A visible and quantitative pEAQ-RUBY system for analysis of transcription factor-promoter regulation in plants

Understanding how transcription factors (TFs) regulate target promoters is central to molecular biology. However, widely used reporter-based assays typically require specialized instrumentation, costly substrates, and labor-intensive procedures. The recently developed RUBY reporter gene, which enables visible betalain accumulation, offers a simple and equipment-free alternative for monitoring gene expression. Here, we established a pEAQ-RUBY reporter system for efficient analysis of TF-mediated promoter regulation. We first optimized a quantitative method for betalain extraction in tobacco and identified 40% ethanol as a safe and effective solvent. We then constructed pGreenII 0800-RUBY reporter vectors by replacing the LUC coding sequence with RUBY while preserving the multiple cloning site for flexible promoter insertion. Four commonly used effector vectors were evaluated, and pEAQ exhibited the highest expression efficiency. Using this system, we confirmed the positive regulation of SARD1 on the SA biosynthetic gene promoter ICS1 and MYC2 on the JA biosynthetic gene promoters AOS, LOX2, and OPR3. We also validated the negative regulation of TCP20 and TCX8 on the LOX2 promoter. All results were consistent with previous reports. Collectively, the pEAQ-RUBY reporter system provides a simple, visible, and safe alternative to conventional luciferase-based assays for studying transcriptional regulation in plants.