Physiological and Proteomic Shifts in Pfaffia glomerata Under Drought and Elevated CO₂

Drought is a major constraint to plant growth and productivity, and rising atmospheric CO₂ concentrations are expected to modify plant responses to water limitation. However, how CO₂ enrichment reshapes the physiological, metabolic, and molecular regulation of specialized metabolite production under drought remains poorly understood. Here, we investigated the combined effects of elevated CO₂ (e[CO₂]) and drought on plant morphophysiology and on the production of 20-hydroxyecdysone (20E), a phytoecdysteroid of therapeutic relevance whose accumulation may be influenced by environmental stress, in Pfaffia glomerata . Plants were grown under ambient (± 400 µmol mol⁻ 1 ; a[CO₂]) or elevated (± 800 µmol mol⁻ 1 ; e[CO₂]) CO₂ concentrations combined with sufficient or limited water supply. Elevated CO₂ promoted a metabolic shift under drought, characterized by increased investment in osmoregulatory compounds, including soluble sugars, myo-inositol, and glutamate. In contrast, drought reduced photosynthetic pigments and several primary metabolites regardless of CO₂ level. Drought induced the expression of lignin biosynthesis genes ( PgC4H , PgCCoAOMT , and PgCAD ) under both CO₂ conditions; however, PgCCR was specifically upregulated under e[CO₂] combined with drought, with consistently higher transcript levels than under ambient CO₂. Proteomic analyses revealed that e[CO₂] attenuated the accumulation of many canonical drought-responsive proteins, while selectively promoting the accumulation of the cytochrome P450 CYP72A219-like protein, which was associated with increased whole-plant 20E production. Together, these findings show that drought drives 20E biosynthesis, while elevated CO₂ modulates drought response intensity and allocation, ultimately affecting total 20E yield per plant and its relevance under future climate scenarios.