Effects of NH₄⁺ and K⁺ enrichments on carbon and nitrogen metabolism, life history and asexual reproduction of Vallisneria natans L. in aquarium experiments

Abstract

Ammonium (NH₄⁺) is an important nitrogen nutrition for plant growth; however, it is toxic to many plants at high concentration. Eutrophication induces NH₄⁺enrichment in shallow lakes and increases risk of NH₄⁺ toxicity to submersed macrophytes. Potassium (K⁺) was known as an alleviant to the toxic syndromes of plant under NH₄⁺ stress. In the present study, we examined the single effects and co-effects of NH₄⁺ and K⁺ enrichments (∼0.65 mg L⁻¹ NH₄–N, ∼45 mg L⁻¹ K⁺) in water column on submersed macrophyte Vallisneria natans L. with focus on carbon (C) and nitrogen (N) metabolism, life history and asexual reproduction of the plant in outdoor aquariums lasting for about five months. We found that the NH₄⁺ enrichment alone increased growth and decreased death rate of the plant, leading to higher biomass, longer survivorship and more tuber production and ramets of the plants, indicating that the NH₄⁺ enrichment was not stressful to the plant. The combination of NH₄⁺ and K⁺ enrichments enhanced the plant growth to less extent as did the NH₄⁺ enrichment alone, and reduced the plant death rate. The NH₄⁺ enrichment increased the concentrations of free amino acids and nitrogen, and decreased the concentrations of soluble carbohydrate in leaves and tubers of the plant irrespective of the K⁺ enrichments. The K⁺ enrichments alone did not affect growth, death rates and C–N metabolism of the plant. The K⁺ alone or together with NH₄⁺ enrichments had negligible effect on tuber production and tuber C–N metabolism of the plant. The present results indicated that the K⁺ enrichment had negligible effects on the plant; the NH₄⁺ enrichment (0.65 mg L⁻¹ NH₄–N) benefited the plant growth, while it might negatively affect the asexual reproduction due to low carbohydrate contents of the tubers particularly under low light stress in eutrophic lake.

Keywords:

• NH₄⁺
• K⁺
• Vallisneria natans L.
• life history
• asexual reproduction
• carbohydrate
• free amino acids

Introduction

Ammonium (NH₄⁺) is an important nitrogen nutrition for plant. Moderate NH₄⁺ availability benefits plant growth; however, high NH₄⁺ concentration is toxic to many plants (Britto & Kronzucker 2002; Cao et al. 2007). In shallow eutrophic lakes, high NH₄⁺ concentration in water column is not uncommon due to overloading of sewage, agricultural runoff and depletion of oxygen in the lake, and is considered as one main cause for the succession and/or decline of submersed vegetation (Farnsworth-Lee & Baker 2000; Smolders et al. 2006; Cao et al. 2007; Zhang et al. 2011; Su et al. 2012).

Submersed macrophytes can take up NH₄⁺ and NO₃⁻ by roots and shoots and prefer NH₄⁺ over NO₃⁻ when both are equivalently available (Nichols & Keeney 1976; Short & McRoy 1984; Barko et al. 1991). In experimental studies, NH₄⁺ supplement in water column induced the accumulation of nitrogen and free amino acids (FAA) and depletion of soluble carbohydrates (SC) in the tissues of submersed macrophytes (Best 1980; Smolders et al. 1996; Kohl et al. 1998; Cao et al. 2009), and stimulated plant growth in oligotrophic waters but inhibited plant growth in eutrophic waters (Cao et al. 2007). In mesocosm experiments, NH₄⁺ supplement in water column led to low SC contents in rhizomes of Vallisneria natans L. and seagrass Posidonia oceanica (Invers et al. 2004; Cao et al. 2007), and inhibited ramet production of V. natans possibly due to low SC contents in the rhizomes (Cao et al. 2007).

