DOI: 10.1007/s11099-012-0046-2 PHOTOSYNTHETICA 50 (3): 437-446, 2012

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Photosynthetic response of Vallisneria natans (Lour.) Hara (Hydrocharitaceae) to increasing nutrient loadings X.L. CAI*,**, G. GAO*,+, X.M. TANG*, J.Y. DAI*,**, and D. CHEN*,** State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China* Graduate University of Chinese Academy of Sciences, Beijing 100049, China** Abstract To explore the effects of water column nutrient loading on photosynthesis of the submerged macrophyte Vallisneria natans (Lour.) Hara during the growth season (June to October), we determined the diurnal and seasonal variation in rapid light curves of plants cultivated under 4 different nutrient concentrations (N-P [mg L–1]: (1) 0.5, 0.05; (2) 1.0, 0.1; (3) 5.0, 0.5; (4) 10.0, 1.0). Nutrient concentration significantly affected the magnitude of the rapid light curves of V. natans, but not the direction of their diurnal variations. At low nutrient conditions (N-P 1 [mg L–1]: 0.5, 0.05), the maximum relative electron transport rate (rETRmax) and minimum saturating irradiance (Ek) derived from rapid light curves were significantly lower than those of other treatments, and their seasonal variations were suppressed. These results indicated that photosynthesis of V. natans was inhibited by the lack of nutrients in water column. At high nutrient conditions (N-P 4, [mg L–1]: 10.0, 1.0), there was an increase in photosynthetic rate in the light-limited region of rapid light curve (α), and a decrease in rETRmax and Ek, relative to moderate nutrient conditions (N-P 2, [mg L–1]: 1.0, 0.1). In addition, at high nutrient concentrations, the rapid light curves of V. natans reached a plateau, and then markedly declined compared with those at the lower nutrient levels, especially in July and August. These results suggested that V. natans were adapted to low-light environments in the high-nutrient loading treatment. Additional key words: diurnal variation; nutrient loading; rapid light curves; shading effect; Vallisneria natans. Introduction Nitrogen (N) and phosphorus (P) can play diverse roles in aquatic ecosystems; although these elements are often the primary limiting nutrients for growth of aquatic plants (Duarte 1990, Romero et al. 2006), they can also cause eutrophication of water bodies. Typically, submerged macrophytes decline with increasing eutrophication (Hosper and Jagtman 1990, Jeppesen et al. 1990, Klein 1993). Despite the frequency with which such loss of submerged macrophytes is observed, the mechanisms that lead to the loss of submerged macrophytes at high N and P concentrations are not well understood. Some resear-chers hold that the nutrients have a direct toxic effect on submerged macrophytes (Best 1980, Burkholder et al.

1992, Cao et al. 2004, Nimptsch and Pflugmacher 2007). Others suggest that the loss of hydrophytes is related to increased shading and competition from the epiphyton and/or phytoplankton (Phillips et al. 1978, Hough et al. 1989, Daldorph and Thomas 1995, Li et al. 2008).

Plants are easily influenced by environment, and have a series of mechanisms for responding to environmental changes (Wu et al. 2007). To understand the mechanisms that lead to the loss of macrophytes, it is necessary to have a deep understanding of the responses from sub-merged macrophytes during eutrophication. In studies on plant physiology and ecology, chlorophyll fluorescence analysis has been widely used as a noninvasive and rapid

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Received 27 March 2012, accepted 5 June 2012. +Corresponding author; tel.: + 86 25 86882187, e-mail: guanggao@niglas.ac.cn Abbreviations: Ek – minimum saturating irradiance; F – instantaneous fluorescence of light-adapted state; Fm’ – maximal fluorescence of light-adapted state; N – nitrogen; P – phosphorus; PAR – photosynthetically active radiation; PSII – photosystem II; rETR – relative electron transport rates; rETRmax – maximum relative electron transport rate; T – water temperature; α – photosynthetic rate in light-limited region of rapid light curve; ФPSII – effective quantum yield of photosystem II. Acknowledgments: We are grateful to Bo Zhang, Gongsheng Tao, Hui Tang, Linjin Yu, Lizhen Liu, Meng Sun, Qing Li, Weiwei Wang, Ying Chen and Yingying Zhang for help in the experiment. We also thank Sarah Poynton for her linguistic improvements. This work was supported by the National Basic Research Program of China (No. 2008CB418103), the National Water Science and Technology Projects (No. 2009ZX07101-013) and the National Natural Science Foundation of China (No. 41071341).

