GRS is one of the most common diseases at present, which has a great impact on the quality of ginseng and seriously threatens development of the ginseng industry. Previous studies have suggested that GRS is associated with the accumulation of metal ions and the metabolism of phenolic substances in the root epidermis . Scholars have argued that the red substance in the epidermis of ginseng root may have been formed by a complexation reaction. Additionally, GRS involves microbial infestation . The occurrence of GRS is a complex mechanism, which is not clear at present.
In this study, metabolites and total RNAs were extracted from the same variety of 5-year-old ginseng planted in the same ginseng farm (higher rusty root index) were prepared from healthy and diseased ginseng tissues. The purpose was to ensure that ginseng samples experienced the same field management and climate change in the growth process .
Metabolome and transcriptome analysis methods have provided a basis for the study of many plant diseases, and a large number of previous studies have also found the response of metabolic and transcription levels to abiotic stress and biological infection in plants [21–25]. Therefore, we conducted UHPLC–MS/MS and RNA-Seq analysis to the diseased ginseng and HG. Our results showed that in GRS, there were 939 metabolites differentially accumulated and 9451 genes significantly differentially expressed.
Metabolite accumulation differences in diseased tissues compared with HG were analyzed by metabolomics. The results showed that the metabolite expression patterns of the two ginseng tissues were significantly different. Among the metabolites up-regulated to accumulate in diseased tissues, including organic acids, alkaloids, alcohols, and phenols, they have been closely associated with plant responses to biotic and abiotic stresses. Our study revealed that many organic acids produced significant changes in diseased tissues of ginseng (Table 1). Organic acids are essential intermediates in plant energy balance and flow and are involved in plant response to abiotic stresses. For example, α-linolenic acid is a jasmonic acid precursor compound, which plays a vital role in plant resistance by forming jasmonic acid and generating methyl jasmonate through enzymatic reactions[27, 28]. However, considering that most organic acids act as intermediate metabolites, it is difficult to determine their specific stress response role . Alkaloids are structurally diverse and have many biological activities that affect plant growth. The accumulation of alkaloids is a vital defense strategy for plants in response to biotic stresses and is a crucial class of compounds involved in plant chemical defense. In diseased tissues, we also found an upregulation of the accumulation of multiple alkaloid metabolites. The KEGG enrichment results of differential metabolites also enriched multiple alkaloid biosynthetic pathways (Fig. 2C). This result suggests that alkaloid metabolites are essential for the stress response of ginseng. Chemical defense involving alkaloids may occur in diseased tissues of ginseng against pathogenic bacteria and abiotic stresses. Besides, we also noted that multiple alcohol metabolites showed higher accumulation in diseased tissues. It was shown that the accumulation of alcohols usually plays a role in the fight against oxidative stress. They can act as intermediates of redox reactions with good scavenging capacity for free radicals and superoxide[32, 33]. It can be speculated that ginseng accumulates more alcohols in diseased tissues to cope with the oxidative stress in itself to improve its resistance. Besides, we found that various phenolics were also induced in diseased tissues in response to external stress. The phenolic content is often considered closely related to the total antioxidant capacity of plant tissues. Studies have shown that flavonoids are also one of the main chemical defense substances in plants and can reduce various forms of reactive oxygen species in plant cells, which are usually produced in response to external environmental stresses such as light intensity, temperature, and soil moisture. Besides, it is also an active substance involved in plant-microbe interactions. In our results, a significant upregulation of a large number of flavonoids was found. Significant enrichment of the flavonoid biosynthetic pathway was also found by KEGG enrichment analysis (Fig. 2C). Overall, these compounds accumulate in diseased tissues of ginseng and are jointly involved in the oxidative stress response and chemical defense of ginseng.
Terpenoids, especially saponins, are the main active substances in ginseng. We found significant accumulation or down-regulation of several saponin substances in diseased tissues, such as Ginsenoside Ro, 20(S)-Ginsenoside Rh2, and Pseudoginsenoside F11. In a previous study, ginsenosides had inhibitory effects on pathogens. It is speculated that GRS may impact ginseng terpene metabolism, and further studies are needed to determine whether saponins play a role in disease response.
