Response Of The Contents Of Free Amino Acids And Nitrogen Isotopes Of Phragmites Australis Leaves To Emergence

Amino acid in plants tissue is important primary nitrogen metabolites.Concentrations and nitrogen isotopic composition of amino acid have been widely used to understand the nitrogen metabolism mechanism of plants under different environmental pressures?such as high temperature,heavy metals,drought,flooding?change.Amino acid plays an important role in plants,not only regulate the metabolic balance in plants,but also reduce the environmental damage caused by plants and ensure the survival of plants under environmental stress.At present,many researches focus on studying the flood-fighting mechanisms of plants,using free amino acid concentrations in plant tissue with nitrogen isotopes to study the response of plants to flooding time and depth of flooding are relatively weak.Therefore,the responsed of free amino acids in plants to flooding and traced with flooding stress by nitrogen isotopic compositions of amino acid were analyzed to clarify the adaptation mechanism of nitrogen metabolism under flooding stress and provide scientific basis for the protection of plants.Research focusing on the following two aspects.On the one hand,the establishment of standard amino acid solution concentration and nitrogen isotope measurement and correction,which can provide the basis for the determination of plant sample concentration and its nitrogen isotope.On the other hand,in order to explore nitrogen metabolism mechanism of Phragmites australis?Cav.?Trin.ex Steud.leaves under different flooding conditions,the P.australis collected from the Poyang Lake Wetland and used to design a submerging P.australis under different depths for the 11days.The total carbon and nitrogen contents and their isotopic compositions,as well as the free amino acids concentrations and their isotopic compositions in P.australis leaves were analyzed.The results are presented as follows:?1?Variable concentrations of amino acid standard mixtures?from 0.04 to 1nmol/?L?were separately evaporated,derivatized,and subsequently analyzed.The calibration plots constructed from peak area data provided by GC-MS-IRMS versus the original standard concentration showed excellent linearity for all the compounds tested,confirming the accuracy of our method.The linear determination coefficients?R²?for the standard curves of all derivatives ranged between 0.9900 and 0.9971,the relative standard deviation of from0.003%to 0.014%,with an average of 0.008%.Analysis of stable nitrogen isotope composition of 0.04 to 1nmol/?L tert-Butyldimethylsilylation?tBDMSi?amino acid standard shows the reproducibility of?¹⁵N is between 0.05‰to 1.16‰,the mean reproducibility of?¹⁵N is 0.48‰.This indicated that this method can be used to determine the concentration and nitrogen isotope composition of amino acids.?2?In order to circumvent all the derivatization issues and to assess the extent of fractionation,the?¹⁵N values of the commercially available amino acids and their analogous tBDMS derivatives were measured by EA-IRMS 253Plus and compared with the values obtained by GC-MS-IRMS.The difference????obtained between the native isotope value?true value?and the predicted value?corrected value?were less than 1.64‰,indicated that this method can calibrate the nitrogen isotope fractionation caused by the GC system and the derivatization procedure.?3?The total nitrogen isotopic composition was gradually ¹⁵N-depleted with the increase of flooding time?P<0.05?,the protein nitrogen isotopic composition was obviously changed with the increase of flooding time?P<0.05?,the amino acid?¹⁵N values of flooding and unflooding shows that the slope for each graph is deviation from 1?y=x?,indicated that nitrogen metabolism in P.australis leaves can respond to environmental moisture.?4?There was no obviously changes in total nitrogen contents?P>0.05?in P.australis leaves after 11 days flooding.However,total free amino acids concentration decreased significantly after 4 days of flooding?P<0.05?,indicated that free amino acids are more sensitive than total nitrogen under flooding stress.?5?Short-term flooding promoted the glycolysis process,serine derived from glycolysis process can be used in indicating short-term stress in P.australis leaves.Long-term flooding inhibited the TCA cycle in the P.australis leaves,the free amino acids?asparagine and aspartic acid?associated with the TCA cycle can be used in indicating long-term flooding stress in P.australis leaves.?6?The nitrogen isotope compositions of glutamine,serine,aspartic acid,and phenylalanine in the leaves of P.australis were significantly different after 11 days of flooding,individual amino acids responded in a diverse way to flooding stress,suggesting a differential sensitivity of each biosynthetic pathway to this stress.