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method for monitoring the functional state of photosyn-thetic organisms (Krause and Weis 1991, Maxwell and Johnson 2000, Adams and Demmig-Adams 2004). This technique yields a wealth of information on photosyn-thesis. Detailed information on the photosynthetic performance and photoadaptive state of a plant is derived from the rapid light curves, which describe the relation-ship between electron transport rate (ETR) and photo-synthetically active radiation (PAR) (Schreiber 2004, Ralph and Gademann 2005). So the rapid light curves could be used to reflect the status of plants. However, in situ measurements of rapid light curves are strongly dependent on time of day and recent light history

(Edwards and Kim 2010). Consequently, measurements of rapid light curves using the Pulse-Amplitude-Modulation (PAM) fluorometry should consider fluctua-tions in ambient light conditions. To address this topic, we therefore investigated the diurnal and seasonal varia-tion in the rapid light curves of the macrophyte V. natans (Lour.) Hara with a wide distribution in China cultivated under different N and P concentrations during the growth season. We sought to: (1) determine how the photosyn-thetic behavior of V. natans was affected by inorganic nutrients in the water column, and (2) increase under-standing of the decline of this submerged macrophyte during eutrophication.

Materials and methods Plants: Winter buds of V. natans (fresh mass 1.02 ± 0.37 g) were planted in ten plastic containers (length 50 cm, width 38 cm, depth 25 cm) with 20 cm of tap water and 5 cm of sand for culture on 7 March 2010, and main-tained under greenhouse conditions. After two months, the growing buds at the stage of four to five leaves (about 20 cm of height) were transplanted into sand in individual plastic pots (diameter 7 cm, depth 10 cm). Twenty-one pots (each planted with a single shoot) were then transferred to each of the 16 high-density polyethylene containers (volume 100 L, top diameter 51 cm, bottom diameter 40 cm, depth 63 cm) containing full tap water (as experimental medium). During the first 2 weeks after all the individual pots were prepared, all plants were maintained under the same conditions in the greenhouse for acclimation prior to the experiment.

Experimental design: The experiment was conducted in 16 high-density polyethylene containers in the green-house and was started on 29 May 2010. We used the nutrient concentration in the water column as the experimental factor, with four increasing levels of N-P, as follow [mg L–1]: N-P 1 0.5, 0.05; N-P 2 1.0, 0.1; N-P 3 5.0, 0.5; and N-P 4 10.0, 1.0. This experiment was a completely random design, replicated 4 times. To main-tain the constant concentration of nutrients throughout the experiment, the total N and total P in the water were determined every 2 weeks (Jin and Tu 1990), and the nutrients were added as concentrated solutions of KNO3 and KH2PO4. Water lost to evaporation was replenished with tap water (total N 2.274 ± 0.012 mg L–1, total P 0.032 ± 0.001 mg L–1) to maintain the original water volume. Considering the total water volume in each bucket, nutrients added with tap water were insignificant.

Measurements of rapid light curves: Photosynthetic activity of V. natans was assessed using rapid light curves (White and Critchley 1999, Ralph and Gademann 2005). The rapid light curve measurements were made six times per day (at 7:00, 9:00, 11:00, 13:00, 15:00, and 17:00 h) on each of 4 days (4 June, 20 July, 11 August and

19 September). Measurements were taken under water on one representative (i.e., of typical appearance and inter-mediary age) leaf per container (French and Moore 2003), approximately 5 cm from the leaf top, using a Diving-PAM fluorometer (Walz, GmbH, Effeltrich, Germany).

