Transcriptome and metabolome analysis reveals mechanism of light intensity modulating iridoid biosynthesis in Gentiana macrophylla Pall

Abstract

Light intensity is a key factor affecting the synthesis of secondary metabolites in plants. However, the response mechanisms of metabolites and genes in Gentiana macrophylla under different light intensities have not been determined. In the present study, G. macrophylla seedlings were treated with LED light intensities of 15 µmol/m²/s (low light, LL), 90 µmol/m²/s (medium light, ML), and 200 µmol/m²/s (high light, HL), and leaves were collected on the 5th day for further investigation. A total of 2162 metabolites were detected, in which, the most abundant metabolites were identified as flavonoids, carbohydrates, terpenoids and amino acids. A total of 3313 and 613 differentially expressed genes (DEGs) were identified in the LL and HL groups compared with the ML group, respectively, mainly enriched in KEGG pathways such as carotenoid biosynthesis, carbon metabolism, glycolysis/gluconeogenesis, amino acids biosynthesis, plant MAPK pathway and plant hormone signaling. Besides, the transcription factors of GmMYB5 and GmbHLH20 were determined to be significantly correlated with loganic acid biosynthesis; the expression of photosystem-related enzyme genes was altered under different light intensities, regulating the expression of enzyme genes involved in the carotenoid, chlorophyll, glycolysis and amino acids pathway, then affecting their metabolic biosynthesis. As a result, low light inhibited photosynthesis, delayed glycolysis, thus, increased certain amino acids and decreased loganic acid production, while high light got an opposite trend. Our research contributed significantly to understand the molecular mechanism of light intensity in controlling metabolic accumulation in G. macrophylla.

Keywords: Gentiana macrophylla Pall.; Iridoid biosynthesis; Light intensity; Metabolome analysis; Transcriptome analysis.

Fig. 1

The growth conditions of G. macrophylla seedlings under different light intensity treatments on the 5th, 10th and 15th days, respectively. LL, ML and HL indicates the low, medium and high light treatment group, respectively

Fig. 2

The contents of chlorophylls and carotenoid under different light intensity treatments at 5-day in G. macrophylla seedlings leaves. (a) Chlorophylls content. (b) Carotenoid content. *Statistically significant differences (* P < 0.05, ** P < 0.01, and *** P < 0.001)

Fig. 3

HPLC content determination of four major iridoid metabolites at 5-day. (a) Black, pink, and blue represents chromatogram of ML, LL, and HL, respectively, while, 1, 2, 3 and 4 represents chromatographic peaks of loganic acid, swertiamarin, gentiopicroside and sweroside, respectively. (b) Determination of levels of four major iridoid metabolites. *Statistically significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001)

Fig. 4

Untargeted metabolome analysis of the metabolic composition in G. macrophylla seedling leaves treated with different light intensities at 5-day. (a) Bar diagram depicts metabolites classification. The x-axis and y-axis represents the numbers and the classes of metabolites, respectively. (b) Hierarchical clustering heatmap analysis of iridoid compounds by z-score. The color represents the scale of iridoids contents. Red indicates upregulation, while, green indicates downregulation. Lowercase letters represent a significant difference of p ≤ 0.05 in the same iridoid metabolite among the treatments

Fig. 5

Untargeted metabolome analysis of differentially accumulated metabolites (DAMs) in the ML-vs.-LL, ML-vs.-HL, and LL-vs.-HL comparisons at 5-day. (a) Numbers of DAMs. Red indicates upregulation, while, blue indicates downregulation. (b-d) KEGG enrichment analysis of the DAMs

Fig. 6

The contents of the 41 identified DAMs refer to KEGG pathway. The color represents the scale of metabolites contents. Red indicates upregulation, while, green indicates downregulation. Lowercase letters represent a significant difference of p ≤ 0.05

Fig. 7

Analysis of differentially expressed genes (DEGs) in the ML-vs.-LL, ML-vs.-HL, and LL-vs.-HL comparisons at 5-day. (a) Numbers of DEGs. Red indicates upregulation, while, blue indicates downregulation. (b-d) Bar diagrams of GO classifications of DEGs

Fig. 8

Enrichment analysis of DEGs in the ML-vs.-LL, ML-vs.-HL, and LL-vs.-HL comparisons. (a-c) GO enrichment analysis of the DEGs. (d-f) KEGG enrichment analysis of the DEGs

Fig. 9

The expression of DEGs involved in iridoid biosynthesis at 5-day. (a) Heatmap of DEGs expression levels (normalized via the z-score). The color shading represents the expression levels. (b) Venn diagram depicting comparison of the DEGs numbers in the iridoid biosynthesis pathway

Fig. 10

Determination of selected DEGs expression via RNA-seq and RT-qPCR comparison. The x-axis, left y-axis and right y-axis indicates the three treatment groups, RT-qPCR expression levels and the RNA-seq data with FPKM, respectively. Error bars indicate the standard deviation of three independent replicates

Fig. 11

Analysis of transcription factors (TFs). (a) Distribution of TFs. (b) Venn diagram depicting comparisons of the differential TFs numbers. (c) Heatmap of correlations between differential TFs and iridoid compounds, R ≥ 0.8. (d) Correlation network diagram among the DEGs involved in iridoid biosynthesis, GmbHLH20 and GmMYB5, R ≥ 0.8. Red lines indicate a positive correlation, while blue lines indicate a negative correlation. Line thickness indicates the strength of the correlation

Fig. 12

A schematic overview: working model of light regulation on iridoid biosynthesis in G. macrophylla