maximum quantum yield for primary photochemistry quantum yield for reduction of end electron acceptors (RE) at the PSI acceptor side ψ E0 ≡ ET 0 /TR 0 = (1 − V J ) efficiency/probability that an electron moves further than Q A − δ R0 ≡ RE 0 /ET 0 = (1 − V I )/(1 − V J ) efficiency/probability that an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side Specific energy flux (per active, i.e., per Q A -reducing PSII reaction centre -RC), in ms

Chlorophyll (a, b) Content
Chlorophyll a and Chl b content was determined at the end of the experiment (day 10th), according to [44]. The chlorophyll concentration was expressed as mg/g wet biomass (wb) of a leaf. The extraction was carried out in a dark room using 10 mL of 90% acetone and clear sand and the centrifugation lasted 10 min at 1500 rpm. Specimens' absorbance was measured at the wavelengths of 647, 664, and 750 nm using a Shimadzu UV-1800 spectrophotometer, Kyoto, Japan. The chlorophyll content was calculated according to the following equations: Chl a (µg mL −1 ) = 11.93E 664 − 1.93E 647 , Chl b (µg mL −1 ) = 20.3E 647 − 4.68E 664 , where E = corrected absorbency (absorbency at the wavelength − absorbency at 750 nm). The final chlorophyll content was expressed as mg Chl g −1 wb using the volume of acetone used to extract the pigments, divide by the wb of the leaf.

Relative Growth Rate
The relative growth rate (RGR) was calculated as RGR (day −1 ) = (LnWB t2 − LnWB t1 )/(t 2 − t 1 ), where WB t2 was the wet biomass on the experimental day 10th, and WB t1 was the wet biomass on the experimental day 1st.

Statistical Analysis
All statistical analyses were conducted using the software STATISTICA for Windows (version 7.1; StatSoft and TIBCO Software Inc., Palo Alto, CA, USA). Data were presented as mean (n = 6) ± standard error (SE) for each sample. Normality was tested by the Shapiro-Wilk's W test, while Levene's test tested the homogeneity. The effects of salinity (fixed factor, two levels: 9 and 19) and irradiance (fixed factor, two levels: 100 and 400 µmol photons m −2 s −1 ) on JIP-test parameters were analyzed with the parametric repeated measures analysis of variance (two-way repeated-measures ANOVA) and on chlorophyll content and RGR with parametric factorial analysis of variance (two-way ANOVA). The multiple comparisons were tested by Duncan's post hoc test. In the present study, samples did not have normal distribution and homogeneity of variance; thus, the significance level (p-value) was defined as 0.01 [45]. Redundancy analysis (RDA) was used to quantify the irradiance and salinity effects on the variation of chlorophyll fluorescence (day 8th), chlorophyll content (day 8th), and RGR (day 10th) parameters using Canoco 5.1 (Microcomputer Power, Ithaca, NY, USA). Statistical tests were run using the Monte Carlo permutation procedure.

(a) Normalizations and Subtractions
The chlorophyll a fluorescence polyphasic O-J-I-P kinetics of dark-adapted samples grown under C1, C2, C3, and C4, for 1, 5, and 8 days are evident in Figure 1. Each kinetic is the average of six replicates, after each of the kinetics Ft = f(t) was normalized on its initial value; i.e., the averages are plotted as F t /F 0 = f(t). This normalization was permitted as the minor variations observed among the raw F 20µs values were random and not depending on the treatment. Hence, the experimental F 20µs was reasonably taken as the real F 0 . The kinetics demonstrated clearly that high light levels (C3 and C4) result in a pronounced suppression of the variable fluorescence (F t − F 0 ) throughout the entire time course of the 1 s.

(b) The JIP-Test Parameters
The trapped energy flux per RC (TR 0 /RC) was not affected by irradiance or salinity and their interaction ( Figure 2A, Table S1), but the effect of irradiance was significant on RCs per PSII antenna Chl a (RC/ABS; p < 0.001, Table S1). Mean RC/ABS values ( Figure 2B) were higher at 100 µmol photons m −2 s −1 (C1, C2) than at 400 µmol photons m −2 s −1 (C3, C4). The maximum RC/ABS values were observed at C2 and at C1 on the 5th day, while the minimum values were observed at C3 on the 5th day and at C4 on the 1st day. Post hoc comparisons (Table S2) showed that RC/ABS values were significantly lower in C3 and C4 than in C1 and C2.

