Glutathione Involvement in Potato Response to French Marigold Volatile Organic Compounds

Abstract

To elucidate the involvement of glutathione in the mitigation of induced oxidative changes and the sequestration of perceived volatiles in cells, we exposed potato plants to French marigold essential oil. The formation of short-lived radicals, the determination of scavenging activity towards ascorbyl and DPPH radicals, and the assessment of the potato plants' overall intra/extracellular reduction status were performed using electron paramagnetic resonance spectroscopy (EPR). The results showed the presence of hydroxyl radicals in potatoes, with significantly reduced accumulation in exposed plants compared to the control group after 8 h. However, the kinetics of EPR signal intensity change for the pyrrolidine spin probe (3CP) in these plants showed very low reducing potential, suggesting that the antioxidant system acts lethargically and/or the probe has been reoxidized. Total glutathione and its reduced/oxidized form ratio, determined spectrophotometrically, showed that the exposed plants initially had lower glutathione levels with diminutive, reduced form compared to the control. Still, after 8 h, both characteristics were similar to those of the control. RT-qPCR analysis revealed that the volatiles altered the expression of glutathione metabolism-involved genes, especially that of glutathione-S-transferase, after 8 h. Glutathione metabolism was affected by volatiles in the initial response of potato plants exposed to French marigold essential oil, and glutathione molecules were involved in the mitigation of induced oxidative burst.

Keywords: antioxidative system; essential oil; plant-to-plant communication; priming defense; reactive oxygen species.

Figure 2

Short-lived radical production and assessed antioxidant scavenging activity of potato plants exposed to French marigold EO. (A) EPR signal intensities of DEPMPO/OH adducts corresponding to the amount of hydroxyl radical (^•OH) trapped upon their production in the potato leaves. Small amounts of carbon-centered and hydrogen radicals were also detected (inserted spectrum). Scavenging activity (%) of potato leaf MeOH extracts towards (B) ascorbyl radicals (^•Asc) and (C) DPPH radicals (representative EPR spectra of ^•Asc and DPPH radicals are presented as inserts). The DEPMPO adduct EPR signal intensities were measured from intact leaves, and the radical scavenging activities were measured from leaf extracts obtained from potato plants exposed to French marigold essential oil (EO) for 2, 4, 6 or 8 h, or in unexposed control plants (C). EPR signal intensities were quantified by measuring the area under the spectra. Values are the means of three biological replicates (n = 3).

Figure 3

The capacity of potato leaf tissues to alter the EPR signal of pyrrolidine spin probes. Kinetics of change in the EPR signal intensity of spin probes 3CxP and 3CP in strips of potato leaf after 2 h ((A) and (B), respectively) and 8 h ((C) and (D), respectively) of treatment with EO (red circles) and the corresponding controls (black squares). The arrangement of leaf strips from control (C) and EO exposed (EO) plants on tissue cell, along with the direction of 1D EPR imaging, is illustrated on the right side. Values are the means of three biological replicates (n = 3).

Figure 4

Spatiotemporal visualization of the potato leaf tissues’ capacity to alter the EPR signal of a pyrrolidine spin probe. Typical potato leaf with highlighted section used for the 2D EPR imaging experiment ((A), upper panel). Schematic representation of control (C) and treated (T) leaf samples, positioned in the ZY-plane within the EPR tissue cell ((A), lower panel). Two-dimensional EPR images of control (C) and treated (EO) potato leaf samples after 2 or 8 h of treatment with FM-EO, followed by incubation in the spin probe 3CP (B).

Figure 1

Experimental setup. (A) Young soil-grown potato plants were exposed to French marigold essential oil (EO) for different time periods in tightly closed glass jars, while controls without EO were maintained under the same conditions. (B) Potato material was collected immediately after single exposure to EO for 2, 4, 6 and 8 h in order to examine the effect of EO on antioxidant scavenging activity and glutathione metabolism. (C) For the analyses of French marigold EO’s priming effect on the expression of genes related to glutathione metabolism, material was collected immediately (0 d), 5 days (5 d) and 10 days (10 d) after single exposure for 8 h (8 h) or three-times-repeated 8 h long (3 × 8 h) EO exposure treatment. (D) The phytochemical composition (%) of French marigold essential oil was used in this study. The complete list of compounds has previously been published [29].

