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

**Background:** Trans-cinnamaldehyde (TC) is a phenylpropanoid secondary metabolite from cinnamon (Lauraceae) with known fungicidal, antimicrobial, and anticancer activities. Despite its use in commercial pesticides, its phytotoxic mode of action in plants was poorly understood. This study investigated TC's effects on Arabidopsis thaliana morphology, metabolism, and gene expression to elucidate its mechanism as a potential bioherbicide.

**Methods:** A dose-response curve was established using 0–1200 μM TC to calculate IC50 (46 μM) and IC80 (87 μM) for root growth inhibition. Root structure and ultrastructure were examined by light and transmission electron microscopy after 7 and 14 days of IC50 treatment. Mitochondrial membrane potential was assessed using JC-1 fluorochrome with confocal microscopy. Reactive oxygen species (H2O2 via DAB staining; O2− via DHE fluorescence) were quantified. Lipid peroxidation was measured as MDA content. Hormone quantification (benzoic acid, salicylic acid, IAA) was performed by GC-MS. Molecular docking and 200 ns molecular dynamics simulations modeled TC binding to ALDH2B4. qPCR analyzed expression of 9 ALDH genes in roots and shoots. An antiauxin (PCIB) reversion bioassay tested auxin involvement.

**Key Results:** TC strongly inhibited root growth (IC50 = 46 μM, IC80 = 87 μM) without affecting germination except at 800–1200 μM. Treated seedlings developed adventitious roots, ectopic root hairs, and disorganized root meristems. Ultrastructurally, TC caused plasma membrane detachment from cell walls, thickened cell walls, increased mitochondrial numbers with active division, and after 14 days, numerous vacuoles and highly active Golgi apparatus with abundant vesicles. JC-1 staining showed mitochondrial membrane depolarization at IC80 (7 days) and both IC50 and IC80 (14 days). H2O2 increased significantly in all TC treatments (65–70% of root area stained vs. <3% in controls, p ≤ 0.001). O2− increased significantly in the elongation zone at all TC treatments except IC50 at 14 days (p ≤ 0.01–0.001). MDA content showed no significant difference. GC-MS revealed benzoic acid increased ~900%, salicylic acid ~200%, and IAA ~200% (p ≤ 0.01–0.001). PCIB partially reverted adventitious root formation but did not restore normal root length. Molecular docking showed TC binding in a hydrophobic pocket near the NAD+ cofactor of ALDH2B4, with hydrogen bonding to Asn-170 and π–π stacking with Phe-460 and Tyr-171. MD simulations confirmed stable binding over 200 ns with the aldehyde group positioned near catalytic Cys-303. In shoots, ALDH3I1, ALDH3H1, ALDH2B4, ALDH3F1, ALDH11A3, and ALDH2C4 were significantly overexpressed (~1.5-fold, p < 0.05). In roots, only ALDH22A1 was significantly repressed (~1.5-fold, p < 0.05).

**Clinical Implications:** This study demonstrates that TC acts through a multi-target mechanism involving ALDH-mediated conversion to cinnamic acid, driving massive benzoic acid accumulation, auxin dysregulation, and oxidative stress that ultimately triggers programmed cell death. The very low IC50 (46 μM) and IC80 (87 μM) values indicate potent phytotoxicity. The compound's natural origin, multiple modes of action (reducing resistance risk), and specific effects on root architecture make TC a promising candidate for bioherbicide development. The differential ALDH gene expression between roots and shoots suggests tissue-specific metabolism that could be exploited for targeted weed control.