(b) The JIP-Test Parameters
The trapped energy flux per RC (TR0/RC) was not affected by irradiance or salinity and their interaction ( Figure 2A, Table S1), but the effect of irradiance was significant on RCs per PSII antenna Chl a (RC/ABS; p < 0.001, Table S1). Mean RC/ABS values ( Figure  2B) were higher at 100 μmol photons m −2 s −1 (C1, C2) than at 400 μmol photons m −2 s −1 (C3, C4). The maximum RC/ABS values were observed at C2 and at C1 on the 5th day, while the minimum values were observed at C3 on the 5th day and at C4 on the 1st day. Post hoc comparisons (Table S2) showed that RC/ABS values were significantly lower in C3 and C4 than in C1 and C2.
The effect of irradiance was significant on the light absorption flux of antenna chlorophyll molecules per RC (also a measure of PSII apparent antenna size-ABS/RC; p < 0.001, Table S1). Mean ABS/RC values ( Figure 2C) were higher at 400 μmol photons m −2 s −1 (C3, C4) than at 100 μmol photons m −2 s −1 (C1, C2). The maximum values of ABS/RC were observed at C3 on the 5th day and at C4 on the 1st day, while the minimum values were observed at C1 on the 5th day and at C2 on the 8th day. Post hoc comparisons (Table S3) showed that ABS/RC values were significantly lower in C1 and C2 than in C3 and C4 conditions. There was a significant interaction between salinity and treatment time on ABS/RC (p < 0.01, Table S1). At salinity 19 (C2, C4), no significant differences were observed. At 400 μmol photons m −2 s −1 (C3), ABS/RC values increased from the 1st to the 5th day, but they decreased on the 8th day.
The effect of irradiance was significant on the dissipated energy flux per RC (DI0/RC; p < 0.001, Table S1). Mean DI0/RC values were higher at 400 μmol photons m −2 s −1 (C3, C4) than at 100 μmol photons m −2 s −1 (C1, C2). The maximum of DI0/RC were observed at C3 on the 5th day and at C4 on the 1st day, while the minimum values were observed at C2 on the 8th day and at C2 on the 1st day. Post hoc comparisons (Table S4) showed that DI0/RC values were significantly lower in C1 and C2 than in C3 and C4 conditions. Values are means ± standard error, n = 6. Statistical differences from the post hoc test (p < 0.01) are represented using different Latin letters. For further information see Table 1.
The effect of irradiance was significant on φP0, i.e., the efficiency by which PSII transforms excitation energy to oxido-reduction energy (reduction of the primary electron quinone acceptor QA to QA − ) (p < 0.01, Table S1). Mean φP0 values ( Figure 3A) were higher at 100 μmol photons m −2 s −1 (C1, C2) than at 400 μmol photons m −2 s −1 (C3, C4). The maximum φP0 values were observed at C1 on the 1st day and at C2 on the 8th day, while the minimum values were observed at C3 on the 5th day and at C4 on the 1st day. Post hoc comparisons (Table S5) showed that φP0 values were significantly lower in C3 and C4 than in C1 and C2. There was also a significant interaction between salinity, irradiance and treatment time on φP0 (p < 0.01, Table S1). At salinity 9 and irradiance 400 μmol photons m −2 s −1 (C3), φP0 values decreased on the 5th day, but they partly recovered on the 8th day. In the other conditions (C1, C3, C4), φP0 did not exert significant changes over time.  Table 1.
The effect of irradiance was significant on the light absorption flux of antenna chlorophyll molecules per RC (also a measure of PSII apparent antenna size-ABS/RC; p < 0.001, Table S1). Mean ABS/RC values ( Figure 2C) were higher at 400 µmol photons m −2 s −1 (C3, C4) than at 100 µmol photons m −2 s −1 (C1, C2). The maximum values of ABS/RC were observed at C3 on the 5th day and at C4 on the 1st day, while the minimum values were observed at C1 on the 5th day and at C2 on the 8th day. Post hoc comparisons (Table S3) showed that ABS/RC values were significantly lower in C1 and C2 than in C3 and C4 conditions. There was a significant interaction between salinity and treatment time on ABS/RC (p < 0.01, Table S1). At salinity 19 (C2, C4), no significant differences were observed. At 400 µmol photons m −2 s −1 (C3), ABS/RC values increased from the 1st to the 5th day, but they decreased on the 8th day.
The effect of irradiance was significant on the dissipated energy flux per RC (DI 0 /RC; p < 0.001, Table S1). Mean DI 0 /RC values were higher at 400 µmol photons m −2 s −1 (C3, C4) than at 100 µmol photons m −2 s −1 (C1, C2). The maximum of DI 0 /RC were observed at C3 on the 5th day and at C4 on the 1st day, while the minimum values were observed at C2 on the 8th day and at C2 on the 1st day. Post hoc comparisons (Table S4) showed that DI 0 /RC values were significantly lower in C1 and C2 than in C3 and C4 conditions.
