Trans-cinnamaldehyde-related overproduction of benzoic acid and oxidative stress on Arabidopsis thaliana

Abstract

Introduction: Trans-cinnamaldehyde is a specialised metabolite that naturally occurs in plants of the Lauraceae family. This study focused on the phytotoxic effects of this compound on the morphology and metabolism of Arabidopsis thaliana seedlings.

Material and methods: To evaluate the phytotoxicity of trans-cinnamaldehyde, a dose-response curve was first performed for the root growth process in order to calculate the reference inhibitory concentrations IC50 and IC80 (trans-cinnamaldehyde concentrations inducing a 50% and 80% inhibition, respectively). Subsequently, the structure and ultrastructure of the roots treated with the compound were analysed by light and electron microscopy. Based on these results, the following assays were carried out to in depth study the possible mode of action of the compound: antiauxinic PCIB reversion bioassay, determination of mitochondrial membrane potential, ROS detection, lipid peroxidation content, hormone quantification, in silico studies and gene expression of ALDH enzymes.

Results: Trans-cinnamaldehyde IC50 and IC80 values were as low as 46 and 87 μM, reducing the root growth and inducing the occurrence of adventitious roots. At the ultrastructural level, the compound caused alterations to the mitochondria, which were confirmed by detection of the mitochondrial membrane potential. The morphology observed after the treatment (i.e., appearance of adventitious roots) suggested a possible hormonal mismatch at the auxin level, which was confirmed after PCIB bioassay and hormone quantification by GC-MS. The addition of the compound caused an increase in benzoic, salicylic and indoleacetic acid content, which was related to the increased gene expression of the aldehyde dehydrogenase enzymes that can drive the conversion of trans-cinnamaldehyde to cinnamic acid. Also, an increase of ROS was also observed in treated roots. The enzyme-compound interaction was shown to be stable over time by docking and molecular dynamics assays.

Discussion: The aldehyde dehydrogenases could drive the conversion of trans-cinnamaldehyde to cinnamic acid, increasing the levels of benzoic, salicylic and indoleacetic acids and causing the oxidative stress symptoms observed in the treated seedlings. This would result into growth and development inhibition of the trans-cinnamaldehyde-treated seedlings and ultimately in their programmed-cell-death.

Keywords: ALDHs; Arabidopsis; cinnamic acid; hormones; oxidative stress; trans-cinnamaldehyde.

Figure 1

Arabidopsis thaliana roots under magnifier after 14 days of growth treated with 0 (A), 50 (B), 100 (C), 200 (D) and 400 µM (E) of trans-cinnamaldehyde.

Figure 2

Images of transmission electron microscopy (TEM) of control Arabidopsis thaliana roots. (A) Straight cell wall separating two cells. (B, C) Symmetric control cells with spheric nuclei and regular cell walls. (D) Plant cell finishing dividing. (E) Statoliths in root cell. (F) Control cell with some mitochondria. (n, nucleus; m, mitochondria; cw, cell wall; st, statoliths). Scale bars: (A) 1 µm; (B–E) and (F) 2 µm; (C) 5 µm.

Figure 3

Images of transmission electron microscopy (TEM) of Arabidopsis thaliana roots treated with IC₅₀ of TC for 7 days. (A, B) Cells with deposits between the membrane and wall. (C, D, H) Cells with dividing mitochondria. (E) Cell with a plasma membrane separated from the cell wall. (F, G) Cells with many mitochondria. (n, nucleus; m, mitochondria; cw, cell wall; dcm, detached cell membrane; m*, dividing mitochondria). Scale bars: (A, E, F) 2 µm; (B, G) 5 µm; (C, H) 1 µm; D 0.5 µm.

Figure 4

Images of transmission electron microscopy (TEM) of Arabidopsis thaliana roots treated with IC₅₀ of TC for 14 days. (A–C, G, H) Cells with detached cell membranes and cytoplasm vacuolated. (D–F) Vacuolated cells with active Golgi apparatus. (n, nucleus; cw, cell wall; dcm, detached cell membrane; v, vacuole; g, Golgi apparatus). Scale bars: (A, C, H): 2 µm; (B, G): 5 µm; (D–F): 1 µm.

Figure 5

Confocal microscopy images of JC-1 stained Arabidopsis roots after treatment with IC₀ TC (control), IC₅₀ TC, and IC₈₀ TC for 7 and 14 days. Valinomycin-treated roots are shown as positive control.

Figure 6

Arabidopsis roots after 14 d of PCIB bioassay. (A) Control, (B) IC₅₀ TC, (C) IC₈₀ TC, (D) 15 μM PCIB, (E) 15 μM PCIB + IC₅₀ TC, (F) 15 μM PCIB + IC₈₀ TC, (G) 30 μM PCIB, (H) 30 μM PCIB + IC₅₀ TC, (I) 30 μM PCIB + IC₈₀ TC. Scales bar: 400 μm.

Figure 7

Relative concentration of Arabidopsis plant hormones identified through GC-MS after 14 days of treatment with the IC₅₀ of TC. Asterisks indicate significant differences compared to the control: ** (p ≤ 0.01), *** (p ≤ 0.001); data were analysed through t-test with p ≤ 0.05. Bars indicate standard deviation.

Figure 8

(A) In situ determination of H₂O₂ in A. thaliana seedlings control (C) and treated with the IC₅₀ and IC₈₀ of TC for 7 and 14 days. Scale: 50 μM; (B) quantification of the integrated optical density (IOD) expressed as a percentage of the root area stained with DAB. C-7 (control 7 days), IC₅₀-7 (IC₅₀ of TC after 7 days), IC₈₀-7 (IC₈₀ of TC after 7 days), C-14 (control 14 days), IC₅₀-14 (IC₅₀ of TC after 14 days), IC₈₀-14 (IC₈₀ of TC after 14 days). Asterisks indicate significant differences compared to the control: *** (p ≤ 0.001); (C) in situ determination of O₂ ⁻ in A. thaliana seedlings treated with IC₅₀ and IC₈₀ TC for 7 and 14 days in apex and root elongation zones. Scale: 50 μm; (D) quantification of corrected total cellular fluorescence (CTCF) of roots stained with DHE. C.A (control apex), IC₅₀ A (IC₅₀ apex), IC₈₀ A (IC₈₀ apex), C.E (control elongation zone), IC₅₀ E (IC₅₀ elongation zone), IC₈₀ E (IC₈₀ elongation zone). Asterisks indicate significant differences compared to the control: ** (p ≤ 0.01), *** (p ≤ 0.001). Bars indicate standard deviation.

Figure 9

(A) Trans-cinnamaldehyde in the binding site of homology model (ALDH2). (B) Distances of π−π stacking and hydrogen-bond interactions in molecular docking studies. (C) Distance between the hydrogen HD1 in TC and the center of mass of Cys-303 throughout the MD simulation.

Figure 10

Relative gene expression of different ALDH genes in Arabidopsis seedlings treated with the IC₅₀ TC in roots (A) and shoots (B) for 14 d. Asterisks indicate significant differences compared to the root control for treated roots and to the shoot controls for treated shoots: ** (p ≤ 0.05). Bars indicate standard error.