Mechanism of NH₄⁺ toxicity to plants is far from clear, possible mechanisms including acidification of growth media, uncoupling of photosynthetic phosphorylation, oxidative stress, futile recycling of NH₄⁺ across membrane and unbalance of C–N metabolism of plants (Kohl et al. 1998; Britto et al. 2001; Britto & Kronzucker 2002; Nimptsch & Pflugmacher 2007; Cao et al. 2011). Potassium (K⁺) is similar to NH₄⁺ with respect to molecular radius, electron charge and hydration energy, implying that they share some common components in membrane transport (Tsay et al. 2011). Addition of K⁺ can alleviate NH₄⁺ toxicity to terrestrial plants in both experimental and field studies (Barker et al. 1967; Lips et al. 1990; Feng & Barker 1992; Cao et al. 1993; Britto & Kronzucker 2002). In roots of rice and barley, high concentration of K⁺ reduced the influx and efflux of NH₄⁺ across cytosol membrane, decreased the efflux-to-influx ratio of NH₄⁺ and alleviated the futile NH₄⁺ recycling across cytosol membrane (Szczerba et al. 2008; Balkos et al. 2010). Submersed macrophytes and seagrass are capable of taking up K⁺ through their roots and shoots (Nichols & Keeney 1976; Short & McRoy 1984; Barko et al. 1991; Zhong et al. 2013). It is not clear whether K⁺ supplement in cultural or cultivation solution could alleviate NH₄⁺ toxicity to submersed macrophytes when both K⁺ and NH₄⁺ are absorbed by the shoots of macrophytes, in contrast to the uptaking of K⁺ and NH₄⁺ only by the roots in terrestrial plants.

In a field survey of 31 lakes along the lower reaches of the Yangtze River, Cao et al. (2007) found that the submersed macrophyte V. natans preferring mesotrophic over eutrophic lakes. V. natans is a common submersed rosette macrophyte in lakes along the lower reaches of the Yangtze River, and declines in most of these lakes due to serious eutrophication (Zhang et al. 1999). V. natans produces tubers in early winter when its aboveground part is dying, and seedlings will germinate from both seeds and tubers in the coming spring, but seedlings germinating from tubers are stronger and more effective in clonal growth than those from seeds (Xiong & Li 2000). In eutrophic lakes, reproduction of V. natans depends mainly on overwintering tubers (Xiong & Li 2000; Chen et al. 2006). In experimental studies, NH₄⁺ enrichment in water column disturbed C–N metabolism of V. natans, but affected its growth either positively or negatively as dependent on NH₄⁺ concentration and light availability (Cao et al. 2007, 2009, 2011). Cao et al. (2007) suggested 0.56 mg L⁻¹ NH₄–N in water column as an upper threshold for growth of V. natans in eutrophic lakes. As tuber production is a key linkage between individual growth and reproduction of V. natans, examining effects of NH₄⁺ enrichment on quantity and quality of the tuber would help to understand population dynamics of this species in eutrophic lakes.

In the present study, we examined the single effects and co-effect of NH₄⁺ and K⁺ enrichment in water column on V. natans in an aquaria experiment lasting for about five months, which covered the key stages in the plant life history (i.e. tuber production and death of the aboveground part), with focus on C–N metabolism, growth and tuber production of the plant; and tested if high K⁺ concentrations in water column could alleviate stressful effects of NH₄⁺ enrichment on the plant.

Materials and methods

The experiments were conducted in Donghu Experimental Station of Lake Ecosystem (30°32′53.41″N, 114°21′15.63″E) from 19 July to 4 December in 2011. Seedlings of V. natans were collected from the mesotrophic Tanglinhu area of Lake Donghu. The seedlings selected for the experimental treatments were similar in size and healthy in appearance, with an average dry weight of 0.37 g per individual plant. Sediment for culturing the plant was collected from deep bottom near to the stands of V. natans in the lake and then mixed thoroughly in a big tank.

Experimental design and procedures

The experiment was a full factorial design with NH₄⁺ and K⁺ enrichments in water column as two factors, including four treatments: tap water being the control (indicated by C) and supplement with (NH₄)₂SO₄ (NH₄⁺ treatment, A for short), and supplement with K₂SO₄ (K⁺ treatment, K for short), and both (AK for short), respectively. Each treatment had five replicates.

One hundred and twenty of the seedlings were uniformly transplanted into 20 aquaria (50 × 50 × 80 cm) that contained 18-cm sediment in the bottom, with six seedlings in each aquarium, and then the aquaria were filled with tap water to 60-cm depth. The aquaria were placed outdoor under a black net roof, which shade about 80% of sun light, and the plants were kept for acclimation in tap water for 10 days before the supplements of NH₄⁺ and K⁺ were added. Concentrations of PO₄–P, NH₄–N, NO₃–N and K⁺ in water column of each aquarium were measured in every three days; stocking solutions of (NH₄)₂SO₄ and K₂SO₄ were added to the water column to achieve appropriate concentrations of NH₄–N and K⁺ in every 3 and 15 days, respectively. During the experiment, water in each aquarium was renewed nine times in order to minimize algal growth in the water column.

