Transcriptome and Metabolome Analysis of Low-Pressure Regulation in Saussurea involucrata Leaves

Abstract

Saussurea involucrata, an endangered medicinal plant, thrives in high mountain regions at altitudes ranging from 3500 to 5000 m. Being a plant that grows at high altitudes means it possesses unique physiological mechanisms and stress-responsive genes that regulate and adapt to the high-altitude environment. While many cold-resistant genes have been cloned and their mechanisms studied, the genes and molecular mechanisms involved in adaptation to hypobaric hypoxia remain largely unexplored. This study conducted transcriptomic and metabolomic analyses on the leaves of S. involucrata under normal atmosphere (101 kPa) and low pressure (60 kPa). A total of 2383 differentially expressed genes (DEGs) and 336 differentially accumulated metabolites (DAMs) were identified utilizing RNA-seq and UPLS-MS techniques. The results indicated that S. involucrata exhibits responses to hypobaric hypoxia environments by engaging in DNA repair, membrane transport, hypoxic response, reproductive processes, and various metabolic activities associated with nutrient uptake and the effective utilization of chemical components. It is worth noting that under low-pressure treatment, flavonoids are predominantly negatively regulated, whereas terpenoids are primarily positively regulated. These findings identify key genes and metabolites in S. involucrata that respond to hypobaric hypoxia treatment, providing a theoretical basis for the development of its medicinal value and for low-altitude cultivation.

Keywords: S. involucrata; flavonoid regulation; hypobaric hypoxia; metabolomics; oxidative stress; transcriptomics.

Figure 1

Phenotypic observations of S. involucrata in the NN group and HH group after three weeks of treatment: left: normobaric normoxic, right: hypobaric hypoxia.

Figure 2

Transcriptome data-quality assessment and analysis of DEGs. (A) Pearson correlation test of 6 samples; (B) PCA of 6 samples, with different colors in the figure denoting distinct samples (NN: normobaric normoxic, HH: hypobaric hypoxia); (C) histogram of S. involucrata DEGs; (D) volcano map of DEGs in S. involucrata.

Figure 3

DEG enrichment analysis scatter plot and circle plot. (A) Scatter plot of KEGG enrichment analysis of DEGs; (B) circle plot of KEGG enrichment analysis of DEGs; (C) scatter plot of GO enrichment analysis of DEGs. The color of the dots indicates the enrichment significance, while the size of the dots reflects the number of DEGs that are enriched.

Figure 4

Assessment of metabolome data quality and analysis of DAMs. (A) The metabolite categories of S. involucrata are represented in a ring diagram. Each color corresponds to a specific metabolite category, with the area of each color block indicating the proportion of that category; (B) PCA of metabolites across 6 samples; (C) Pearson correlation test of S. involucrata treated with NN vs. HH for each metabolite expression level between the groups; (D) volcano plot of DAMs in S. involucrata.

Figure 5

Scatterplot of DAM screening and KEGG enrichment analysis. (A) The types of DAMs in S. involucrata under NN vs. HH treatment. Blue indicates down-regulated DAMs, while green denotes up-regulated DAMs. (B) The top 20 DAMs from the S. involucrata leaves. The horizontal axis represents the log2FC of the differential metabolites, and the vertical axis displays the differential metabolites. (C) KEGG pathway enrichment of S. involucrata, Where the symbol superscripted after the compound means that the substance has isomers.

Figure 6

Correlation analysis of DAMs and DEGs. (A) Nine-quadrant map of genes and metabolites; (B) the co-enrichment of DEGs and DAMs, with blue representing the transcriptome and orange representing the metabolome; (C) correlation network diagram for involvement in sesquiterpene and triterpene biosynthesis; (D) correlation network diagram for involvement in the biosynthesis of flavones aglycones I, with red denoting genes, green denoting metabolites, solid lines indicating positive regulation, and dotted lines representing negative regulation.

Figure 7

Flavonoid metabolic pathways in response to HH in S. involucrata. (A) The DEGs and DAMs linked to the flavonoid pathway, where solid lines represent direct regulation and dotted lines indicate indirect regulation. The up-regulated DAMs are highlighted by a red rectangle, whereas down-regulated DAMs are marked with a green rectangle. Up-regulated DEGs are shown in a purple ellipse, down-regulated DEGs are illustrated in a blue ellipse, and bidirectionally regulated DEGs are denoted by a yellow ellipse. (B) A clustering heatmap analysis of DEGs related to flavonoid biosynthesis derived from RNA-seq data.

Figure 8

The α-linolenic acid pathway in response to HH in S. involucrata. (A) DEGs and DAMs associated with the α-linolenic acid pathway. The red rectangle indicates up-regulated DAMs, the purple ellipse denotes up-regulated DEGs, the blue ellipse represents down-regulated DEGs, and the yellow ellipse illustrates bidirectionally regulated DEGs. (B) A cluster heatmap analysis of DEGs related to α-linolenic acid metabolism derived from RNA-seq data.

Figure 9

WGCNA analysis of S. involucrata genes. (A) The results of the cluster tree diagram of the S. involucrata DEG expression module, with 10 distinct modules represented in various colors; (B) analysis of the relationship between gene modules and 11 flavonoids, with the intensity of the red color indicating a stronger positive correlation, and the intensity of the blue color signifying a stronger negative correlation; (C) positive correlation network diagram of hub genes and flavonoids; (D) negative correlation network diagram of hub genes and flavonoids, with green circles representing flavonoids, orange diamonds denoting hub genes, blue solid lines indicating promotion of synthesis, and yellow solid lines representing inhibition of accumulation.