Differential effects of sodium nitroprusside, salicylic acid, and simvastatin on growth, leaf anatomy, biochemistry, and essential oil composition of Thymus vulgaris L.

Abstract

This study evaluated how foliar application of sodium nitroprusside (SNP), salicylic acid (SA), and simvastatin (SIM) applied singly and in all pairwise combinations, affects the growth, leaf anatomy, biochemistry, and essential-oil profile of greenhouse-grown Thymus vulgaris L. SNP + SA produced the most pronounced vegetative response, increasing shoot fresh weight by 43.5% and shoot dry weight by 24.9%, and root dry weight by 86.6%. Plant height was greatest (38.7 cm) under SA applied alone, glandular trichome density was raised only by SNP applied alone (+ 10.8%), capitate trichome density was highest under SIM (+ 26.6%), and glandular trichome diameter tended to decrease under every treatment relative to the control, although this effect was not significant ( P = 0.117). SIM produced the highest total soluble protein content (0.364 ± 0.010 mg g⁻¹, an increase of 53.6% compared to control), indicating mild stress-like signaling, while soluble carbohydrate was highest under SNP + SA (1.39-fold) and SNP + SIM (1.32-fold). Total flavonoid content rose by 107.7% under SNP + SA. Total phenolics were highest under SNP + SA (115.3 mg GAE g⁻¹ FW) and SA + SIM (112.4 mg GAE g⁻¹ FW), whereas SNP applied alone lowered them to 75.4 mg GAE g⁻¹ FW against a control value of 105.1 mg GAE g⁻¹ FW. Chlorophyll a was elevated only under SA + SIM (+ 14.3%), the treatment that also gave the highest chlorophyll b (+ 40.3%), whereas SNP applied alone reduced chlorophyll a by 49.5% and chlorophyll b by 41.8%. Essential-oil yield increased under every treatment except SIM applied alone and peaked under SNP (1.83 ± 0.04 mL 100 g⁻¹ DW, an increase of 32.5%). In the essential oil profile, 30–34 compounds were identified per treatment, accounting for 99.6–100.0% of total peak area. Thymol was the dominant constituent in every treatment and peaked under SNP alone (52.24 ± 1.09%), with γ-terpinene showing the inverse pattern. At the class level, SNP shifted the profile toward oxygenated monoterpenes (67.6 vs. 54.5%) at the expense of monoterpene hydrocarbons; SIM elevated sesquiterpene hydrocarbons to 9.7% (vs. 3.4%) and uniquely induced phenylpropanoid accumulation, with trans-anethole reaching 3.2% under SIM and 7.3% under SA + SIM. (+)-4-Carene emerged as a strong SA-responsive marker (5.98% under SNP + SA). These results demonstrate that SNP, SA, and SIM exert distinct, target-specific effects on T. vulgaris : SNP + SA promotes biomass and flavonoid production; SNP alone maximizes both oil yield and thymol enrichment; and SIM induces a unique sesquiterpene–phenylpropanoid signature. These findings support targeted foliar-elicitor use for directing essential-oil composition and plant performance.