Measurements of environmental indices

Photosynthetically active radiation (PAR) was measured just below water surface in each aquarium at noon in every three days using an underwater irradiance sensor (UWQ-7893) connected to a data logger (Li-1400, Li-Cor Company, Lincoln, NE, USA). Water samples from each aquarium were filtered through GF/C glass fiber membrane (1.2 μm in pore diameter; Whatman company, Middlesex, UK), and used for the analysis of PO₄–P, NH₄–N, NO₃–N and K⁺ concentrations. NH₄–N was analyzed by the Nessler method and NO₃–N by the UV spectrophotometric method (Eaton et al. 1995). PO₄–P was measured following the method described by Golterman (Golterman & Clymo 1969). K⁺ was measured by a Dionex DX-100 ion chromatograph (DIONEX, Sunnyvale, CA, USA) equipped with an IonPac CS12A analytical column (250 × 4 mm). At the beginning of the experiment, five replicates of 50-mL sediment from the mixed sediment were centrifuged at 5000g, and the supernatants were collected for examining the concentrations of PO₄–P, NH₄–N, NO₃–N and K⁺ following the methods described as above.

Measurements of C–N metabolites, growth and tuber production of V. natans

In each aquarium, ramet numbers (N_R) of V. natans were counted and 3–5 mature leaves from the 3–5 robust plants were randomly collected every 15 days. The leaves from each aquarium were separately oven dried at 80 °C to a constant weight for further biochemical analysis. At the end of the experiment, all tubers of V. natans buried in the sediment were collected in each aquarium, clearly washed by distilled water, counted and weighted. Ten tubers randomly chosen from each aquarium were oven dried at 80 °C to a constant weight for further biochemical analysis.

The oven-dried leaves and tubers were ground into fine powders separately. About 50 mg of the powder was extracted twice with 5 mL 80% ethanol at 80 °C for 20 min, the extracts were pooled together and centrifuged at 10,000g for 15 min, and the supernatant was used to examine FAA and SC concentrations using alanine and glucose as standards, respectively (Yemm & Willis 1954; Yemm et al. 1955). The residue was used to examine starch content following the method of Dirk et al. (1999). Concentrations of C and N were measured by an elemental analyzer (Flash EA 1112, CE Instruments, Italy).

As biomass of V. natans is linearly correlated to its ramet numbers (unpublished data of Cao et al.), relative growth rate (RGR) of V. natans was calculated from the changes in N_R of the plant in each aquarium using the following formula: where N_R1 and N_R2 are the ramet numbers counted at the date of T₁ and T₂, respectively. Negative value of RGR means death of the plants, and hereafter is expressed as death rate of the plant.

Data analysis

Analysis of data was conducted with R software running in Win 7 [R Core Team (2012). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.]. Data were tested for normality and equality of variance, and when necessary, transformed by log₁₀(x) or sqrt(x), prior to analysis. Significant differences of tuber properties between different treatments were verified through one-way analysis of variance (ANOVA) followed by a Tukey honestly significant difference (HSD)'s test. Two-way ANOVA was then conducted to examine the effects of NH₄⁺ (A) and K⁺ (K) enrichments in water column and their combination on growth, tuber production and biochemical parameters of V. natans. Spearman's rank correlation was used to evaluate the relationships between biochemical parameters in tubers and those in the leaves of V. natans. Partial correlation was used to examine the relationships between maximum number of the ramets during the experiment (N_M), death rate of V. natans and the total tuber biomass.

Results

Environmental parameters of the treatments

PAR was 34–296 μmol m⁻² s⁻¹ and was not significantly different among the treatments. Concentrations of PO₄–P, NH₄–N and NO₃–N in the sediment pore water were 0.26, 1.3 and 0.16 mg L⁻¹, respectively, at the beginning of the experiments (Table 1). Addition of NH₄⁺ and K⁺ to the water column significantly increased their concentrations, with 0.04, 0.04, 0.63 and 0.66 mg L⁻¹ NH₄–N and 1.3, 45, 1.2 and 45 mg L⁻¹ K in the C, K, A and AK treatments, respectively. NO₃–N concentrations increased from 0.66 to 1.54 mg L⁻¹ caused by supplying NH₄⁺ to the water column. PO₄–P concentration was 7.5–8.8 μg L⁻¹ in the water column and did not differ significantly among the treatments (Table 1).