The effect of irradiance was significant on ϕ P0 , i.e., the efficiency by which PSII transforms excitation energy to oxido-reduction energy (reduction of the primary electron quinone acceptor Q A to Q A − ) (p < 0.01, Table S1). Mean ϕ P0 values ( Figure 3A) were higher at 100 µmol photons m −2 s −1 (C1, C2) than at 400 µmol photons m −2 s −1 (C3, C4). The maximum ϕ P0 values were observed at C1 on the 1st day and at C2 on the 8th day, while the minimum values were observed at C3 on the 5th day and at C4 on the 1st day. Post hoc comparisons (Table S5) showed that ϕ P0 values were significantly lower in C3 and C4 than in C1 and C2. There was also a significant interaction between salinity, irradiance and treatment time on ϕ P0 (p < 0.01, Table S1). At salinity 9 and irradiance 400 µmol photons m −2 s −1 (C3), ϕ P0 values decreased on the 5th day, but they partly recovered on the 8th day. In the other conditions (C1, C3, C4), ϕ P0 did not exert significant changes over time. Values are means ± standard error, n = 6. * = statistically significant interaction. For further information see Table 1 and Figure  2.
The effect of irradiance was significant on the efficiency/probability that an electron moves further than QA − (ψE0; p < 0.01, Table S1). Mean ψE0 values ( Figure 4A) were higher Values are means ± standard error, n = 6. * = statistically significant interaction. For further information see Table 1 and Figure 2. The effect of irradiance was significant on the quantum yields for electron transport to the plastoquinone (PQ) pool (ϕ E0 ; p < 0.01, Table S1). Mean ϕ E0 values ( Figure 3B) were higher at 100 µmol photons m −2 s −1 (C1, C2) than at 400 µmol photons m −2 s −1 (C3, C4). The maximum ϕ E0 values were observed at C2 and at C1 on the 1st day, while the minimum values were observed at C3 on the 5th day and at C4 on the 1st day. Post hoc comparisons (Table S6) showed that ϕ E0 values were significantly lower in C3 and C4 than in C1 and C2. The effect of treatment time was significant on ϕ E0 (p < 0.01, Table S1). ϕ E0 values in C3 decreased on the 5th day, but they partly recovered on the 8th day.
The effect of irradiance was significant on the quantum yields for electron transport to the electron acceptors at the PSI acceptor side (ϕ R0 ; p < 0.01, Table S1). Mean ϕ R0 values ( Figure 3C) were higher at 100 µmol photons m −2 s −1 (C1, C2) than at 400 µmol photons m −2 s −1 (C3, C4). The maximum ϕ R0 values were observed at C2 on the 1st day and at C2 on the 5th day, while the minimum values were observed at C3 and at C4 on the 5th day. Post hoc comparisons (Table S7) showed that ϕ R0 values were significantly lower in C3 and C4 than in C1 and C2. There was also a significant interaction between irradiance and treatment time on ϕ R0 (p < 0.01, Table S1). At irradiance 400 µmol photons m −2 s −1 (C3, C4), ϕ R0 decreased from the 1st to the 5th day, but they partly recovered on the 8th day, while at 100 µmol photons m −2 s −1 (C1, C2), ϕ R0 decreased over time.
The effect of irradiance was significant on the efficiency/probability that an electron moves further than Q A − (ψ E0 ; p < 0.01, Table S1). Mean ψ E0 values ( Figure 4A) were higher at 100 µmol photons m −2 s −1 (C1, C2) than at 400 µmol photons m −2 s −1 (C3, C4). The maximum ψ E0 values were observed at C1 on the 1st day and at C2 on the 1st day, while the minimum values were observed at C3 on the 5th day and at C4 on the 5th day. Post hoc comparisons (Table S8) showed that ψ E0 values were significantly lower in C3 and C4 than in C1 and C2. The effect of treatment time was significant on ψ E0 (p < 0.01, Table S1). Ψ E0 values decreased on the 5th day, but they partly recovered on the 8th day.
The effect of irradiance was significant on the efficiency/probability that an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side (δ R0 ; p < 0.01, Table S1). Mean δ R0 values ( Figure 4B) were higher at 400 µmol photons m −2 s −1 (C3, C4) than at 100 µmol photons m −2 s −1 (C1, C2). The maximum δ R0 values were observed at C4 on the 1st day and at C3 on the 5th day, while the minimum values were observed at C2 and at C1 on the 8th day. Post hoc comparisons (Table S9) showed that δ R0 values were significantly higher in C3 and C4 than in C1 and C2.
The effect of irradiance was significant on the connectivity of PSII photosynthetic units (M 0 '/M 0 ; p < 0.01, Table S1). Mean M 0 '/M 0 values ( Figure 4C) were higher at 400 µmol photons m −2 s −1 (C3, C4) than at 100 µmol photons m −2 s −1 (C1, C2). The maximum M 0 '/M 0 values were observed at C3 and at C4 on the 5th day, while the minimum values were observed at C1 on the 1st day and at C2 on the 8th day. Post hoc comparisons (Table S10) showed that M 0 '/M 0 values were significantly lower in C3 and C4 than in C1 and C2. Differences due to salinity and time are found not to be statistically significant.
values were observed at C3 and at C4 on the 5th day, while the minimum values were observed at C1 on the 1st day and at C2 on the 8th day. Post hoc comparisons (Table S10) showed that M0'/M0 values were significantly lower in C3 and C4 than in C1 and C2. Differences due to salinity and time are found not to be statistically significant.

