English Title:insights into the mechanism of multi-walled carbon nanotubes phytotoxicity in arabidopsis through transcriptome and m6a methylome analysis
Chinese Title:Transcriptome and M6A methylome analysis were used to further understand the phytotoxicity mechanism of multi-walled carbon nanotubes (MWCNTs) to Arabidopsis
Name of the journal：science of the total environment
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Author's Affiliation:Institute of Hemp, Chinese Academy of Agricultural Sciences
Proness provides services:tem, liveDetection of sexual oxygen and antioxidant enzyme activity
Foreword
Carbon nanotubes (CNTs) are cylindrical nanostructures of carbon atoms arranged in a hexagonal lattice. Carbon nanotubes are divided into single-walled (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) according to the number of concentric layers of rolled graphene sheets in the structure. Due to their special physicochemical and mechanical properties, carbon nanotubes are considered promising materials for various applications in the fields of electronics, optics, biomedical engineering, and biosensors. With the increase in the production and widespread application of carbon nanotubes (CNTs), they are inevitably released into the natural environment and ecosystems, where plants are the main primary producers. Therefore, it is imperative to understand the toxic effects of carbon nanotubes on plants.
Although studies have begun to reveal the effects of carbon nanotubes on plants at the morphological and physiological levels, few have studied the interaction between carbon nanotubes and plants at the gene expression level, and the molecular mechanisms of carbon nanotubes' toxic effects on plants are not well understood. In this study, we investigated the phytotoxic effects of CNT in Arabidopsis thaliana and the plant response to CNT exposure by detecting physiological indicators, RNA-Seq, and M6A-Seq to reveal the mechanism behind its toxicity.
Research routes
Grouping and processing of samples
Findings:
1. Phenotypic analysis Compared with MWCNT-treated seedlings, the growth trend of the untreated control group was better (Fig. 1A). MWCNT treatment inhibited root and leaf growth compared to the control group (Fig. 1B, C, D). Moreover, the inhibitory effect is dose-dependent. These observations suggest that MWCNTs have a deleteful effect on the growth of Arabidopsis thaliana seedlings, indicating its phytotoxicity to plant growth.
2. Absorption and translocation of MWCNTs in Arabidopsis thaliana No MWCNTs were observed in root tissue and leaf histiocytes in the control group by TEM image analysis (Fig. 2A,D). Fibrous MWCNTs can be found in the vacuoles and cell walls of root and leaf cells of seedlings treated with MWCNTS (Fig. 2b, c, e, f). These findings suggest that MWCNTs are absorbed by roots from the growth medium and transferred to the leaves.
Figure 2: TEM image of MWCNT distribution in Arabidopsis thaliana.
Accumulation of MWCNTs in Arabidopsis roots (top) and leaves (bottom).
3. Compared with the control, the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in all three treatments were significantly increased by MWCNT-induced accumulation of reactive oxygen species (ROS). Superoxide anion (O2-) levels were significantly increased when treated with 1500 mg L and 2500 mg L MWCNT, but decreased at 3500 mg L MWCNT, and 1500 mg L MWCNTs induced the highest levels of H2O2, O2- and MDA, while these levels decreased with increasing MWCNT concentrations. These results suggest that MWCNTs induce oxidative stress in plants, which may contribute to their phytotoxicity.
4. Antioxidant response to MWCNT Compared with the control seedlings, the SOD activity of MWCNT-treated seedlings was significant and dose-dependently increased. CAT activity decreases at 1500 and 2500 mg L MWCNT and increases at 3500 mg L MWCNT. POD activity decreased slightly under MWCNT treatment. As for anthocyanins, the activity of mwcnts was significant and dose-dependently increased. These results suggest that the ROS clearance mechanism is activated to remove MWCNT-induced excess ROS.
Figure 4: SOD, CAT and POD activities and anthocyanin content in MWCNTS-treated Arabidopsis thaliana seedlings.
5. RNA-Seq and M6A-Seq correlation analysis.
Regression analysis showed that M6A methylation levels were inversely correlated with gene expression levels in all three MWCNT treatments (Figure 8A). Through a combined analysis of M6A-Seq and RNA-Seq data, we identified 35 genes that exhibited significant changes in M6A modification and mRNA expression (fold change >> 2, p < 0.).05）。Based on their methylation and transcription levels, the 35 genes are divided into four categories: hypermethylated, downregulated genes (hyperdownregulated);Hypermethylated, upregulated genes (hyperup);Hypomethylation, down-regulation of genes (hypo-downregulation);and hypomethylated, upregulated genes (hypoupregulated);Seven genes were over-lower, 4 genes were over-the-top, 7 genes were low, and 17 genes were over-the-top (Figure 8b). Total 636% (7 11) of hypermethylation genes are down-regulated, 708% (17, 24) of the hypomethylation genes were upregulated. Thus, the percentage of super-rise and low-rise genes (24 35) is significantly higher than the percentage of super-rise and low-rise genes (11 35). These results suggest that M6A modification may negatively regulate gene transcription in Arabidopsis thaliana after MWCNT treatment.
Figure 8Combined analysis of differentially methylated genes and DEG.
Discussion and summaryThe results of this study showed that high concentrations of MWCNTs significantly inhibited plant growth in a dose-dependent manner. MWCNTs can be absorbed by root cells and transferred to shoots, where they inhibit leaf expansion. At the physiological level, MWCNT exposure induces oxidative stress in plant cells, manifested by overproduction of ROS (H2O2, O2-) and MDA. These observations reflect the general adverse effects of high concentrations of carbon nanotubes on plants and imply that the accumulation of carbon nanotubes in the environment poses a risk to ecosystems. Transcriptome and M6A methylome analysis demonstrated that MWCNTs inhibit auxin signaling and photosynthesis. Therefore, the toxicity of carbon nanotubes may be attributed to oxidative stress, inhibition of auxin signaling and photosynthesis. In response to carbon nanotube exposure, a variety of defense mechanisms, such as activating the antioxidant system against MWCNTs-induced oxidative stress, ROS metabolism, toxin metabolism and plant response to pathogenic bacteria were also enhanced. This study provides new insights into the underlying mechanisms of carbon nanotube phytotoxicity and helps us understand the possible mechanisms of plant defense in response to carbon nanotubes.