Exogenous application of hydrogen peroxide, hydrogen sulfide, and potassium phosphite enhances rosmarinic acid accumulation and modulates key biosynthetic gene expression in Melissa officinalis L

Melissa officinalis L. (lemon balm) is a well-known medicinal plant traditionally valued for its pharmacological properties. Its aerial parts are rich in bioactive compounds such as rosmarinic acid (RA), which play key roles in the plant defense system and contribute to its antispasmodic, sedative, and memory-enhancing activities. However, the natural concentrations of these secondary metabolites are typically low in medicinal plants. This study investigated the influence of exogenous elicitors, hydrogen peroxide (HP, 5 and 10 mM), hydrogen sulfide (HS, 0.25 and 0.5 mM), and potassium phosphite (PP, 0.5 and 1 g/L) on the accumulation of photosynthetic pigments, antioxidant enzyme activity, phenolic content, RA production, and the expression of RA biosynthesis-related genes. All elicitors significantly increased chlorophyll a, b, and carotenoid levels. Treatment with HP and HS notably elevated total phenolic and flavonoid contents. The activity of CAT and APX enzymes varied depending on the elicitor type and concentration applied. Elicitation enhanced RA accumulation by enhancing PAL activity and modulating the expression of key biosynthetic genes (PAL, 4CL, TAT, and HPPR). HS treatment upregulated genes in both phenylalanine and tyrosine pathways, whereas HP was more effective in stimulating the tyrosine pathway. PP elicitation led to differential gene expression depending on its concentration. These results demonstrate that HP, HS, and PP can modulate key metabolic and signaling pathways, thereby boosting RA production and potentially enhancing the medicinal value of M. officinalis in a safe and environmentally friendly manner.