Table 1. Concentrations of PO₄–P, NH₄–N and NO₃–N in the water column (measured every three days during the experimental period) and the sediment pore water (measured at the beginning of the experiments) of the treatments.

Treatments	NH4–N (mg L^−1)	K^+ (mg L^−1)	NO3–N (mg L^−1)	PO4–P (μg L^−1)
Tap water (C)	0.04 ± 0.01	1.36 ± 0.10	0.67 ± 0.02	7.5 ± 1.2
K^+-enriched water (K)	0.04 ± 0.01	45.44 ± 1.61	0.66 ± 0.05	8.4 ± 0.8
NH4^+-enriched water (A)	0.63 ± 0.02	1.21 ± 0.27	1.60 ± 0.12	7.6 ± 1.0
NH4^+- and K^+-enriched water (AK)	0.66 ± 0.02	44.97 ± 1.44	1.47 ± 0.14	8.8 ± 1.9
Sediment pore water	1.30 ± 0.07	8.25 ± 0.18	0.16 ± 0.05	262.0 ± 38.0

Note: The values are expressed as the mean ± SD.

Clonal growth and tuber production of V. natans in response to NH₄⁺ and K⁺ enrichments

According to RGR values of V. natans, its growth could be divided into three stages: rapid growth stage (RGS, 1st–41st day), maintenance stage (MS, 42nd–93rd day) and dying stage (DS, 94th–137th day) during the experimental period (Figure 1).

Figure 1. Temporal changes in ramets number of V. natans grown in different treatments. C: the control with tap water only; A: NH₄⁺ enriched tap water; K: K⁺ enriched tap water; and AK: NH₄⁺ and K⁺ enriched tap water. Values are the mean ± SD (n = 5).

Compared with the control, the NH₄⁺ enrichment alone (A treatment) increased clonal growth of V. natans and prolonged its survivorship period by about three weeks, resulting in 21% higher N_R at the MS stage. The K⁺ alone (K⁺ treatment) or together with NH₄⁺ enrichments (AK treatment) marginally increased the N_R by 6%. The NH₄⁺ enrichment decreased the plant death rates (negative RGR) irrespective of the K⁺ treatments, consequently still having 67% of the maximum N_R at the end of the experiment in the treatments with NH₄⁺ enrichment (Figure 1, Table 2).

Table 2. Percentage of explained variance in ANOVA results for growth, asexual reproduction and biochemical parameters of V. natans in response to NH₄⁺ (A) and K⁺ (K) enrichments in water column of the different treatments.

	Percentage of the sum of squares
	A	K	A × K	Error
RGR	1	1	2	96
Death rate	82***	0	1	17
Maximum ramets number	18*	3	22*	57
In the leaves
Carbon content at RGS	42**	2	3	53
Carbon content at MS	63***	15**	0	22
Carbon content at DS	72***	12**	1	15
Nitrogen content at RGS	70***	0	2	18
Nitrogen content at MS	93***	1	0	6
Nitrogen content at DS	85***	2	1	12
SC content at RGS	63***	5	0	32
SC content at MS	96***	1*	0	5
SC content at DS	17*	3	24*	56
FAA content at RGS	69***	3	1	27
FAA content at MS	72***	0	2	26
FAA content at DS	65***	9*	1	25
Starch content at RGS	5	11	0	84
Starch content at MS	24	1	2	73
Starch content at DS	15	1	2	82
In the tubers
Tuber number	58***	1	5	36
Total tuber biomass	48***	1	5	46
Individual tuber biomass	7	10	1	82
Tuber carbon content	18	1	0	81
Tuber nitrogen content	84***	0	0	16
Tuber SC content	52***	0	0	48
Tuber FAA content	77***	1	3	19
Tuber starch content	49**	0	0	51

Note: Significance is indicated by *<0.05, **<0.01 and ***<0.001.

The NH₄⁺ and/or K⁺ enrichments affected tuber production and biomass of V. natans by means similar to those of the plant N_R at the MS stage, with the tuber number and biomass ranged by A > AK and K > the control. The K⁺ enrichments marginally decreased individual tuber biomass of V. natans irrespective of the NH₄⁺ treatments (Figure 2, Table 3).

