2,4-Epibrassinolide Enhances Drought Tolerance in Prunella vulgaris by Improving Photosynthesis, Redox Homeostasis, and Secondary Metabolism

As a species of significant traditional medicinal importance, <i>Prunella vulgaris</i> is severely limited by drought stress, given its high sensitivity to this environmental constraint. 24-epibrassinolide (EBR) has shown promise in enhancing plant stress resilience and secondary metabolite production, yet its efficacy in mitigating drought effects on <i>P. vulgaris</i> requires further elucidation. In this study, foliar application of EBR (0, 0.01, 0.05, 0.1, 0.2 μmol·L^-1) was applied to drought-stressed <i>P. vulgaris</i> seedlings (maintained at 60% ± 5% field capacity, FC, for 20 days during the flowering stage; control at 75% ± 5% FC). The results showed that drought inhibited the growth and development of <i>P. vulgaris</i>. Compared with the control group, malondialdehyde, hydrogen peroxide, and superoxide anion increased by 77.82%, 27.47%, and 44.95%, respectively. The total chlorophyll content and the coordination between photosystem I and photosystem II decreased by 42.33% and 46.62%, respectively. Additionally, the net photosynthetic rate and biomass of <i>P. vulgaris</i> significantly decreased by 45.12% and 34.66%, respectively. In contrast, the 0.1 μmol·L^-1 EBR significantly enhanced the antioxidant and osmoregulation systems. Compared with drought stress treatment, the activities of SOD, POD, CAT, APX and GPX increased by 10.78%, 45.86%, 48.44%, 40.58% and 63.37%, respectively; soluble sugar, soluble protein and proline contents increased by 53.38%, 29.09% and 45.95%, respectively; and malondialdehyde, hydrogen peroxide and superoxide anion levels decreased by 28.37%, 15.77% and 25.73%, respectively. Total chlorophyll content, photosystem coordination and net photosynthetic rate increased by 55.68%, 43.08% and 45.88%, respectively, along with a significant 42.23% increase in total biomass. Furthermore, EBR upregulated the transcription levels of key phenylpropanoid pathway genes and elevated secondary metabolite contents. The expression of <i>Pv4CL</i>, <i>PvC4H</i>, <i>PvPAL</i> and <i>PvTAT</i> increased by 26.97%, 90.42%, 35.52% and 84.35%, respectively. Accordingly, total phenolic content, caffeic acid, ferulic acid, rosmarinic acid and hyperoside increased by 36.44%, 121.01%, 100.27%, 72.38% and 80.77%, respectively. Lower EBR concentrations (0.01 μmol·L^-1) had no significant effect on most indices, while 0.2 μmol L^-1 EBR showed weakened effects. In summary, under 60% ± 5% field capacity (FC) drought, 2,4-epibrassinolide (EBR) enhances drought adaptation, medicinal yield, and quality of <i>P. vulgaris</i>, with 0.1 μmol L^-1 EBR as the optimal concentration. This improvement is driven by enhanced antioxidant capacity, optimized photosynthesis, promoted root-shoot growth, and activated biosynthesis of medicinal compounds.