ZmbHLH164 confers mercury tolerance in maize by modulation of mercury accumulation and ROS homeostasis

Mercury (Hg) is a highly toxic heavy metal that poses a significant threat to crop productivity. Although bHLH transcription factors are known to regulate plant stress responses, their functions in maize (Zea mays) under Hg stress remain largely unexplored. Here, we identify ZmbHLH164, a maize bHLH gene transcriptionally induced by Hg exposure, as a key regulator of Hg tolerance. Overexpression of ZmbHLH164 significantly reduced Hg accumulation in roots and root‑to‑shoot translocation, thereby enhancing Hg tolerance in maize seedlings. Conversely, knockdown of ZmbHLH164 led to increased Hg accumulation and translocation, with a concomitant loss of tolerance. Mechanistically, using DAP‑seq in combination with yeast one‑hybrid and dual‑luciferase reporter assays, we show that ZmbHLH164 directly binds to the promoter of the catalase gene ZmCAT1 and activates its transcription. This activation enhances CAT activity, promotes H₂O₂ scavenging, and sustains reactive oxygen species (ROS) homeostasis. In parallel, DAP‑seq profiling and yeast one‑hybrid assays revealed that ZmbHLH164 also directly associates with the promoters of multiple metal transporter genes, and qRT‑PCR analysis confirmed that their expression is suppressed in ZmbHLH164‑overexpressing lines, correlating with reduced cellular Hg uptake and root‑to‑shoot translocation. Together, these findings establish that ZmbHLH164 confers Hg tolerance through a dual mechanism-activating ROS detoxification while restricting Hg transport. This regulatory module not only provides mechanistic insights into plant Hg tolerance but also highlights ZmbHLH164 as a promising target for breeding low‑Hg‑accumulating crops to enhance food and environmental safety.