Ecotype-dependent drought responses in Ziziphora clinopodioides: from trichome development to pulegone biosynthesis

Drought is a major abiotic stress affecting plant growth and secondary production, particularly in medicinal and aromatic plants. This study investigated the effects of drought stress on glandular trichome density, phenolic compounds, essential oil production across five ecotypes of Ziziphora clinopodioides . In addition, the expression of key genes involved in pulegone biosynthesis was analyzed in a selected ecotype. Plants were subjected to three irrigation regimes: well-watered conditions, irrigation withheld at the vegetative stage (long-term drought), and irrigation withheld at the flowering stage (short-term drought). Long-term drought markedly increased glandular trichome density across all ecotypes, with the greatest responses observed in the ecotypes of Velian and Taleghan. These structural changes were accompanied by increased total phenolic and flavonoid content and a 63% increase in essential oil content, reaching 1.93% (v/w), whereas essential oil yield remained largely unaffected. The ecotype of Galugah exhibited the highest essential oil content and yield among the evaluated ecotypes. Drought also induced pronounced, ecotype-dependent changes in essential oil composition. Pulegone content increased in most ecotypes, reaching 53.02% in Taleghan under long-term drought, whereas the Kabudarahang ecotype maintained a distinct 1,8-cineole dominant chemotype with contrasting responses to drought stress. Furthermore, long-term drought was associated with increased transcript abundance of Limonene Hydroxylase ( LH ) and Isopiperitenol Dehydrogenase ( IPD ) genes in the Velian ecotype, coinciding with increased glandular trichome density, enhanced essential oil content, and greater pulegone accumulation. Collectively, these findings suggest that drought induces associated concurrent structural, biochemical, and transcriptional changes, contributing to ecotype-specific variation in metabolic traits while maintaining essential oil productivity. The superior performance of the Galugah, Velian, and Taleghan ecotypes highlights the potential of Z. clinopodioides as a promising species for climate-smart cultivation in water-limited environments and as a valuable genetic resource for breeding programs targeting drought resilience and enhanced monoterpene production.