The leaf of V. natans was clamped in a dark leaf clip (Diving-LC, Walz, Effeltrich, Germany), and the rapid light curves were measured using increasing actinic irradiances of 10-s duration (White and Critchley 1999, French and Moore 2003, Ralph and Gademann 2005), each separated by a 0.8-s saturating pulse (2,400–3,000 μmol m–2 s–1). The relative electron transport rate (rETR) was calculated as rETR = ФPSII × PAR × 0.84 × 0.5, where ФPSII = (Fm’ – F)/Fm’ is the effective quantum yield of photosystem II (PSII), Fm’ is the maximal fluorescence of light-adapted state, F is the instantaneous fluorescence of light-adapted state; PAR is the photosynthetically active radiation generated by the internal halogen lamp of the Diving PAM, 0.84 is the instrument default absorption coefficient, and 0.5 assumes that the photons absorbed are equally partitioned between the two photosystems (Genty et al. 1989). Because the accuracy of the internal halogen light source was sensitive to changes in battery power (Hanelt et al. 2003, Edwards and Kim 2010) and the measurement time was long, we analyzed all samples with a Diving-PAM plugged into an AC power source. The PAR immediately adjacent to the sample was measured with the microquantum sensor of the Diving-PAM, that had been calibrated against a Li-Cor quantum sensor (Li-Cor, Lincoln, NE, USA).

Data analysis: To determine the three photosynthetic parameters (α – photosynthetic rate in light-limited region of rapid light curve; rETRmax and Ek), and to quantitatively compare the rapid light curves, data from the rapid light curves were fitted to the exponential equation of Platt et al. (1980), as recommended by Ralph and Gademann (2005).

P = Ps × [1 – exp(–α × Ed/Ps)] × exp(–β × Ed/Ps) (1)

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Ps is a scaling parameter defined as the maximum potential rETR; α is measured by the initial slope of the rapid light curve before the onset of saturation; Ed is the downwelling irradiance (wavelength 400–700 nm); β is the negative slope of the rapid light curve for high irradiances. In the absence of photoinhibition (β = 0), Eq. 1 becomes a standard rectangular hyperbola, with an asymptotic maximum rETR value (Harrison and Platt 1986), and the equation loses exp(–β × Ed/Ps) (Edwards and Kim 2010). The parameters rETRmax and Ek were estimated using the following equations:

rETRmax = Ps × [α/(α + β)] × [β/(α + β)]β/α (2)

Ek = rETRmax/α (3)

Curves were fitted to the experimental data using Sigmaplot 10.0. The photosynthetic parameters (α, rETRmax, Ek), derived from curve-fitting, were analyzed using SPSS 11.5, and subjected to repeated measures ANOVA, with treatment as the main factor and period (time and date; time nested within date) as the repeated measure. To correct for violations of sphericity, the Greenhouse-Geisser adjustment was used. Significant differences among treatment means (p<0.05) were deter-mined by Bonferroni test. Relationships among variables were examined using Pearson’s correlation.

Results The diurnal variation in ambient PAR had a bimodal pattern on each of the 4 sampling dates, but with different absolute values and peak positions (Fig. 1). Daily mean PAR was 137, 131, 110, and 99 μmol m–2 s–1 for June, July, August, and September, respectively. The diurnal variation in water temperature showed an increase and then a decline after reaching the maximum in June, July and August, and a gradual increase in September (Fig. 1). The daily mean water temperature was 23.0, 30.9, 31.2, and 27.5oC for June, July, August, and September, respectively.

There were significant effects of water column

nutrient concentration, determination date (month), and time, on the photosynthetic parameters of V. natans, as determined from the rapid light curves (Table 1, Fig. 2). There was a significant positive correlation (p<0.01) between rETRmax and PAR, as well as between Ek and PAR. There was a significant negative correlation (p<0.01) between α and PAR (Table 2).

