FAST-TRACE: delineating tissue-specific cis-regulatory activities in the cadmium/zinc hyperaccumulator Sedum plumbizincicola.

FAST-TRACE is a transient expression system for rapid homologous promoter analysis in Sedum plumbizincicola, enabling identification of critical regulatory regions and a MYB transcription factor, SpTRB4, that activates the SpHMA3 promoter. Investigating the SpHMA3 promoter in Sedum plumbizincicola is essential to elucidate the molecular regulatory mechanisms underlying the above-ground metal detoxification. Given that generating stably transformed plants is time-consuming and costly, an efficient, low-cost method for the preliminary promoter activity assessment is needed. Here, we present FAST-TRACE (Fast Agrobacterium-mediated Spatio-Temporal Regulatory Activity and Cis-element Evaluation), a rapid transient transformation method for leaf tissues mediated by Agrobacterium tumefaciens. Following optimization (OD600= 0.8), qualitative and quantitative measurements of promoter activity can be obtained within 2-3 days. Using FAST-TRACE, we systematically evaluated the promoter responses to multiple stimuli, including abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), and light/dark conditions. The LUC/REN ratios showed strong correlation with the transcript levels of endogenous SpHMA3, SpCAX3, and SpNRAMP3, confirming the accuracy of this system in reflecting native expression dynamics. Truncation analysis identified two critical regions: deletion of the P1-P2 segment (- 1303 to -1054) reduced activity by 2.5-fold, indicating the presence of positive regulatory elements, while the P5-P6 region (- 303 to -54) displayed highly activity and is enriched in core promoter and regulatory motifs, including GATA motifs, MYB and MYC binding sites, suggesting its critical role in transcriptional regulation. Notably, yeast one-hybrid screening using the P1-P2 region as bait identified a MYB transcription factor, SpTRB4, which directly activated the SpHMA3 promoter by approximately 3.7-fold in homologous S. plumbizincicola leaves. FAST-TRACE was successfully applied to Sedum alfredii and Nicotiana tabacum, and extended to subcellular localization. In conclusion, the scalability and simplicity of FAST-TRACE make it a practical attractive alternative for both qualitative and quantitative promoter analysis using luciferase reporters.