Table 3. Spearman's correlation coefficients of relationships between the biochemical parameters in tubers and those in the leaves of V. natans at the MS and DS stages.

Spearman's correlations	Nitrogen content in MS	Nitrogen content in DS	SC content in MS	SC content in DS	FAA content in MS	FAA content in DS	Starch content in MS	Starch content in DS
Tuber nitrogen content	0.6987**	0.7606***	−0.7420***	−0.7523***	0.7915***	0.6821**	−0.3994	0.3416
Tuber SC content	−0.7709***	−0.6987**	0.7214**	0.7007**	−0.7853***	−0.7234**	0.5810**	−0.3271
Tuber FAA content	0.6780**	0.7110***	−0.7337***	−0.7276***	0.7812***	0.6698**	−0.4180*	0.0506
Tuber starch content	−0.5212*	−0.5996**	0.4840*	0.4923*	−0.4840*	−0.6058**	0.3416	−0.1992

Note: Significance is indicated by *<0.05, **<0.01 and ***<0.001.

Figure 2. Total tuber number, individual tuber biomass and total tuber biomass of V. natans grown in different treatments at the end of the experiment. C: the control with tap water only; A: NH₄⁺ enriched tap water; K: K⁺ enriched tap water; and AK: NH₄⁺ and K⁺ enriched tap water. Values are the mean ± SD (n = 5). Different letters indicate significant differences (p < 0.05).

Carbon and nitrogen metabolism of V. natans in response to NH₄⁺ and K⁺ enrichments

The NH₄⁺ enrichments greatly enhanced N contents in the leaves of V. natans. The N contents of the plant leaves increased in the A and AK treatments gradually with the experimental time until the DS stage; however, in C and K treatments, it increased only slightly at the early RGS stage and then gradually decreased. The K⁺ enrichments did not affect the N contents irrespective of the NH₄⁺ treatments. At the end of the experiment, the N contents were around 39.7 and 26.1 mg g⁻¹ in the treatments with (A and AK) and without NH₄⁺ addition (C and K), respectively (Figure 3, Table 2). Carbon contents in the leaves of V. natans were around 370 mg g⁻¹, maintained relatively stable values and did not differ among the treatments during the RGS and MS stages. The NH₄⁺ enrichments enhanced the C contents but the K⁺ enrichments reduced the C contents at the DS stage as compared to the control (Figure 3, Table 2).

Figure 3. Temporal changes in nitrogen and carbon contents in leaves of V. natans grown in different treatments. RGS: rapid growth stage; MS: maintenance stage; DS: dying stage; C: the control with tap water only; A: NH₄⁺ enriched tap water; K: K⁺ enriched tap water; and AK: NH₄⁺ and K⁺ enriched tap water. Values are the mean ± SD (n = 5).

FAA contents in the leaves of V. natans increased gradually with the experimental time in the treatments with NH₄⁺ enrichment and reached 2 mg g⁻¹ at the end of the experiment, while maintained relatively stable values (0.65 mg g⁻¹) during the experimental period in the treatments without NH₄⁺ enrichment (Figure 4(a), Table 2). SC contents in the leaves of V. natans decreased gradually from 32 to 11.53 mg g⁻¹ during the experiment in the A and AK treatments, and maintained to be stable (42.83 mg g⁻¹) in the control and K treatments until the late DS stage when the SC contents increased subsequently (Figure 4(b), Table 2). The K⁺ enrichments did not affect the FAA and SC contents. Starch contents in the leaves of V. natans increased at the early RGS stage and then gradually decreased in all the treatments (Figure 4(c), Table 2).

Figure 4. Temporal changes in contents of free amino acids (FAA), soluble carbohydrate (SC) and starch in leaves of V. natans grown in different treatments. C: the control with tap water only; A: NH₄⁺ enriched tap water; K: K⁺ enriched tap water; and AK: NH₄⁺ and K⁺ enriched tap water. Values are the mean ± SD (n = 5).

The NH₄⁺ enrichments enhanced FAA and N contents and decreased SC and starch contents in the tubers of V. natans, with 1.80, 1.65, 2.71 and 3.04 mg g⁻¹ FAA, 20.16, 20.01, 28.47 and 28.26 mg g⁻¹ nitrogen, 29.43, 29.47, 22.18 and 22.94 mg g⁻¹ SC and 675.10, 678.44, 615.28 and 612.79 mg g⁻¹ starch in the control, K, A and AK treatments, respectively. The K⁺ enrichments did not affect the biochemical indices. Carbon contents of the tubers did not differ among the treatments (Figures 5 and 6).

