Jianhong SHU, Jiahai WU, Taoying QIN, Xiaoli WANG

Guizhou Institute of Prataculture, Guiyang 550006, China

Nitrogen is one of the macro-elements essential for plants. It exists in the soil mainly in the forms of inorganic nitrogen (including ammonium nitrogen and nitrate nitrogen) and organic nitrogen (including amino acid nitrogen). Crops have a large demand for nitrogen, but the bio-available nitrogen content in soil is very low, which leads to the massive and wide application of chemically synthesized nitrogen fertilizer in farmlands. In China, the current utilization efficiency of nitrogen is about 30%[1].To ensure crop yields, nitrogen fertilizer is usually applied excessively, and is even abused. Too much or too little application of nitrogen fertilizer all will produce adverse effects on crop yield and quality. Even worse, the nitrogen loss in soil will cause serious threats to ecology and human health, such as soil pollution, eutrophication and excessive nitrate accumulation in agricultural products[2].

Qiancao No.1 (Festuca arundinacea cv. Qiancao No.1) is a local variety in Guizhou Province. It has loose requirements for soil, but it has drought resistance, high temperature resistance,wide adaptability,trampling resistance and long green period.Qiancao No.1 is suitable to be planted in low mountains,hills,plains and other similar ecological zones in middle and upper reaches of Yangtze River.It has high-content nutritional ingredients, and is favored by cattle, horses and donkeys.Moreover,Qiancao No.1 is also an excellent grass variety for environmental greening and ecological management.In this study, the physiological and biochemical changes in protein content, chlorophyll content,enzyme activities and amino acids contents in tall fescue under nitrogen stress were analyzed so as to provide certain reference for rational utilization and ecological potential exploration of Qiancao No.1 in the future.

Materials and Methods

Materials

Plant material The full tall fescue seeds were soaked in water overnight at 50 ℃. They were soaked in 75%ethanol for 30 s along with continuous stirring and rinsed three times withsterile water. Subsequently, they were soaked in 0.1% HgCl for 4 min and rinsed 5-6 times with sterile water. After laying four layers of filter paper,the petri dishes were sterilized. In the sterilized petri dishes,certain amounts of sterile distilled water were added with a pipette, and then certain amounts of tall fescue seeds were placed on the filter paper. After adding certain amounts of water, the petri dishes were placed in a light incubator(25 ℃, 12-h lighting/d). After the germination, the seeds were transferred to self-made hydroponic cups containing Hoagland nutrient solution.The culture was carried out under conditions of temperature of 25 ℃and light/dark cycle of 16/8. The nutrient solution was replaced once every three days.After a 30-d culture,the tall fescue plants were transplanted. Total two treatment groups were arranged,including normal nitrogen supply treatment (CK) and nitrogen stress treatment. After 7 d, the upper parts of the plants were sampled for analysis.

Hoagland nutrient solution The Hoagland nutrient solution was composed as follows:1.25 mM KNO3,1.25 mM Ca (NO3)2·4H2O,0.5 mM MgSO4·7H2O, 0.25 mM KH2PO4, 11.6 μM H3BO3, 4.6 μM MnCl2·4H2O, 0.19 μM ZnSO4·7H2O,0.12 μM Na2MoO4·2H2O,0.08 μM CuSO4·5H2O and 10 μM Fe( Ⅲ)-EDTA. In the nitrogen stress treatment groups, the NO3-in the Hoagland nutrient solution was replaced by Cl-.

Methods

Chlorophyll content determination A certain amount (0.2 g)of each plant sample was shredded (2 mm) and ground in liquid nitrogen. It was transferred to a 50-ml centrifuge tube and mixed with 25 ml of 95% ethanol. The tube was then bathed in water at 50 ℃for 2-3 h till the leaf sample turned white. Subsequently, the extract was removed, cooled to room temperature and diluted to a certain volume. The absorbances of the solution at 645 and 663 nm were determined. There were three replicates for each treatment.The chlorophyll contents were modified using the Amon method[3].

Chlorophyll a = (12.71 A663-2.59 A645)V/W×1 000;

Chlorophyll b = (22.88 A645-4.67 A663)V/W×1 000;

Total chlorophyll content = (8.04 A663+20.29 A645)V/W×1 000.

Amino acid analysis The standards and conventional reagents were all purchased from the Sigma Cooperation. The gradient concentrations of sample solutions, mixed with external standard, were mixed with QC in 1.5-mp EP tubes. A certain amount (120 μg) of protein supernatant was added to each of the tubes.All the tubes were vortexed for 1min and then centrifuged at a high speed for 2 min. The supernatants were transferred to other new tubes for use.

A certain volume (50 μl) of each supernatant was mixed with certain amounts of buffer (120 μl) and derivative solution(30 μl).The mixtures were vortexed for 1 min and centrifuged instantly. Subsequently, the tubes were bathed in water (or dry bath) at 55 ℃for 15 min. They were cooled in a refrigerator and then centrifuged instantly for determination.

The determination was performed by LC-MS/MS (Shimadzu LC20A HPLC; API 4000 QQQ). The chromatographic conditions were as follows:AAA C18 column(5 μM,150×4.6 mm); mobile phase A, water (containing 0.01% TA and 0.1% TB); mobile phase B, acetonitrile (containing 0.01%TA and 0.1%TB);flow rate,0.8 ml/min;column temperature,50 ℃;injection volume,5 μl.

Enzyme extraction The PBS buffer was composed as follows: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4and 2 mM KH2PO4. The sample solutions were diluted by 10 times with PBS buffer. They were centrifuged at 8 000 r/min for 10 min. The supernatants were used for determination of soluble protein content and SOD,POD and GR activities with kits (Nanjing Jiancheng Bioengineering Institute).