Chlorophyll Content
There was a significant interaction between irradiance and salinity on leaf Chl a and Chl b content (p < 0.001, Table S11). Mean Chl a and Chl b values are shown in Figure 5A,B and were maximal at C3 and minimal at C1. Post hoc comparisons showed that Chl a (Table S12) and Chl b (Table S13) values under C1 and C4 were significantly lower than those under C3. There was not any significant effect of irradiance and salinity on Chl a/b ratio (Table S2). Mean values of the Chl a/b ratio are shown in Figure 5C. The maximum mean Chl a/b values were at C2 and the minimum values were at C4. and were maximal at C3 and minimal at C1. Post hoc comparisons showed that Chl a (Table S12) and Chl b (Table S13) values under C1 and C4 were significantly lower than those under C3. There was not any significant effect of irradiance and salinity on Chl a/b ratio (Table S2). Mean values of the Chl a/b ratio are shown in Figure 5C. The maximum mean Chl a/b values were at C2 and the minimum values were at C4.

Relative Growth Rate
There was no significant effect of salinity and irradiance on RGR (Table S14). Mean RGR values ( Figure 6) were maximal at C3 and minimal at C2.

Relative Growth Rate
There was no significant effect of salinity and irradiance on RGR (Table S14). Mean RGR values ( Figure 6) were maximal at C3 and minimal at C2.