Figure 5. Contents of FAA, SC of and starch in tubers of V. natans grown in different treatments at the end of the experiment. C: the control with tap water only; A: NH₄⁺ enriched tap water; K: K⁺ enriched tap water; and AK: NH₄⁺ and K⁺ enriched tap water. Values are the mean ± SD (n = 5). Different letters indicate significant differences (p < 0.05).

Figure 6. Contents of nitrogen and carbon in tubers of V. natans grown in different treatments at the end of the experiment. C: the control with tap water only; A: NH₄⁺ enriched tap water; K: K⁺ enriched tap water; and AK: NH₄⁺ and K⁺ enriched tap water. Values are the mean ± SD (n = 5). Different letters indicate significant differences (p < 0.05).

Relationships between C–N metabolites, growth and tubers production of V. natans

The FAA and N contents in the tubers of V. natans positively correlated with their contents in the leaves and negatively correlated with the SC contents in the leaves at the MS and DS stages (p < 0.01 for all). The SC and starch contents in tubers positively correlated with the SC contents and negatively correlated with the FAA and N contents in the leaves at the MS and DS stages (p < 0.05 for all). The starch contents in the leaves positively correlated with the SC contents and negatively correlated with the FAA contents in the tubers at the MS stage (p < 0.05 for all). No significant relationship was found between the biochemical indices of the tubers and the starch contents in the leaves at the DS stage (Table 3).

The total tuber biomass of V. natans significantly positively correlated with the maximum N_R in the MS stage (r = 0.633, p = 0.004), and negatively correlated with the death rates (r = −0.500, p = 0.029; Table 4).

Table 4 The partial correlation of total tuber biomass, maximum number of the ramets (N_M) and death rate of V. natans during the experiment.

Variables 1	Variables 2	Control variables	Partial coefficient
Total tuber biomass	NM	Death rate	0.633*
Total tuber biomass	Death rate	NM	−0.500*

Note: Significance is indicated by *<0.05.

Discussion

In the present study, a moderate NH₄⁺ enrichment (ca. 0.65 mg L⁻¹ NH₄–N) in water column enhanced ramet production and reduced the death rate of V. natans, and consequently resulted in more tubers of the plant as compared to those in the treatments without NH₄⁺ enrichment at the end of the experiment. Water column NH₄–N concentration in the present study is slightly higher than the concentration (0.56 mg L⁻¹ NH₄–N) toxic to V. natans in a field survey reported by Cao et al. (2007); however, it did not inhibit the plant growth as observed by Cao et al. (2007). Considering low PAR commonly occurring in eutrophic lakes along the middle-lower reach of the Yangtze River (Wu et al. 2012), the high PAR and the high carbohydrate contents might have alleviated NH₄⁺ toxicity to the plant in our experiment (Cao et al. 2009, 2011).

As NH₄⁺ toxicity to submersed macrophytes depended on both of NH₄⁺ concentration and light availability (Ariz et al. 2011; Cao et al. 2011); high NH₄–N concentration of 7.5 mg L⁻¹ in water column was not toxic to Vallisneria spinulosa at high PAR (Li et al. 2008), while 0.25 mg L⁻¹ NH₄–N inhibited the growth of Ceratophyllum demersum and Myriphyllum spicatum at low PAR (Cao et al. 2011); and species-specific NH₄–N concentrations were reported toxic to different submersed macrophytes, with an NH₄–N threshold of ca. 0.25–3.7 mg L⁻¹ (Van Katwijk et al. 1997; Cao et al. 2007, 2011; Li et al. 2007). In our experiments, the PAR of 245 μmol m⁻² s⁻¹ reached photosynthetic saturation light intensity of V. natans (unpublished data of Cao et al.) and Vallisneria americana (Madsen et al. 1991), and was much higher than 120 μmol m⁻² s⁻¹ at which 0.81 mg L⁻¹ NH₄–N in water inhibited the growth of V. natans (Cao et al. 2007). Therefore, the high PAR might have enhanced the tolerance of V. natans to NH₄⁺ enrichment in the present study (Cao et al. 2009, 2011). Death of V. natans initiated at almost the same time in all treatments, but the NH₄⁺ enrichment reduced the death rates and prolonged the survivorship, which is in consistent with the results usually observed in annual herbaceous crops with supplement of N fertilizer (Dingkuhn et al. 1992; Gutiérrez-Boem et al. 2004).

