Foliar proline alleviates drought stress in peppermint via consistent growth and essential oil benefits, with stress-dependent regulation of ion balance and abscisic acid

This study examined whether exogenous proline (Pro, 10 mmol/L, foliar) uniformly alleviates drought stress in peppermint (Mentha × piperita L.) or whether its benefit intensifies with increasing stress severity. Plants were grown under four water field capacity (WFC) levels (95, 75, 50, 25%), with or without Pro, in a 4 × 2 factorial randomised design. Two-way ANOVA (Levene's test confirmed homogeneous variance) partitioned each variable into WFC and Pro main effects and their interaction, followed by Tukey's HSD (P < 0.05). Water deficit reduced growth and relative water content, increased osmolytes, disrupted ionic homeostasis (higher Na+ and Na+/K+; lower K+, Ca2+, Mg2+), activated antioxidant defences, and altered hormone levels (higher abscisic acid (ABA); lower indole-3-acetic acid (IAA), gibberellic acid (GA3)). Pro significantly improved growth, water status, most osmolytes, antioxidants, IAA, GA3, and essential oil (EO) yield, with no significant WFC × Pro interaction detected for these variables, providing no statistically detectable evidence that the magnitude of the effect differed among WFC levels. Pro's effect on Na+, the Na+/K+ ratio, and ABA instead showed a significant WFC × Pro interaction (P = 0.040, 0.046, 0.001), intensifying under severe deficit. EO yield showed a biphasic response, rising at 75% and 50% WFC and falling at 25%, with Pro further increasing yield and menthol content. Pro thus acts mainly as a consistent protectant with no detected WFC × Pro interaction, alongside a distinct, stress-dependent role in ion and ABA regulation under severe drought.