Redundancy Analysis (RDA)
Analysis of the correlations between the physiological parameters and stress factors (irradiance and salinity) was performed using an RDA forward selection model ( Figure  7). According to the RDA results, irradiance was the only statistically significant factor that added 98.2% (Monte Carlo permutation test: pseudo F = 17.4, p = 0.001) to the explanatory power. The first axis accounted for 44.2% of the variation (Monte Carlo permutation test: F = 0.88) and was correlated with the irradiance, while the second axis accounted for 0.38% of the variation. The third axis accounted for 9.2% of the variation, while the fourth axis accounted for 3.9% of the total variation. While DI0/RC, ABS/RC, δR0 and M0'/M0 were

Redundancy Analysis (RDA)
Analysis of the correlations between the physiological parameters and stress factors (irradiance and salinity) was performed using an RDA forward selection model (Figure 7). According to the RDA results, irradiance was the only statistically significant factor that added 98.2% (Monte Carlo permutation test: pseudo F = 17.4, p = 0.001) to the explanatory power. The first axis accounted for 44.2% of the variation (Monte Carlo permutation test: F = 0.88) and was correlated with the irradiance, while the second axis accounted for 0.38% of the variation. The third axis accounted for 9.2% of the variation, while the fourth axis accounted for 3.9% of the total variation. While DI 0 /RC, ABS/RC, δ R0 and M 0 '/M 0 were positively correlated with Axis 1, ψ E0 , ϕ E0 , ϕ P0 , and RC/ABS were negatively correlated ( Table 2). The experimental points were distributed rather homogeneously on the PC1/PC2 plane, with PC1 to clearly separate low light from high light conditions. This indicates that the irradiance was by far the most important factor affecting the studied physiological parameters of S. pectinata. Figure 6. Effects of salinity and irradiance on relative growth rate (RGR). Values are means ± standard error, n = 6. For further information see Figure 2.

Redundancy Analysis (RDA)
Analysis of the correlations between the physiological parameters and stress factors (irradiance and salinity) was performed using an RDA forward selection model ( Figure  7). According to the RDA results, irradiance was the only statistically significant factor that added 98.2% (Monte Carlo permutation test: pseudo F = 17.4, p = 0.001) to the explanatory power. The first axis accounted for 44.2% of the variation (Monte Carlo permutation test: F = 0.88) and was correlated with the irradiance, while the second axis accounted for 0.38% of the variation. The third axis accounted for 9.2% of the variation, while the fourth axis accounted for 3.9% of the total variation. While DI0/RC, ABS/RC, δR0 and M0'/M0 were positively correlated with Axis 1, ψE0, φE0, φP0, and RC/ABS were negatively correlated ( Table 2). The experimental points were distributed rather homogeneously on the PC1/PC2 plane, with PC1 to clearly separate low light from high light conditions. This indicates that the irradiance was by far the most important factor affecting the studied physiological parameters of S. pectinata. Figure 7. Triplot from the redundancy analysis (RDA) that shows the relationships between the JIP-test parameters, chlorophyll content and relative growth rate (RGR) parameters of Stuckenia pectinata. The length of the arrows indicates the strength of representation and contribution of each parameter to the PC axes. MI = moderate irradiance, HI = high irradiance, 9 and 19 correspond to salinity values. For further information, see Tables 1 and 2. The first axis accounted for 44.2% of the variation, while the second axis accounted for 0.38%.