Data statistics and analysis The test data were processed and analyzed using Excel 2003 and SPSS 13.0.

Table 1 Effect of nitrogen stress on leaf chlorophyll content in tall fescue mg/g

Table 2 Effects of nitrogen stress on activities of antioxidases in tall fescue

Results and Analysis

Effect of nitrogen stress on leaf chlorophyll content in tall fescue

Chlorophyll plays an important ole in photosynthesis, and its content is one of the important physiological indicators reflecting the growth conditions of plants[4]. The chlorophyll contents varied in tall fescue plants under nitrogen stress. As shown in Table 1,compared with those in the control group,the leaf total chlorophyll content in tall fescue under nitrogen stress was reduced (P ＜0.01); the chlorophyll a content in the nitrogen stress treatment group was reduced(P＜0.01);the chlorophyll b content in tall fescue under nitrogen stress was not changed(P＞0.05).

Effects of nitrogen stress on soluble protein content and antioxidase activities in tall fescue

As shown in Table 2, compared with those in the control groups, the activities of POD, SOD and GS in tall fescue under nitrogen stress were all increased (P ＜0.01); but the soluble protein content in the nitrogen stress treatment group was reduced(P＜0.01).

Effects of nitrogen stress on contents of 18 main kinds of amino acids in tall fescue

Table 3 showed that the total amino acid content in tall fescue under nitrogen stress was reduced significantly(P＜0.05).Among the 18 kinds of analyzed amino acids, the contents of asparagine,serine,glycine,glutamine,glutamic acid,aspartic acid, threonine,alanine, arginine, methionine, valine,isoleucine and leucine were significantly reduced in tall fescue under nitrogen stress (P＜0.05), but the tryptophan content was increased(P＜0.05).

Table 3 Contents of free amino acids in tall fescue under conditions of normal nitrogen supply and nitrogen stress μg/g

Conclusions and Discussion

Nitrogen is closely related to chlorophyll content and chloroplast development, as well as photosynthetic enzyme activities[5].Nitrogen can indirectly affect leaf number and leaf area, increasing photosynthetic area.In addition, nitrogen can increase chlorophyll content and improve leaf nitrogen content. Particularly, nitrogen can affect the contents of photosynthetic enzymes that are directly associated in photosynthesis and promote the smooth proceeding of dark reaction. So, nitrogen has a direct impact on photosynthetic capacity[6]. In rice,the chlorophyll content is reduced gradually with leaf senescence, and chlorophyll a is reduced faster than chlorophyll b[7]. The yellowing in mature and senescent plant leaves is caused by degradation of chlorophyll[8].During the mature and aging processes,the degradation of chlorophyll a is faster than that of chlorophyll b. Under nitrogen stress, the chlorophyll a to chlorophyll b ratio was reduced from 2.194 to 1.682, suggesting that nitrogen stress inhibits chlorophyll a synthesis or accelerates chlorophyll a degradation. Therefore, in agriculture,reasonable application of nitrogen fertilizer can improve the photosynthetic ability of plants, thereby improving crop yields.

SOD is an antioxidant enzyme for scavenging superoxide anion radicals,and is also an induced enzyme typically affected by external environment.SOD is a scavenger against harmful substances in plants. Under stresses,SOD can protect plant cells. Its increased activity can improve the plants’ adaptability to stress. The higher the SOD activity is,the stronger the plant resistance is[9]. POD activity reflects the antioxidant capacity and injury degree in plants. POD is one of the components of antioxidant enzyme protection system in plants. Within a certain range, POD is tolerant to stresses. The responses and protection mechanisms of SOD and POD mitigate the damage of stresses to plants. In this study, the SOD and POD activities in the nitrogen stress treatment group were higher than those in the normal nitrogen supply treatment group, which also demonstrates the conclusion above.

In the growth and development process of plants, glutamine and glutamate are nitrogen donors for the synthesis of main nitrogen-containing compounds in plants. GS stores ammonia in the amide group of glutamine,and it is a donor for ammonia, as well as for other nitrogen-containing compounds in plants[10]. Glutamine synthesis is associated with nitrogen deficiency, drought and salt tolerances in plants. The increased activity of GS improves the ammonia assimilation efficiency and accelerates the nitrogen metabolism[11]. In this study, under nitrogen stress,the increased GS activity enhanced the nitrogen deficiency tolerance of tall fescue.

Protein is the embodiment of life activities, and is the material basis of life. The average protein content is 16%,which is also the important basis for protein content determination by Kjeldahl method. Soluble proteins are important active substances, and are also important permeation regulating substances in plants. They have an important role in maintaining normal physiological functions of plants under stresses. The results of this study showed that the nitrogen stress significantly inhibited the synthesis of soluble proteins (P ＜0.01), and the soluble protein content in the nitrogen stress treatment group was only 47.2% of that in the control group, which were consistent with the study results of Wang et al[12]. It suggests that nitrogen plays an important role in soluble protein content in tall fescue.

Amino acids are the basic structural units of proteins, and their contents directly affect the synthesis of proteins, thereby affecting many metabolic activities.Zhou et al.[13]studied the variations in contents of amino acids in gametophytic blades of Porphyra haitanensis under low nitrogen and phosphorus stress, and among the 13 kinds of detected amino acids,the contents of 10 kinds were significantly reduced. In this study, among the 18 kinds of analyzed amino acids,the contents of 14 kinds were significantly reduced; but the tryptophan content was significantly increased,which still need further studies.

In short,in tall fescue under nitrogen deficiency stress, the activities of SOD, POD and GS were increased;the contents of leaf chlorophyll and soluble proteins were reduced; the contents of most amino acids were reduced, but the content of tryptophan was increased(P＜0.01).

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