Figure 5

Glutathione content in potato plants exposed to French marigold essential oil. Total glutathione content with share of reduced GSH and oxidized GSSG forms and percentage portion of GSH and GSSG in total glutathione, in (A) potato plants exposed to French marigold essential oil (EO) for 2, 4, 6 and 8 h, and corresponding controls (C), and in (B) plants after exposure to French marigold EO for single (1 × 8 h) or three-times-repeated 8 h long (3 × 8 h) periods immediately (0 days) or 5 and 10 days after repeated exposure. The results are presented as means ± standard errors (n = 3) and separated by Fisher’s least significant difference (LSD) test. Bars with different letters denoted statistically different values at p ≤ 0.05.

Figure 6

Dynamic visualization of gene expression onto KEGG glutathione metabolism pathway diagram for potato (sot00480). Map shows the different proteins involved in (A) glutathione biosynthesis, (B) GSH-GSSG redox cycling and (C) GSTs involved glutathionylation. Green squares are hyperlinked to genes in the potato reference pathway. Genes with statistically significant fold change (FC ≥ 2, p ≤ 0.05) are highlighted with red asterisks, and gene abbreviation and value for FC are written next to the square.

Figure 7

RT-qPCR-obtained expression profiles of potato genes involved in the GSH metabolism. Expression of genes involved in (A) biosynthesis of GSH, (B) redox conversion of GSH and (C) conjugating GSTs after exposure to French marigold EO for different time periods (4, 6, 8 and 12 h). The fold change (FC) of the gene’s expression was obtained after ΔΔCt normalization to the expression of reference 18S gene and to the expression in unexposed controls (log₂FC = 0) for each time point, and presented after log2 transformation (log₂FC). Means of three biological replicates (n = 3) were compared with one-way ANOVA and presented with standard errors. Letters above the bars denote significant differences according to Fisher’s LSD post-hoc test at p ≤ 0.05. For each gene at each time point, Student’s t-test was used for comparison between treatment and corresponding control, and statistically different means are denoted with one asterisk (*) for p ≤ 0.05 or with two asterisks (**) for p ≤ 0.10.

Figure 8

RT-qPCR obtained expression profiles of potato genes involved in GSH metabolism. Expression of genes involved in the (A) biosynthesis of GSH, (B) the redox conversion of GSH, and (C) conjugating GSTs was examined in plants immediately (0 days) after exposure to French marigold EO for single (8 h) or three-times-repeated 8 h long (3 × 8 h) periods, or after 5 and 10 days. The fold change (FC) of the gene’s expression was obtained after ΔΔCt normalization to the expression of reference 18S gene and to the expression in unexposed controls (log₂FC = 0) for each time point, and presented after log₂ transformation (log₂FC). Means of three biological replicates (n = 3) were compared with one-way ANOVA and presented with standard errors. The letters above the bars denote significant differences according to Fisher’s LSD post-hoc test at p ≤ 0.05. For each gene at each time point, a Student’s t-test was used for comparison between treatment and corresponding control, and statistically different means are denoted with one asterisk (*) for p ≤ 0.05 or with two asterisks (**) for p ≤ 0.10.

Figure 9

Schematic model summarizing the most significant results obtained when studying potato plants’ early responses to French marigold essential oil (FM-EO) within 2 and 8 h. The measurement of the presence of ^•OH adducts, total glutathione (Glu) content, the ratio between reduced (GSH) and oxidized (GSSG) forms and the expression of genes involved in Glu biosynthesis, Glu redox cycle and glutathionylation showed the relationship between FM-EO-induced oxidative stress and Glu-related antioxidant response.