α, rETRmax and Ek showed significant differences among the different determination times (Table 1, Fig. 2), representing significant diurnal variation in the rapid light curves (Figs. 3,4,5,6). For example, measurements of rapid light curves on 4 June (Fig. 3) revealed that

Fig. 1. Diurnal variation in water temperature (T) (solid line) and photosynthetically active radia-tion (PAR) (dashed line) on 4 June, 20 July, 11 August, and 19 September 2010.

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Table 1. Results of ANOVA testing the effects of different nutrient concentration treatments, determination date and time on photosynthetic rate in light-limited region of rapid light curve (α), maximum relative electron transport rate (rETRmax), and minimum saturating irradiance (Ek) in Vallisneria natans.

* – p<0.05; ** – p<0.01; and *** – p<0.001.

Parameters Factors and interactions F value P value

α Treatment 1.335 0.323 Date 37.050 <0.001*** Time 70.988 <0.001*** Treatment × Date 1.078 0.410 Treatment × Time 2.348 0.014*

rETRmax Treatment 7.053 0.008** Date 1.826 0.200 Time 75.096 <0.001*** Treatment × Date 0.943 0.472 Treatment × Time 1.275 0.253

Ek Treatment 14.789 0.001** Date 24.695 <0.001*** Time 60.313 <0.001*** Treatment × Date 3.338 0.037* Treatment × Time 1.382 0.197

the diurnal variation of α under the different nutrient treatments was similar, but with different values, especially at 9:00 and 11:00 h. The changes of Ek and ETRmax were similar to those observed in PAR, following a bimodal pattern.

α, rETRmax and Ek showed significant differences

among determination dates (months) (Fig. 7). For α, there was a similar trend among different nutrient treatments from June to September, but with different values; the value of α in the N-P 4 treatment was higher than that in other treatments. For rETRmax, there were different seasonal trends among the nutrient treatments. In treatment N-P 1, containing the lowest concentrations of N and P, there was a little change in rETRmax during the plant growth season. In contrast, in N-P 2, N-P 3, and N-P 4, there were seasonal changes of rETRmax, with a similar unimodal pattern, and a maximum in July. Additionally, the rETRmax in N-P 1 and N-P 4 treatments in different months were lower than those in N-P 2 and N-P 3 treatments. The seasonal changes in Ek were similar to those of rETRmax.

There were differences in the shapes of the rapid light curves among the different nutrient treatments. The rapid light curves of N-P 4 treatment, at 7:00, 9:00 and 11:00 h in July, reached a plateau and then markedly declined (Fig. 4A–C). Five phenomena similar to this were observed for the rapid light curves of NP-4 treatment in August (Fig. 5A–E). The rapid light curves of N-P 3 treatment at 7:00, 9:00, 13:00, and 15:00 h in August showed a similar pattern to those of N-P 4, but the curves of N-P 3 declined more slowly than those of N-P 4 treatment (Fig. 5A,B,D,E). In September, the rapid light curves of different treatments measured at 9:00, 11:00, and 13:00 h tended to decline, with those of the N-P 4 treatment declining more rapidly than those of others (Fig. 6B–D).

Discussion Significant diurnal variations in the rapid light curves were observed in each treatment. The direction of diurnal variations of rapid light curves was not markedly influenced by water column nutrient concentration. The study of α, rETRmax, and Ek determined from rapid light curves has led to better understanding of the changes in rapid light curves and their relationship with environ-mental factors. In the present experiment, there was a sig-nificant positive correlation between rETRmax and PAR, as well as between Ek and PAR (Table 2). These results were in agreement with Silva and Santos (2003) who showed that diurnal variations in rETRmax and Ek deter-mined by Diving-PAM had a similar trend as the diurnal patterns of PAR. Our results also agree with those of previous researchers (Belshe et al. 2007, 2008; Edwards and Kim 2010) indicating that photosynthetic parameters are influenced by the recent light history of the plants.

