RNA Sequencing of Arabidopsis thaliana Seedlings after Non-Thermal Plasma-Seed Treatment Reveals Upregulation in Plant Stress and Defense Pathways

Abstract

Not all agricultural practices are sustainable; however, non-thermal plasma treatment of seeds may be an eco-friendly alternative to improve macroscopic plant growth parameters. Despite the numerous successful results of plasma-seed treatments reported in the literature, there is a large gap in our understanding of how non-thermal plasma treatments affect seeds, especially due to the plethora of physical, chemical, and biological variables. This study uses RNA sequencing to characterize the changes in gene transcription in Arabidopsis thaliana (L.) Heynh. seeds 6 days after exposure to surface dielectric barrier discharge plasma treatment. Here, we provide an overview of all pathways that are differentially expressed where few genes are upregulated and many genes are downregulated. Our results reveal that plasma treatment time is a parameter that can activate different pathways in plant defense. An 80 s treatment upregulates the glucosinolate pathway, a defense response to insects and herbivores to deter feeding, whereas a shorter treatment of 60 s upregulates the phenylpropanoid pathway, which reinforces the cell wall with lignin and produces antimicrobial compounds, a defense response to bacterial or fungal plant pathogens. It seems that plasma elicits a wounding response from the seed in addition to redox changes. This suggests that plasma treatment can be potentially applied in agriculture to protect plants against abiotic and biotic stresses without discharging residues into the environment.

Keywords: Arabidopsis thaliana; RNA sequencing; glucosinolates; non-thermal plasma; oxidation-reduction; phenylpropanoids; plant defense; secondary metabolism; wounding.

Figure 1

Germination rate results of plasma-treated Arabidopsis thaliana (L.) Heynh. seeds, demonstrating that 80 s had the strongest effect followed by the 60 s treatment. Control experiments, i.e., no plasma treatment, are indicated with 0 s. Shown as an average of triplicates performed twice independently for a total of 6 replicates. Asterisks denote statistical significance where * signifies p < 0.05; ** is p < 0.01; and *** is p < 0.001.

Figure 2

Principal component analysis (PCA) conducted on the normalized gene expression values of the 60 s (left) and 80 s (right) samples. X− and Y−axes show PC1 and PC2, respectively, with the amount of variance contained in each component, which is 41% and 24% for 60 s and 41% and 21% for 80 s, respectively. Each point in the plot represents a biological replicate, representing 30 seedlings, with a total of 6 biological replicates in the plot. Symbols of the same colors are replicates of the same experimental group where orange represents the control, which are untreated A. thaliana seeds grown into seedlings, and blue represents either 60 or 80 s plasma-treated A. thaliana seeds grown into seedlings.

Figure 3

Heat map of the expression patterns (Z-scaled reads per kilobase of exon per million (RPKM) values) of the full transcriptome for 60 s (left) and 80 s (right). Hierarchical clustering of the relative expression profile of the top 2000 variable genes selected based on the lowest standard deviation using Euclidean distance. Individual samples are shown in columns and genes in rows. The upper axis shows the clusters of samples, and the left vertical axis shows clusters of genes. The color scale represents the relative expression of genes: green indicates low relative expression levels; red indicates high relative expression levels; black indicates zero (no change). The overall trend shows that genes are mainly downregulated after plasma exposure compared to the control, untreated samples.

Figure 4

The MA plot shows the relationship between the average normalized expression on the x−axis and the significance of the differential expression test expressed as log2FC on the y−axis for each gene in the genome. It illustrates the number of DEGs for 60 s (left) and 80 s (right). Gray dots represent the genes that are not significantly differentially expressed, while red and green dots are the genes that are significantly up− and downregulated, respectively, based on their p−values (not shown here).

Figure 5

Gene enrichment analysis for upregulated genes after 60 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 5

Gene enrichment analysis for upregulated genes after 60 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 6

Gene enrichment analysis for downregulated genes after 60 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 6

Gene enrichment analysis for downregulated genes after 60 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 7

Gene enrichment analysis for upregulated genes after 80 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 8

Gene enrichment analysis for downregulated genes after 80 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 8

Gene enrichment analysis for downregulated genes after 80 s plasma treatment. Lollipop diagrams provide information about GO fold enrichment, significance (FDR in log10), and number of genes in each pathway. From top to bottom, GO categories are in the following order: (A) biological process, (B) cellular component, (C) molecular function, and (D) KEGG pathway.

Figure 9

A tentative hypothesis summarizing the findings in this study where 60 s in our study was considered as mild plasma exposure and 80 s as moderate plasma exposure and resulted in phenylpropanoid or glucosinolate biosynthesis, respectively.

Figure 10

Surface dielectric barrier discharge (SDBD) plasma source enclosed in the plasma-seed treatment reactor. (a) Stainless steel reactor with high-voltage coaxial cable connection; (b) schematic of the interior with the inverted SDBD positioned above the seed substrate; (c) photograph of the high-voltage stripe SDBD electrode printed on an alumina dielectric plate. The ground electrode is an aluminum plate behind the dielectric.