In our experiment, the NH₄⁺ enrichment changed C–N metabolism of V. natans greatly, led to increase contents of FAA and N and decreased contents of SC in the leaves and tubers, and decreased the starch contents in the tubers. The similar results were also observed in the leaves and/or rhizomes of many submersed macrophytes under NH₄⁺ stress condition, i.e. Stratiotes aloides, C. demersum, Potamogeton crispus and M. spicatum (Smolders et al. 1996; Cao et al. 2009, 2011). As the leaves of submersed macrophytes cannot prevent the absorption of NH₄⁺ from water column (Van Katwijk et al. 1997; Britto et al. 2001), to avoid NH₄⁺ accumulation in their tissue, the plants incorporate NH₄⁺ into nitrogenous organic compounds instead of transporting it out of the tissue, processes that both are energy and thus carbohydrate demanding (Britto et al. 2001; Britto & Kronzucker 2002; Cao et al. 2009). Reduction of SC contents of V. natans had been found to inhibit the ramet production as a result of NH₄⁺ stress coincident with low PAR (Cao et al. 2007). In the study of Cao et al. (2007), the leaf SC contents dropped to about 50% by the NH₄⁺ enrichment, a value twofold than that examined in the present study. The differences in SC responses of V. natans to NH₄⁺ enrichments in the present study and the study of Cao et al. (2007) implied various photosynthetic carbohydrate production of the plant at different PAR in the two experiments, with more carbohydrate production at high PAR and thus compensating for the SC consumed by the NH₄⁺ enrichment and ensuring carbohydrate requirement for the production of ramets and tubers in our experiment. In eutrophic lakes, stands of V. natans are accessible to high PAR by moving from pelagic to littoral zone, where moderate NH₄⁺ enrichment in water column benefits the expansion of V. natans stands by producing more tubers, in contrast to the decline of the plant stands induced by low PAR and NH₄⁺ enrichment in the pelagic zone.

In the present study, the K⁺ enrichment did not affect C and N metabolism of V. natans as indicated by the contents of FAA, SC, starch, N and C in the leaves and tubers irrespective of the NH₄⁺ enrichment, indicating that the K⁺ enrichments did not further prevent the uptake of NH₄⁺ by the plant. Plants absorb K⁺ by high-affinity transporting system (HATS) and low-affinity transporting system (LATS), which function at low and high external K⁺ concentrations, respectively (Szczerba et al. 2008). The high K⁺ concentrations in our experiment fall within the working range of K⁺ concentration of LATS (Szczerba et al. 2008) and might not compete with NH₄⁺ for LATS when the concentrations of both K⁺ and NH₄⁺ were high.

The NH₄⁺ or K⁺ enrichments enhanced ramet production of V. natans separately at the MS stage; however, their combination decreased the ramet production as compared with the NH₄⁺ enrichment alone. A plausible mechanism for the lower ramet production induced by the K⁺ and NH₄⁺ enrichments was that massive influx of NH₄⁺ and K⁺ into the plant cell might have caused H⁺ to efflux from the cell (Taylor & Bloom 1998; Hoopen et al. 2010; Marschner & Marschner 2012), inducing deionization of NH₄⁺ and accumulation of NH₃ in the cell (Hoopen et al. 2010), which is about two orders of magnitude more toxic to plants than NH₄⁺ ion (Körner et al. 2003; Nimptsch & Pflugmacher 2007).

Conclusion

This study aims to examine long-term single effects and co-effects of NH₄⁺ and K⁺ enrichment in water column on carbon and nitrogen metabolism, life history and asexual reproduction of submersed macrophyte. We found that the K⁺ enrichment had negligible effects on the plant, and the NH₄⁺ enrichment (0.65 mg L⁻¹ NH₄–N) benefited the plant growth but reduced the tuber carbohydrate contents, and their interaction effect on the plant was negligible.

Additional information

Funding

This study was supported by the National Science Foundation of China [grant number 41230853], [grant number 31270508]; National High Technology Research and Development Program of China [grant number 2012ZX07105-004]; State Key Laboratory of Freshwater Ecology and Biotechnology of China [grant number 2014FBZ02].