Exogenous 24-Epibrassinolide Enhances Aluminum Tolerance in Oriental Melon (Cucumis melo var. makuwa), Involving a CmBES1–6–CmSTOP3/5 Regulatory Module

Aluminum (Al) toxicity severely limits crop growth in acidic soils. Although 24-epibrassinolide (EBR), a bioactive brassinosteroid analog, is known to regulate plant stress responses, its role in Al tolerance in oriental melon remains unclear. Here, we investigated the effects of exogenous EBR application on Al stress tolerance in oriental melon seedlings, and the molecular regulatory mechanisms underlying the EBR-induced alleviation of aluminum stress were explored. Seedlings were pretreated with 0, 0.01, 0.1, and 1 μM EBR for 24 h prior to exposure to 100 μM AlCl3. Our results showed that 0.01 μM EBR significantly alleviated Al toxicity by improving root vitality, promoting primary root elongation, reducing hematoxylin staining intensity in root tips, and notably increasing malate exudation. The transcriptional analysis revealed that EBR upregulated CmBES1-6, a core transcription factor in BR signaling, along with Al-responsive genes including CmALMT7, CmALMT8, CmALMT12, CmSTOP3, and CmSTOP5. Silencing of CmBES1-6 though virus-induced gene silencing (VIGS) compromised Al tolerance, as reflected by the reduced expression of these Al-responsive genes. Yeast two-hybrid (Y2H) assays confirmed that CmBES1-6 physically interacts with both CmSTOP3 and CmSTOP5. Furthermore, yeast one-hybrid (Y1H) assays demonstrated that CmBES1-6 directly binds to E-box cis-elements (CANNTG) in the promoters of CmSTOP3 and CmSTOP5. Together, our findings suggest that exogenous EBR promotes BR signaling through CmBES1-6, which is associated with the upregulation of CmSTOP3 and CmSTOP5 and enhanced malate exudation from roots, thereby potentially contributing to improved Al tolerance in oriental melon seedlings. This work implicates a CmBES1-6–CmSTOP3/5 transcriptional module in Al tolerance and provides candidate genes for the genetic improvement of Al resistance in melons cultivated in acidic soils.