Lipids are essential membrane components, and changes in their composition may help plants maintain cellular compartmentalization and membrane integrity. Besides, free radicals are metabolized and produced when cells are under stress or in the process of injury. Their scavenging capacity is still reduced, leading to an imbalance of reactive oxygen species and membrane lipid peroxidation. Therefore, reducing lipid peroxidation has an essential role in plant resilience. In diseased tissues of ginseng, there were more lipid metabolites with significantly altered content. For example, phosphatidylcholine (PE) and phosphatidylethanolamine (PC) are lipids that are present in large amounts in cell membranes. An increase in the PC/PE ratio has been reported to be a marker of the membrane bilayer's structural stability and prevent membrane degradation. Interestingly, we detected a significant increase in several PC levels and a substantial decrease in PE levels in diseased tissues of ginseng. This result may imply that diseased tissues may have a stronger tendency to maintain biofilm integrity and stability than healthy tissues.
GO enrichment analysis helps to reveal the different gene functions of biological processes, molecular functions, and cellular components. GO annotations have been shown to be good predictors of gene function and trends [40, 41]. Our data showed that the genes related to “cell wall organization” (GO:0071554) and “biogenesis response to oxidative stress” (GO:0006979) were abundant on BP. The CC term had the enrichment of “cell wall” (GO:0005618). Additionally, “peroxidase activity” (GO:0004601) was enriched in the MF term. This suggests that there may be a strong oxidation-reduction reaction in the diseased tissues of ginseng, as well as the cell wall organization and oxidative stress.
We found that the biosynthesis of secondary metabolites was significantly enriched at both the metabolic and transcriptional levels, which fully demonstrated that the secondary metabolism of diseased tissue was altered considerably in comparison with that of HG tissues. Among them, metabolic changes of alkaloids and flavonoids in ginseng root tissues may be important chemical defense patterns.
Based on the results of metabolites and DEGs (GO and KEGG) enrichment analysis, we found that plant hormones, cell wall formation, oxidative stress and plant-pathogen interaction were closely related to plant response to disease. Plants produce a variety of hormones during growth. Among them, ABA, SA, and JA play an essential role in mediating plant defense responses to pathogens and abiotic stress[42, 43]. Here, three plant hormones and hormone-related signaling molecules were detected by UHPLC–MS/MS, and all of them were significantly upregulated in diseased tissues (ABA, SA, and MeJA) (Fig S1A). At the transcriptional level, the ABA receptor PYR/PYL/RCAR, negative regulator PP2C, and positive regulator SnRK2 together comprise the regulatory system . In GRS, the genes that regulate ABA signal transduction are differentially expressed and ultimately ABF transcription factor upregulation (Fig. S1B). Under JA-stimulated conditions, JA-Ile, synthesized by JAR1, binds to the receptor COI1, leading to degradation of the repressor protein JAZ, which upregulates the expression level of JA target genes (Fig. S1C) [45, 46]. NPR1 is one of the key regulatory elements of SA-dependent PR gene activation, which interacts with GTA factors to regulate PR gene expression [47, 48]. We found differential expression of genes of the byproducts of NPR1 (BOP2 and NPR3) and multiple genes of TGA transcription factors that culminated in upregulation of PR-1 gene expression (Fig. S1D). Obviously, ABA, SA, and MeJA were fully involved in the stress response of diseased tissues. These changes in crucial hormone levels may be an early response to stress. The affect metabolic processes and eventually lead to changes in growth patterns that are adapted to withstand environmental pressures.
