Untargeted liquid chromatography coupled with mass spectrometry reveals metabolic changes in nitrogen-deficient Isatis indigotica Fortune

Abstract

Isatis indigotica Fortune is a popular herb in traditional Chinese medicine, and various types of metabolites are the basis for its pharmacological efficacy. The biosynthesis and accumulation of these metabolites are closely linked to nitrogen availability; the benefits of low nitrogen application on the environment and herb quality are increasingly prominent. To analyze metabolic changes in the leaves and roots of I.indigotica in nitrogen deficiency conditions, and to identify the pathways and metabolites induced by low nitrogen availability, we used untargeted liquid chromatography coupled with mass spectrometry (UHPLC-TripleTOF) to obtain metabolomics profiling of I.indigotica under two N-deficiency treatments (0 kg/hm²; 337.5 kg/hm²) and normal nitrogen treatment (675 kg/hm²). A total of 447 metabolites were annotated. 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. Our findings showed that nitrogen deficiency modified roots and leaves conditions of I.indigotica, affecting both the primary and secondary metabolism. Moderate nitrogen reduction was beneficial to the accumulation of active ingredients. Our methods and analysis are expected to provide an insight regarding the diversity of metabolites and regulation of their synthesis in low nitrogen application, and better investigate the nitrogen deficiency effect on I.indigotica.

Keywords: Differential metabolites; Isatis indigotica Fortune; Metabolomics; Nitrogen deficiency; Secondary metabolism.

Graphical abstract

Fig. 1

Score scatter plot for PCA model TOTAL with QC of all samples under different nitrogen levels. The abscissa PC [1] and the ordinate PC [2] represent the scores of the first and second ranked principal components, respectively, and the scatter color and shape represent experimental groupings of the samples. The differences between the visible groups are significantly different on the top principal components. The samples are all within the 95% confidence interval (Hotelling's T-squared ellipse).

Fig. 2

Volcano plots of differential metabolites in nitrogen deficiency treatments. Each point in the volcano plot represents a metabolite, the abscissa represents the fold change of the group compared to the substance (take the base 2 logarithm), and the ordinate represents the p-value of the Student's t-test (take the base 10 Logarithm), the size of scatter represents the VIP value of the OPLS-DA model. The larger the scatter is, the larger the VIP value. The scatter color represents the final screening result, the significantly up-regulated metabolites are shown in red, the significantly down-regulated metabolites are shown in blue, and the non-significantly different metabolites are gray.

Fig. 3

The bubble plots of differential metabolic pathway in nitrogen deficiency. Each bubble represents a metabolic pathway respectively, the abscissa and the size of the bubble indicates the size of the influence factor of the pathway in the topological analysis; the larger the size, the larger the influence facto. The ordinate of the bubble and the bubble color indicated the p value (take negative natural logarithm, lnp-value) in the enrichment analysis, the deeper the color, the smaller the p value, and the more significant the enrichment.

Fig. 4

Relative contents of anthranilic acid, epigoitrin and indirubin (indigo) in Isatis leaf and Isatis root under different nitrogen levels. (a) Isatis leaf; (b) Isatis root.