| In this study,the effects of reduced nitrogen application on its growth,physiology and metabolism of Isatis indigotica Fort.from Yuncheng,Shanxi province(S1)and Zhangye,Gansu province(S2)populations were studied,according to the current situation of low nitrogen utilization in the cultivation.The study was conducted by field experiments and pot experiments.There were total four nitrogen treatments in the field experiment study:CK(0 kg/hm2),1/4N(168.75 kg/hm2),1/2N(337.5 kg/hm2),and 1N(675 kg/hm2).For I.indigotica,the growth indexes,photosynthetic pigment contents,photosynthetic parameters,soluble sugars,free amino acids,carbon-nitrogen ratio and the contents of active components in roots and leaves(total flavonoids,indigo and indirubin contents in leaves;epigoitrin content in roots)were analyzed,and effective economic yield analysis was also performed.At the same time,non-targeted liquid chromatography coupled with mass spectrometry(UHPLC-Triple TOF)method was applied to analyze the metabolomics and to investigate the effect of low nitrogen nutrition on the metabolism level and medicinal quality of roots and leaves of I.indigotica in Shanxi populations(which had a larger difference within the group)at 0 kg/hm2(nitrogen deficiency,N-z),337.5 kg/hm2(low nitrogen,N-h),675 kg/hm2(normal nitrogen,N-n)treatments.The pot experiment was conducted with three nitrogen treatment groups:0 mmol/L(nitrogen deficiency,NO),7.5 mmol/L(low nitrogen,N1),15 mmol/L(high nitrogen,N2),and non-target metabolomics methods was also used to discuss the effects of different nitrogen nutrition levels on the metabolism level of I indigotica.The results were as follows:1.Reduced nitrogen application had significant effects on the biomass accumulation and physiological processes of I.indigotica Fort.,and the responses of I.indigotica in Shanxi and Gansu populations to nitrogen were different.Both the fresh and dry biomass of S1 showed an increasing trend with the increase of nitrogen level,and reached a maximum at the normal nitrogen treatment,in which the fresh and dry weight of the plants were respectively 68.4 g,15.3 g;While the fresh and dry biomass of S2 showed a trend of increasing first and then decreasing with the increase of nitrogen levels,the maximum were obtained at the 1/2N treatment,which were 78.5 g and 19.7 g respectively.The application level of nitrogen was positively correlated with the root length,plant height,and main root diameter of I.indigotica.There were no significant differences on the drying rates among the treatments.The net photosynthetic rate and chlorophyll content of S1 and S2 showed an increasing trend with the increase of nitrogen application levels,and the intercellular carbon dioxide concentration showed a decreasing trend.The CK had the highest leaf soluble sugar content and the carbon-nitrogen ratios,and the 1N treatment had the highest free amino acid contents,and there was no significant difference between S1 and S2.2.Reduced nitrogen application was beneficial to the accumulation of active ingredients and the formation of effective economic yield of I.indigotica during harvest stage.The contents of indigo,indirubin and total flavonoids in leaves and epigoitrin in the roots of both S1 and S2 were the highest under the CK treatment,and had decreasing trends with the increase of nitrogen application levels.S2 was more sensitive to nitrogen levels.The nitrogen application level significantly affected the effective economic yield of I.Folium and I.Radix.The effective economic yield of I.Folium under 1/4N and 1/2N treatments were significantly higher than that under the 1N treatment.The effective economic yield of I.Radix of S1 was positively correlated with nitrogen level,with the maximum of 3.31 mg/g under 1N treatment,and that of S2 was under the 1/4N treatment(2.94 mg/g).3.Reduced nitrogen treatment significantly affected the metabolomic profiles of I.indigotica roots and leaves.Principal component analysis separated the three nitrogen treatments.A greater diversity of metabolites was observed in roots than in leaves under N deficiency treatments,suggesting that roots have a more important function in low N tolerance.Differential metabolites were mainly enriched in purine metabolism,phenylpropanoid biosynthesis,the shikimate pathway,tryptophan metabolism,and flavonoid biosynthesis that notably induced only in leaves in low nitrogen stress.Moderate N-deficiency benefits carbohydrate accumulation,whereas accumulation of most amino acids decreases.Uniquely,L-tryptophan was maintained at a high concentration in N-deficiency conditions.Low nitrogen stress induced the accumulation of some specialized metabolites(matairesinol,dictamnine,5-hydroxyindoleacetate(serotonin)in roots and vitexin,xanthohumol,sinapyl alcohol in leaves).N-deficiency also increased the accumulation of adenosine and quality indicators of I indigotica(indirubin-indigo,epigoitrin and anthranilic acid)in a certain degree.4.Nitrogen levels significantly affected metabolic pathways and metabolite levels in Isatis indigotica leaves at seedling stage.The results of metabolome analysis of I.indigotica at different levels of nitrogen nutrition showed that dihydroxyindole,anthranilic acid,N-acetylanthranilic acid significantly discriminated the three treatments.Low nitrogen generally led to the decrease of most amino acids(such as arginine,threonine and valine),while the contents of phenylalanine significantly increased under low nitrogen treatments.The content of methionine was almost unchanged at different nitrogen levels.In addition,this study found that there was a complex relationship between indole compounds and nitrogen application levels.The content of indole and dihydroindole had a significant positive correlation with nitrogen levels,while significant accumulation of indigo and indirubin was observed under low nitrogen treatment Quercetin(one of a flavonoids)was maintained at a high level under N1,but was reduced by about 16 times and 5 times under NO and N2 treatments,respectively.It was worth noting that the content of indole-3-acetic acid(IAA)showed an increasing trend with the increase of nitrogen level,and the pro-compound indole-3-pyruvate significantly accumulated under low nitrogen treatment.It was speculated to be related with low nitrogen tolerance of I.indigotica. |
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