Determination Of Free Amino Acids In Plant Leaves And Their Responses To Atmospheric Nitrogen Deposition

The amino acids in plant tissue are important metabolites in the process of nitrogen metabolism and present in the forms of free amino acids, polypeptide and protein. Although the free amino acids(FAA) in moss samples only account for 2% of the total nitrogen, they play an important role in the growth of mosses, not only in acting as the nitrogen source,but also regulating the balance of plant metabolism to ensure the survival of plants under stress environment. Mosses, having no real root, survive on the absorption of atmospheric deposition to obtain essential nutrients, so we always regard the total nitrogen(TN) of mosses as the indicators of atmospheric nitrogen deposition. FAA, as important metabolite in the process of plant nitrogen metabolism, also has a good response to the atmospheric nitrogen deposition. The nitrogen of FAA has a significant positive correlation with TN, which indicate a good response of FAA to the nitrogen deposition. However, the environmental conditions in city limit the survival of mosses, so the use of FAA in plants, which have a good response to the atmospheric pollution, has theoretical and practical significance.This paper adopted reversed-phase high performance liquid chromatography, determined with O-phthalaldehyde(OPA) and 9-fluorenyl methyl chloroformate(FMOC) derivation, automated online pre-column derivation, fluorescence detector, which can achieve the determination of 21 free amino acids within 19 min, the method of double internal standard with Nva and Sar achieved the accurae analysis of the concentration of Asp, Asn, Arg, Ala, Glu, Gln, Gaba, Gly, His, Thr, Tyr, Va, Met, Phe, Ile, Leu, Lye, Pro, Ser, Trp and Hyp. The limit of quantification of lysine was 2.325 mg /L, and limit of quantification of the other amino acids were below 0.53mg/L. The relative standard deviation of retention time of the chromatogram ranges from 0.07% to 0.55%(n=6), with an average of 0.14%. The relative standard deviation of area of the chromatogram ranges from 1.5% to 5.2%(n=6), with an average of 0.14%. Each amino acids has a good linear correlation coefficient ranging from 0.9983 to 0.9999 at 4.5pmo/μL- 450pmo/μL gradient. These all assure the precision, accuracy of instrument. Therefore, this method can be used to determine the concentration of amino acids.In general, however, plant samples always contain large amounts of impurities such as pigment, sugar and organic acids, which can affect the determination of free amino acids in plant samples and also easy to block the chromatographic column, so inpurities must be removed by means of purification and so on. In this paper, FAA in plant samples were extracted with ultra-pure water and ultrasound, protein precipitation with trichloroacetic acid, and then purified samples with cationic resin, the purification procedure removed a large number of pigments and inorganic salts and other impurities, which alleviated the damage of samples to the liquid chromatographic column. The average recovery was 95.9% and the relative standard deviation of the chromatogram area of FAA in sample range from 0.51% to 4.17%, which assured the reliability and accuracy of the method, so it provided a reliable means for the determination of free amino acids in plants.In this paper, by comparing the different composition and concentration of free amino acids in plant leaves under different atmospheric nitrogen deposition, I found that the concentration of free amino acids in plant leaves have a good response to atmospheric nitrogen deposition. With the increase of atmospheric nitrogen deposition from the suburb to trunk road, the total concentration of FAA in moss samples increased from 1187μg/g to 5610μg/g(4.7-fold), TN in moss increased gradually from 1.1% to 4.9%(4.4-fold). With the same total nitrogen, the moss samples also had almost the same concentration of total FAA; the concentration of total FAA in Cedar leaves increased from 494μg/g to 4027μg/g(8.1-fold), while total nitrogen(TN) increased from 1.1% to 2.6%(2.3-fold). So the total FAA in plant tissue has a good response to atmospheric nitrogen deposition, and more sensitive than TN of plant tissues.Specific FAA in plant tissues has a different response to atmospheric nitrogen deposition. In the place with low nitrogen deposition, Arg was the major FAA as the source of nitrogen, the concentration and percentage of Arg, Asn, Lys, and Ala had a good response to atmospheric nitrogen deposition; In the place with serious nitrogen deposition, Arg accumulation might reach saturation, plants began to accumulate Gln, Gaba, and Lys to store exess nitrogen to reduce the toxic effects of high doses of nitrogen to plants, so the concentration and percentage of Gln, Gaba, Lys and Pro had a good response to atmospheric nitrogen deposition, and the percentage of Arg, Asp and Glu also had a good response to atmospheric nitrogen deposition.In places with the same atmospheric nitrogen deposition, different plants had different FAA composition, Glu, Arg, Lys, Gln and Asn had the maximum differentiation, and the way to store nitrogen was also different. Different plants have different responses to nitrogen deposition. This will provide a theoretical basis in studying the relationship between nitrogen deposition and higher plants.