Effect Of Nitrogen Deficiency And Nitrogen Recovery On Growth And Metabolism Of Isatis Indigotica Fort.at Seedling Stage

In this paper,we studied the effect of nitrogen deficiency and nitrogen recovery on growth,physiology,nutrition and active components of Isatis indigotica,which were grown hydroponically.The tryptophan synthetase alpha chain was cloned in I.indigotica to discuss the expression level at different nitrogen treatments and in different tissues.In addition,we studied the effect of nitrogen recovery level on physiological characteristics and active components of I.indigotica.Shanxi cultivation population seedlings of I.indigotica were used in these studies.In nitrogen deficiency and nitrogen recovery experiment,three treatments were set up:CK(basic nutrient solution cultured for 15 days),nitrogen deficiency(Nitrogen deficiency nutrient solution cultured for 15d),nitrogen recovery(Nitrogen deficiency nutrient solution cultured for 4d after basic nutrient solution cultured for 6d,final basic nutrient solution cultured for 5d):Growth index,nitrogen metabolic enzymes and antioxidant enzymes activities,chlorophyll content and photosynthetic parameters were analyzed.Furthermore,nutritional qualities were researched,such as the contents of free amino acid,soluble protein,soluble sugar,mineral elements.The accumulation of alkaloids ingredient and flavonoids contents was also researched in this experiment.Tryptophan synthetase alpha chain was isolated,and the sequences of DNA,cDNA and predicted protein were analyzed.Gene expression level in different nitrogen treatments and different tissues was studies by real-time quantitative PCR.In nitrogen recovery level experiment,five nitrogen levels were set up:CK(ON),T1(1/3N),T2(1/2N),T3(1N),T4(2N),to study the response of photosynthetic pigment,anti-oxidation index,nutritional quality and active components.These researches could provide a theoretical basis of improving I.indigotica yield and medicinal quality and decreasing the use of nitrogen fertilizer.Results obtained were as follows.1.Nitrogen deficiency and nitrogen recovery on growth and physiological state showed different among I.indigotica seedlings.N deficiency could increase main root diameter,fresh weight and dry weight of leaves and roots,and enhance NR activity.N recovery enhanced the main root diameter,main root length,fresh weight and dry weight of leaves and roots,MDA content,but reduced NR activity.The two treatments both enhanced root activity,while nitrate content,nitrogenous metabolism enzymes activities(GS,GOGAT,GDH),antioxidant enzymes activities(POD,CAT,SOD),content of chlorophyll,and photosynthetic parameters(Pn,Gs,Ci and Tr)were all reduced.2.Different nitrogen treatments affect accumulation of nutrition ingredient and the active ingredients in I.indigotica at seedling stage.N deficiency increased mineral elements K,Ca,Mg,Mn,Fe,Na,B,Ti and Se contents in leaves,promoted the accumulation of fourteen free amino acids except Ala,improved the content of total free amino acids in leaves and soluble polysaccharide in roots,but reduced content of the free amino acid in the root and soluble protein.N recovery enhanced the content of Al,Ba,Hg in leaves,promote the accumulation of twelve free amino acids and soluble protein,reduced the total free amino acids in leaves and soluble polysaccharide in roots.N deficiency and N recovery would decreased the contents of indigo and indirubin in leaves,but improved the total alkaloids in leaves and(R,S)-epigoitrin in roots.In addition,the total flavonoids contents in roots were decreased by N deficiency,but the contents were increased at N recovery treatment.3.By cloning tryptophan synthetase alpha chain sequence in I.indigotica,and bioinformatics analyzing on it,the results showed that the full-length genomic DNA consists 2013 bp with eight exons and seven introns.The length of cDNA is 1074 bp,and the coding sequence consist 936 bp,flanked with 32 bp 5'-UTR and 106 bp 3'-UTR.The predicted protein of IiTSA is 311 amino acids long,with the molecular mass of 32922.1 kDa and theoretical isoelectric point of 6.76.This amino acid sequence of IiTSA has high homology with other plants,and has closer genetic relationship with Isatis tinctoria in evolution.4.IiTSA expression level was different in nitrogen treatments and tissues.IiTSA expression level under the condition of N deficiency was down-regulated more than that of under the treatment of N recovery.The contents of indigo and indirubin showed significant positive correlation with the expression of the IiTSA,so their change trends were the same.Moreover,IiTSA existed in all tissues.The highest expression level was observed in leaves,followed by fruits,stems,roots and flowers,and the lowest was in flowerbuds.5.Physiological indexes response to different nitrogen recovery levels existed large discrepancies in I.indigotica at seedling stage.In the treatments of CK and T4,it is in favor of improving the contents of chlorophyll a,chlorophyll b,carotenoid and total chlorophyll.Chlorophyll a content was the highest in CK while chlorophyll b content was the highest in T4.In T1,T2 and T3,the photosynthetic pigment contents increased with the increasing of nitrogen level.MDA content increased first and then decreased with the increasing of nitrogen recovery level,and SOD change trend was just the opposite.MDA content and POD activity were the highest in CK,and the CAT activity in T2 treatment was the largest.In T1,T2 and T3,POD and SOD activity were increased and MDA content decreased with nitrogen level enhanced.6.Different nitrogen recovery levels influenced on nutritional quality and medicinal quality.Free amino acid content in leaves raised first,then dropped and raised last,the soluble protein content changed on the contrary.Soluble protein content in T1 treatment and free amino acid content in T3 treatment were the highest.Soluble sugar content in root increased gradually,maximizing at the T4 treatment.With the improvement of nitrogen recovery level,indigo and indirubin contents in leaves decreased after rising first,and reached the maximum in T2 treatment;(R,S)-epigoitrin content in roots raised after dropping first,and reached the maximum at T4 treatment.The change trends of total alkaloids and total flavonoids contents were the same,total alkaloids were largest at T4,and total flavonoids were largest at T1 treatment.