Lignin is a highly branched polymer of phenylpropanoid compounds, which is the main component of the plant cell wall . Because lignin is a kind of non-degradable mechanical barrier for most microorganisms, it plays an important role in plant defense. This is considered one of a series of mechanisms for plants to prevent microbial invasion. Lignin biosynthesis is a complex genetic network, in which many enzymes are involved [50, 51]. Among these enzymes, several key enzymes, including PAL, 4CL, CCR, OMT, and CAD were upregulated (Fig. 5) [52, 53]. Based on these analysis, we believe that lignin may accumulate in GRS, which usually occurs when plants are attacked by pathogens. In addition, we detected 9 compounds involved in phenylpropanoid metabolic pathways through UHPLC–MS/MS (Fig. 2D). The relative levels of phenolic compounds, such as eugenol, coniferin and chlorogenate, were far higher in the disease tissues than in HG.
Exposure of plants to various biotic and abiotic stress conditions triggers rapid changes in reactive oxygen species (ROS) production and clearance. ROS play an important role in signaling pathways that regulate inflammatory and defense responses in plants but its accumulation is often harmful to cells. Peroxisomes are subcellular organelles with a basic oxidative type of metabolism and may be the primary site of intracellular ROS production [55, 56]. This organelle is a small, usually spherical body, encased by a lipidic double membrane. Enrichment analysis with KEGG showed that both PXMP2 and MPV17, involved in ROS metabolism, were transcriptionally downregulated (Figure S2). PXMP2 is a perovskite membrane protein with the channel-forming activity that allows free diffusion of compounds, such as H2O2 . MPV17 is a protein involved in the production of ROS . Several PEX family protein genes were differentially expressed, including PEX13 and PEX14, involving matrix protein import, and PEX16 and PEX19, involving membrane protein import [58, 59]. Moreover, although peroxisomes regulate the production and release of ROS and CAT and SOD may also play an antioxidant role, oxidative stress may still be one of the causes of death of epidermal cells in diseased tissues.
Based on the available findings, the plant immune system depends on the cell's innate immunity and systemic signals from the site of infection. Plants have two layers of defense against pathogens, PTI and ETI. Here, compared to HG, there were 96 DEGs in GRS associated with the plant-pathogen interaction pathway. RBOH mediates ROS generation, which is strongly associated with both PTI and ETI [62, 63]. RBOHD and RBOHF are considered to be critical components of plant defense . FLS2 is a pattern recognition receptor (PRR) that can trigger innate immunity in plants and is also involved in endocytosis . WRKY33 plays a vital role as a transcription factor in plant resistance to pathogens . The downregulation of the above protein genes, as well as NOA1 and MEKK1, may indicate that part of PTI is repressed in GRS. Interestingly, ETI seems to be activated in GRS. RPS2 is a resistance (R) protein in plants, while RIN4 is an important negative regulator of natural plant immunity and has a regulatory effect on RPS2. HSP90.1 is required for full RPS2 resistance and is rapidly induced in plants in the face of biological stress . SGT1 positively regulates disease resistance produced by many R proteins . In our analysis, RIN4 was downregulated and PRS2, SGT1, and HSP90.1 were upregulated. Furthermore, NHO1, an essential universal resistance gene in plants, was upregulated in expression . Overall, the immune system of diseased tissues is activated, which will indirectly lead to the hypersensitive response and cell wall reinforcement. Simultaneously, the discovery of many DEGs in the “plant-pathogen interaction” pathway also suggested that GRS is related to pathogen infection.
To further understand the critical aspects of ginseng response to GRS, we combined metabolomic data with DEGs for analysis. Many differential metabolites and DEGs were annotated in the same metabolic pathway (Fig. 7A). Among them, both parts of the data were significantly enriched in the phenylpropane metabolic pathway. Considering that the phenylpropanoid biosynthesis pathway is closely related to phenolic production, flavonoid metabolism, and lignin formation, it is speculated that it may be a critical metabolic pathway in ginseng in response to GRS. Therefore, we constructed a correlation network of differential metabolites and DEGs in this metabolic pathway based on the results of correlation analysis (Fig. 7B), which may play a direct or indirect regulatory role in altering the corresponding metabolites.