Materials and Method

Plant Material

The experimental materials were collected from a eutrophic lake (Nanhu Lake, E 114°21.198, N30°33.118) in Wuhan, China. After removing the mud and epiphyte by washing with 0.5% sodium hypochlorite solution, a few ramets developing from the single clone were selected for pre-incubation, and the growth medium was adapted from one-fourth of Hoagland nutritious solution. Plant materials propagated vegetatively through a few generations in the green house of Wuhan University, to avoid the ramets from different clone. Furthermore, extremely low genetic variation of E. crassipes in China was revealed in our previous study (Li et al., 2006), and thus these plants developed from single clone in the present study might not differ in genotype.

Plants were kept in the culture room and were received to a 12/12 h photoperiod. The photon flux measured was 200^mol m-2 s-1and the temperature was 30/22 °C (day/night) at plantlet level. In order to avoid toxicity of ammonium, an experiment was conducted to detect the effect of ammonium on plant growth. Modified Hoagland solutions, one-fourth strength, were used in order to obtain several concentrations (0.5, 1.0, 3.0, 5.0 and 7.0 mM) of ammonium. The pH of the nutrient solution was kept at 5.8, and was adjusted daily using KOH. No significantly synmpose of plant was observed in each ammonium treatment, and 5.0 mM total nitrogen was selected in further experiments.

Experiment Design

New-generated E. crassipes single clone plants (4-5 leaves) with root length not beyond 1 cm were selected to be incubated for our experiment in greenhouse (Wuhan University). The incubation ramets were transferred to full nutrient solution (2 mM KH2PO4, 0.5 mM MgSO4, 50 ^M FeNaEDTA, 25 ^M H3BO3, 2 ^M MnSO4^O, 1 ^M ZnSO4'7H2O, 0.5 ^M CuSO45H2O, 0.2 ^M NaMoO4) containing 5mM nitrogen source. Nitrogen was supplied with NaNO3, NH4Cl, or NaNO3 plus NH4Cl. Plantlets were grown in the presence of five different NO3-: NH4+ratios: 100:0, 75:25, 50:50, 25:75 and 0:100. The nutrient solution pH value was kept at 5.8, and the growth medium solutions were changed every two days. Each treatment consisted of four experimental replicates.

Harvest

Plants were harvested at 28 days after transfer of single clone plants to grow medium. Numbers of generated clones were recorded in each treatment. Harvested plants were separated into leaves and roots respectively, and four individuals from each treatment were dried at 85 °C for 24 h to measure dry weight and nitrate accumulation. At the same time, other fresh roots and leaves under different growth conditions were used to determine tissue ammonium concentration, NR activity, and GS activity.

Tissue Nitrate and Ammonium Determination

Nitrate content was determined with water extraction of dry tissues. Sample of 100 mg dry tissue was dispersed in double-distilled water at 45 C for 60 min. The suspension was centrifuged at 5000g for 15 min, and the nitrate level was measured in the supernatant using salicylic-acid method according to Cataldo et al. (1975). Absorbance was measured at 410 nm. Tissue ammonium concentration was determined by the indophenol-blue reaction (Solozano, 1969). Fresh plant material was grounded with liquid nitrogen, dissolved in distilled water (1 g fresh weight (FW) to 10 ml of distilled water), and extracts were heated at 70°C for exactly 2 min, centrifuged at 5,000 g for 5 min at 4C and ammonium determined in the supernatant. The amount of ammonium was expressed as ^mol NH4+ g -1 FW.

Enzyme Activity Measurement

In vitro NR activity assays were performed according to a modification of Kaiser and Huber (1997). Plant material (500 mg) were grounded to fine powder with liquid nitrogen, and suspended in 1.5 ml of 50 mM HEPES-KOH buffer (pH 7.5), containing 10 ^M FAD, 5 mM MgCl2, 0.5 mM EDTA, 5 mM DTT, 0.1% Triton X-100 (w/v), 10% glycerol (v/v), 50^M leupeptin, 1% PVPP (w/v), 0.2 mM PMSF. The homogenate was centrifuged for 20 min at 1, 5000 g and 0.25 ml of supernatant was added with 0.75 ml assay mixture consisting of 50 mM HEPES-KOH buffer (pH 7.5), 10 mM KNO3, 0.2 mM NADH, and 2 mM EDTA for NRA and incubated at 30C for 10 min. The reaction was stopped by adding 0.05 ml 0.5M Zn acetate. Excess NADH was destroyed with 10 mM phenazine methosulphate. The amount of nitrite released in 0.5 ml reaction mixture was estimated by adding 1 ml of 1% sulfanilamide in 3 M HCl (w/v) and 1 ml of 0.02% N- (1-naphthyl) ethylene diamine dichorite solution, and the absorbance was read at 540 nm. The amount of nitrite was read from a standard curve of nitrite. NR activity is expressed as ^mol NO2- g -1 FW h -1.

Total GS activity was assayed following the modified method of Rhodes et al. (1978). Fresh tissue (500 mg) was grounded in liquid nitrogen and suspended with 1.5 ml of Tris-HCl buffer (pH 7.6) containing 10 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM MgCl2, 0.5 mM PMSF, 50^M leupeptin, and 10% glycerol (v/v). The homogenates were centrifuged at 15000 g for 30 min. The assay mixture consisted of 100 mM imidazole buffer (pH 7.2), 11.4 mM ATP, 45 mM NH2OH- HCl, 45 mM MgSO4 and the enzyme extract. After incubation at 37 C for 15 min, and the reaction was terminated by adding FeCl3 reagent (10% FeCl3 in 0.2 M

HCl, 24% TCA and 50% (v/v) HCl mixed in the ratio of 1:1:1). Absorbance of the supernatant was measured at 540 nm after centrifugation at 6000 g for 5 min. One unit of enzymatic activity was defined as the formation of ^mol y-glutamyl hydroxamate per hour.

Calculations and Statistics

The relative growth rate (RGR) of E. crassipes based on net dry weight increment was calculated according to Hunt (1982):

where W1 and W2 are initial and finial sample dry weight and t is culture time.

Data were statistically analyzed using the one-way analysis of variance. Tukey's multiple range test was used to compare the means of all nitrogen treatments. Statistical analyses of the data were carried out at P < 0.05 significance level using SPSS software package (release 11.5, SPSS Inc., Chicago, IL, USA).

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