Exogenous melatonin induces phenolic compounds production in Linum album cells by altering nitric oxide and salicylic acid

Abstract

Melatonin is a pleiotropic molecule that can influence various aspects of plant performance. Recent studies have exhibited that it mediates plant defensive responses, probably through managing redox homeostasis. We tried to track the regulatory effects of melatonin on the antioxidant machinery of Linum album cell culture. To this, different concentrations of melatonin were applied, and the oxidative status of cells was investigated by measuring the levels of oxidative molecules and antioxidant agents. The results showed that H₂O₂ content did not change at the low melatonin levels, while it increased at the high concentrations. It can be correlated with the low melatonin dosages capacity to remove excessive amounts of H₂O₂, while the high melatonin dosages exhibit toxicity effects. In contrast, the NO enhancement occurred at 50 μM melatonin, proposing its role in triggering melatonin-induced defensive responses. The MDA results stated that NO led to oxidative stress in melatonin-treated cells at 50 μM melatonin. Antioxidant enzyme POD was activated by melatonin treatment, while SOD enzyme behaved reversely which can explain the changes in the H₂O₂ level. In addition, the analysis of the phenolics profile showed that the contents of phenolic acids, flavonoids, and lignans enhanced following an increase in PAL enzyme activity. The increased level of phenolic hormone SA can indicate that melatonin affects the defensive responses in L. album cells through a SA-dependent pathway. In general, it seems that melatonin, by modulating NO and SA levels, can induce the activity of antioxidant enzymes and the production of phenolics, especially lignans, in L. album cells.

Figure 1

Effects of different concentrations of melatonin on H₂O₂ (A), NO (B), and MDA (C) contents in L. album cells. Means from 3 separate experiments ± SE. Means marked with the same letter were not significantly different according to Duncan's multiple range test (p value ≤ 0.05).

Figure 2

Effects of different concentrations of melatonin on the activities of POD (A), and SOD (B) enzymes in L. album cells. Means from 3 separate experiments ± SE. Means marked with the same letter were not significantly different according to Duncan's multiple range test (p value ≤ 0.05).

Figure 3

Effects of different concentrations of melatonin on the activities of PAL (A), and TAL (B) enzymes in L. album cells. Means from 3 separate experiments ± SE. Means marked with the same letter were not significantly different according to Duncan's multiple range test (p value ≤ 0.05).

Figure 4

Effects of different concentrations of melatonin on secoisolariciresinol (A), lariciresinol (B), matairesinol (C), and podophyllotoxin contents in L. album cells. Means from 3 separate experiments ± SE. Means marked with the same letter were not significantly different according to Duncan's multiple range test (p value ≤ 0.05).

Figure 5

(A) Score (left) and loading (right) plots of the principal component analysis (PCA) conducted on the metabolomics data from samples treated with melatonin concentrations and the control. Each group of samples on the plot is indicated by an ellipse with 95% CI. (B) HCA map was employed for clustering of several metabolites and oxidative status according to Pearson correlations coefficient. Data are obtained from three replicates for each variation at all samples. Positive and negative correlations are described by red and blue colour, respectively. There are 5 basic clusters that are shown 1 to 5 on the picture.

Figure 6

A summary of this research in which NO molecule can play a crucial role in melatonin responses in L. album cells. An increase in NO can affect H₂O₂ as a secondary messenger for inducing the phenylpropanoid pathway through enhancing SA production and the activity of PAL and TAL enzymes. Also, NO can provoke lipid peroxidation after melatonin treatment.

Figure 7

Schematic representation of a hypothetical model of melatonin-induced antioxidant machinery in L. album cell culture.