Although our experiment was maintained in the greenhouse, seasonal changes were evident in PAR and water temperature (Fig. 1). While monitoring the varia-tion in rapid light curves of V. natans during 4 months of the growth season, we observed marked seasonal variation in the mean of each photosynthetic parameter (Fig. 2D–F), in agreement with studies by Dennison

(1987), Herzka and Dunton (1997) and Alcoverro et al. (1998). Water column nutrient concentration significantly affected the magnitude of the rapid light curves of V. natans, and its interaction with other environmental factors also influenced the seasonal variations in the photosynthetic parameters. In the N- and P-limited environment, foliar N and P contents increased with N and P additions (Harrington et al. 2001). Foliar N content is generally correlated with photosynthetic capacity (Evans 1989). The assimilation of N into amino acids required the provision of carbon skeletons and reducing power which could be supplied by photosynthetic processes (Turpin et al. 1990). Therefore, increased N availability may induce photosynthetic carbon fixation and metabolism. P is also a major mineral nutrient which is important for photosynthesis (Rychter and Rao 2005). Increased P availability is essential for the assimilation of photosynthetic carbon in plants (Campbell and Sage 2006). In treatment N-P 1, containing the lowest concentrations of N and P, the rETRmax and Ek were significantly lower than those in N-P 2 and N-P 3 treatments (Fig. 2B,C), and did not show marked seasonal changes (Fig. 7B,C). This was due to nutrient limitation in the water column of the N-P 1 treatment, and increased

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Fig. 2. Effects of nutrient loading treatment, determination date, and measurement time on photosynthetic rate in light-limited region of rapid light curve (α), maximum relative electron transport rate (rETRmax), and minimum saturating irradiance (Ek) (means ± SE, n = 4) of Vallisneria natans. Different letters indicate significant difference at p<0.05 (determined by mean separation with Bonferroni test). availability of N and P nutrients could promote photo-synthesis, in agreement with studies by Agawin et al. (1996), and Lee and Dunton (1999).

However, high nutrient levels can produce adverse effects on V. natans.  In the aquatic environment, in-creased levels of N and P are usually accompanied by an increase in epiphyton and phytoplankton (Schindler 1977, Hecky and Kilham 1988, Russell et al. 2005, Smith 2006, Faithfull et al. 2011), and the blooms of competing primary producers in water can lead to reductions in light available to submerged macrophytes (Borum 1985, Tomasko and Lapointe 1991, Wear et al. 1999, Williams et al. 2002, Roberts et al. 2003). The photosynthetic performance and photoadaptive state of submerged macrophyte during eutrophication could be reflected by the rapid light curves. In our experiment, nutrient enrichment not only induced significant changes in the magnitude of rapid light curves, but also affected the

shape of rapid light curves. Masini and Manning (1997) found that aquatic plants growing at their maximum depth limits had higher efficiency of light capture (α) and lower light requirements for saturated photosynthesis, than did plants growing in shallower water. Ruiz and Romero (2003) reported that lower saturated photosyn-thesis and greater photosynthetic efficiencies were observed in submerged macrophytes inhabiting inner harbors, in comparison to less turbid (higher light avail-ability) outer harbor. The initial slope of the rapid light curve (α) is related to the efficiency of light capture (Schreiber 2004, Ralph and Gademann 2005). Submerged macrophytes can increase their light-capture efficiency at low light conditions (Goldsborough and Kemp 1988, Maberly 1993, Olesen et al. 2002). Therefore, rETRmax

and Ek were lower and α was higher in the N-P 4 treatment than in the N-P 2 and N-P 3 treatments (Figs. 2,7), indicating that V. natans cultured in N-P 4

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Fig. 3. Diurnal variation, on 4 June, in rapid light curves (rETR-PAR), photosynthetic rate in light-limited region of rapid light curve (α), maximum relative electron transport rate(rETRmax), and minimum satura-ting irradiance (Ek) (means ±SE, n = 4) for Vallisneria natansunder 4 different nutrient con-centrations. Increasing levels of N-P [mg L–1]: N-P 1 0.5, 0.05; N-P 2 1.0, 0.1; N-P 3 5.0, 0.5; and N-P 4 10.0, 1.0.