Irradiance Effects
The irradiance significantly affected the photosynthetic performance from the two factors tested, but not the chlorophyll content and RGR of S. pectinata (Figure 7). Acclimation of plants to HI includes regulation of (a) the number of the reaction centers and the size of their light-harvesting antennae, (b) the photosynthetic electron transport chain by dissipation of the excess energy to heat, and (c) the stoichiometry of photosynthetic components such as chlorophylls [46][47][48].
Under HI conditions (C3, C4), the density of active RCs (RC/ABS) per PSII, i.e., the number of Q A -reducing RCs per PSII antenna Chl, as a result of partial inactivity of RCs [49], decreased. This result indicates that some active RCs were converted into heat sinks, enhancing the energy dissipation, as also indicated by DI 0 /RC. DI 0 /RC represents the total dissipation of untrapped excitation energy from all RCs concerning the number of active RCs [35]. As the number of inactive centers under HI increased ( Figure 2B), the DI 0 /RC ratio also increased ( Figure 2D) because the inactive centers could not trap the photon, so the number of untrapped photons increased. Thus, the heat dissipation of excessive excitation energy slowed the overreduction of the photosynthetic electron transfer chain and minimized the potential photooxidative damage. On the other hand, an increase in the absorbance flux per reaction center (ABS/RC) value, as a measure for the average PSII antennae size [49], indicated overloading of PSII RC with electrons [38]. Although, in general, a smaller photosystems antenna size is reported under high light [50], we speculate that this is related to a Chl content increase, at least under the moderate salinity condition (C3).
HI affected negatively the parameters related to yields (ϕ P0 , ϕ E0 , and ϕ R0 ) and efficiency/probability (ψ E0 ) (p < 0.01), indicating that photoinhibition on PSII activity occurred of S. pectinata that considered as a positive acclimation for the downregulation of photosynthetic excitation pressure [51,52]. HI was also observed to reduce the maximum quantum yield in other plants [53,54]. Hu et al. [26] also showed that HI induced the decrease in effective quantum yield of PSII in S. pectinata. In agreement, TR 0 /RC represents the maximal rate by which an exciton is trapped by the RC, resulting in the reduction of Q A − [55], which remained unaffected with all the treatments (Figure 2A). These results confirm that S. pectinata seemed to exhibit a photo acclimation rather than the photodamage of the reaction center complexes when exposed to HI. Among the numerous biophysical parameters derived by the JIP-test, ϕ R0 is theoretically related to the energy supply from PSI towards the Calvin cycle, which is by definition the quantum yield of this supply. Indeed, there was a weak correlation ϕ R0 with RGR ( Table 2, RDA axis 4th). However, the actual energy supply to the Calvin cycle is affected by several other factors in addition to ϕ R0 , and RGR does not depend only on the activity of the Calvin cycle. Thus, in this study, HI did not impair the RGR. As a result, the plants could relocate resources to compensate for any potential costs associated with photoinhibition, repair of photosystem II, and processes of high-light acclimation.
On the other hand, δ R0 increased when S. pectinata was exposed to HI, suggesting an improvement in the efficiency of electron transport, and therefore, energy, from plastoquinol (PQH 2 ) via cytochrome b 6 /f to the PSI end electron acceptors (plastocyanin, ferredoxin) [56]. Thus, overexcitation energy pressure in PSII can be alleviated despite not increasing the sum of energy flux into PSI acceptors. Indeed, δ R0 values increased during the first steps of stress, and only severe stress caused depletion of the efficiency of electron transport up to end electron acceptors of PSI [57]. The result was consistent with those in the stems of Hexinia polydichotoma. It suggests that the electron transport pattern related to PSI can play an important role in the photoprotection of PSII in adverse conditions [58].
Acclimation to HI was also expressed by a decrease in the energetic connectivity of PSII photosynthetic units compared to moderate irradiance treated samples (C1, C2). It is worth remembering that, in separate units, the initial fluorescence rise is exponential and that the bigger the degree of connectivity, the more the curves deviate towards sigmoidal [35]. Though none of the kinetics in the present study appear sigmoidal, the deviation from exponential could not be excluded, and it was evaluated by comparing M 0 ' with M 0 (see Table 1) since the higher is their ratio, the more towards the exponential curve is the fluorescence rise.

