Melatonin and calcium modulate the production of rosmarinic acid, luteolin, and apigenin in Dracocephalum kotschyi under salinity stress

Melatonin (Mel) and calcium (Ca²⁺) have a regulatory role in the induction of specialized metabolites production and defensive responses against stresses. Therefore, in this study, the effects of Mel and Ca²⁺ and the possible relationship between them in the increase of the production of phenolic compounds in Dracocephalum kotschyi Boiss. under both control and salinity stress conditions were investigated. The results showed that 75 mM NaCl reduced shoot dry biomass but elevated H₂O₂ content, electrolyte leakage (EL) level, total phenolic and flavonoid contents (TPC and TFC), and DPPH scavenging capacity. Salinity stress also upregulated gene expression of phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS), as well as the activities of PAL and tyrosine ammonia-lyase (TAL) enzymes. Pre-treatment of the plants with CaCl₂ and Mel affected these attributes in a dose-dependent manner. Application of 5 mM Ca²⁺ and 100 μM Mel improved shoot dry biomass and reduced the level of EL and H₂O₂ content but enhanced TPC and TFC, DPPH scavenging capacity, PAL and TAL activities, PAL and RAS transcripts, and content of rosmarinic acid (RA), luteolin flavone (LF) and apigenin flavone (AF) under salinity stress. Pre-treatment of D. kotschyi with lanthanum chloride (LaCl₃) as a plasma membrane channel blocker, ethylene glycol tetra-acetic acid (EGTA) as a Ca²⁺ chelator and trifluoperazine (TFP) as a calmodulin (CaM) antagonist, impaired Mel effects on the above attributes under salinity stress. In contrast, pre-treatment with p-chlorophenylalanine (p-CPA), as an inhibitor of Mel biosynthesis, did not impair the impacts of Ca²⁺ on the production of phenolic compounds in salt-exposed plants. These results suggested that the effect of Mel on the induction of phenolic compounds production requires the influx of extracellular Ca²⁺ into the cells and is dependent on Ca²⁺/CaM signaling.

Keywords: Apigenin; Calcium/calmodulin; Dracocephalum kotschyi; Lamiaceae; Luteolin; Melatonin; PAL; RAS; Rosmarinic acid; Salt stress.