Fig. 4. Diurnal variation, on 20 July, in rapid light curves(rETR-PAR), photosynthetic rate in light-limited region of rapid light curve (α), maxi-mum relative electron trans-port rate (rETRmax), and mini-mum saturating irradiance(Ek) (means ± SE, n = 4) for Vallisneria natans under 4 different nutrient concen-trations. Increasing levels of N-P [mg L–1]: N-P 1 0.5, 0.05; N-P 2 1.0, 0.1; N-P 3 5.0, 0.5; and N-P 4 10.0, 1.0.

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Fig. 5. Diurnal variation, on 11August, in rapid light curves(rETR-PAR), α, rETRmax and Ek

(means ± SE, n = 4) for Vallis-neria natans under 4 differentnutrient concentrations. Increas-ing levels of N-P (in mg L–1):NP-1 0.5, 0.05; NP-2 1.0, 0.1;NP-3 5.0, 0.5; and NP-4 10.0, 1.0.

Fig. 6. Diurnal variation, on 19 September, in rapid light curves(rETR-PAR), photosynthetic rate in light-limited region of rapid light curve (α), maximum relative electron transport rate(rETRmax), and minimum satura-ting irradiance (Ek) (means ±SE, n = 4) for Vallisneria natansunder 4 different nutrient noc-centrations. Increasing levels of N-P [mg L–1]: N-P 1 0.5, 0.05; N-P 2 1.0, 0.1; N-P 3 5.0, 0.5; and N-P 4 10.0, 1.0.

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Fig. 7. Seasonal variation in photosynthetic rate in light-limited region of rapid light curve (α), maximum relative electron transport rate (rETRmax), and minimum saturating irradiance (Ek) (means ± SE, n = 4) for Vallisneria natans under 4 different nutrient concentrations. Increasing levels of N-P [mg L–1]: N-P 1 0.5, 0.05; N-P 2 1.0, 0.1; N-P 3 5.0, 0.5; and N-P 4 10.0, 1.0.

Table 2. Correlation analysis (Pearson’s correlation coefficient) of two environmental variables, photosynthetically active radiation (PAR) and water temperature (T) and three photosynthetic parameters, photosynthetic rate in light-limited region of rapid light curve (α), maximum relative electron transport rate (rETRmax) and minimum saturating irradiance (Ek). Correlation is significant at the 0.01 level (2-tailed, Pearson’s correlation).

Parameter PAR T

α –0.595** –0.192 rETRmax 0.632** 0.358 Ek 0.742** 0.285

treatment might be subjected to light limitation and its photosynthesis was also reduced relative to moderate nutrient conditions. Additionally, the rapid light curves of N-P 4 treatment reached a plateau and then markedly declined when compared with those of the other nutrient treatments under similar light intensities (Figs. 4,5).

This phenomenon of decline in the rapid light curves also appeared in the plants subjected to treatment N-P 3 (Fig. 5), further showing that V. natans under high nutrient levels was sensitive to strong light. These results suggested that V. natans were adapted to low-light environments and their photosynthesis was inhibited in the high-nutrient loading treatment. 

Because of the special growth environment, sub-merged macrophytes not only face the concentration changes in chemical substances, but also may suffer from competing primary producers as a result of nutrient enrichment. The changes of the rapid light curves under the high nutrient conditions were likely related to reductions in light availability associated with increasing nutrient loadings. Therefore, our results suggested that the decline of submerged macrophytes during eutrophi-cation is not only related to the concentration changes of nutrients, but also possibly related to the changes of microbes as a result of nutrient enrichment. These results highlight the importance of the changes in microbes as a result of nutrient enrichment in the decline of submerged macrophytes during eutrophication.

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