Interactive Effects between Irradiance and Salinity
The chlorophyll content is associated with acclimation to different irradiance levels. HI or sun-grown plants contain lower chlorophyll than low light-grown or shade plants, which is considered a long-term regulation mechanism that controls light absorption capacity [59]. The results reveal that Chl a and Chl b content in S. pectinata increased significantly (p < 0.01) under HI and low salinity ( Figure 5, Table S2), inconsistent with the general trend. Hu et al. [26] and Pilon and Santamaría [1] showed that the total chlorophyll concentration of S. pectinata was higher at low irradiance, which resulted in higher photosynthetic performance. Obviously, the HI condition used in this study was not too high to make the plants unable to produce more chlorophyll. This result is in line with a previous study focusing on Zostera marina (L) responses to light stress applied within controlled laboratory conditions [60]. Hu et al. [26] also noticed that HS and HI reduced (p < 0.001) the Chl a content in S. pectinata, in agreement with the results of this study, where Chl a content at HI and HS was lower (p < 0.01) than HI and MS.
The Chl a/b ratio indicates the degree of sun/shade acclimation and the structure of the photosynthetic apparatus to improve light capture [61,62]. Chl a/b ratio decreases under low irradiance conditions because the plants develop bigger light-harvesting complexes (LHCs). Both Chl a and Chl b are found in LHCs, yet only Chl a is found in the reaction centers. As a result, bigger LHCs as a response to low light levels lead to increments of both Chl a and Chl b; still, the overall Chl a/b ratio drops as there is no increase in the number of Chl a molecules forming the reaction centers of the photosystems. This pattern was not observed in our study, where the Chl a/b ratio remained constant under all treatments, as in the studies of [38,63] regarding the species of Lemna. Accordingly, the dissipation of energy became extremely important, reducing the risk of photoinhibition (see above). In any case, plants had a light-harvesting antenna size (mean Chl a/b ratios < 6.5) not susceptible to HI [64]. The absence of significant changes in the Chl a/b ratio under irradiance stress also revealed that the irradiance levels were adequate for the plant's growth throughout the experiment [65]. It is well-known that under stressful conditions, the plant may reallocate resources toward increased growth or alterations in morphology at the expense of defensive compounds and reproductive structures [41].

Interactive Effects with Time
Photo-acclimation corresponds to different processes involving many cellular components and occurring over a broad range of time scales, from seconds to days. This study focused on long-term photo acclimation (days) monitored by changes in the number and size of the reaction centers and the photosynthetic electron transport chain. Changes within the eight days of the experiment duration were noticed in all measured parameters but statistical changes in the parameters related to yields (ϕ P0 , ϕ E0 , and ϕ R0 ) and in DI 0 /RC. Besides regulation of irradiance as discussed above, ϕ P0 and DI 0 /RC acclimation is also affected by salinity, indicating that the photosynthetic apparatus could cope with salinity stress, and the plant was tolerant within the range from 9 to 19. The great range of tolerance of S. pectinata to salinity (9-18) has been reported by other studies [3,23,24]. Hu et al. [26] also reported that PSII units and electron flow were not influenced under salinity stress.

Stuckenia pectinata in Vistonis Lake
Ruppia maritima L. is a submerged macrophyte that survives in brackish estuarine and coastal lagoon waters near Vistonis Lake. Following the scenario of the replacement of S. pectinata by R. maritima due to sea level and salinity rise, it seems the lower biomass which would be available to the overwintering waterfowl at Vistonis will impact the structure and function of higher trophic levels [66]. However, based on this study, that is a not near-future scenario as the Vistonis S. pectinata clone tolerated salinity of at least up to 19 and can occasionally survive salinity increases up to 34. High tolerance was also confirmed by the RGR but evidenced a tendency of a decrease at a salinity of 19 ( Figure 6). This is a topic that should be further investigated in the future through long-term experiments.

Conclusions
This study investigates the correlations among parameters representing processes of two different biological levels, from subcellular (photosynthetic) to organismal (growth). It is now clear that the submerged macrophyte S. pectinata had great acclimation potential to present and future stressful irradiance and salinity conditions in hypertrophic Vistonis Lake. Such physiology might also be of great importance for other environmental disturbances, such as heavy metals. However, further research on the role of other abiotic factors, such as temperature and different nutrient forms, is needed to better determine how climate change will influence S. pectinata's abundance. In addition, other species aspects, such as morphology and reproduction, should be further investigated in the future in longterm experiments.