Melatonin Modulates Copper Homeostasis Through NO Dependent Manner in Rice ( <i>Oryza sativa</i> L.)

Melatonin (MT) plays a critical role in regulating rice responses to heavy metal stress. However, the mechanism by which MT alleviates copper (Cu) toxicity in rice remains insufficiently understood. In this study, excessive Cu increased endogenous MT level in rice roots, accompanied by enhanced gene expression involved in MT biosynthesis, suggesting its role in Cu stress responses. Exogenous MT reduced Cu accumulation and restored root growth in rice under Cu stress, thereby mitigating Cu-induced phytotoxicity. Mechanistically, MT increased the root pectin content and its Cu retention, thereby reducing Cu influx into the cytoplasm. Additionally, exogenous MT upregulated the expression of Heavy Metal ATPase 4 (OsHMA4) involved in vacuolar sequestration, as well as Cu efflux transporter genes OsHMA6 and OsHMA9. Conversely, it downregulated the plasma membrane Cu uptake-associated genes Copper Transporter (OsCOPT1, OsCOPT2, and OsCOPT3), the vacuolar Cu exporter OsCOPT7, and the xylem loading transporter gene OsHMA5, thereby alleviating Cu toxicity in rice. Furthermore, MT elevated nitric oxide (NO) levels in root tips, and application of the exogenous NO scavenger, 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), attenuated the mitigative effect of MT on excess Cu-stressed rice. In summary, MT alleviates Cu toxicity by reducing Cu binding to root cell walls, thereby limiting Cu uptake and translocation from roots to shoots. This process may depend on NO accumulation and provides new insights into the molecular mechanisms underlying MT-induced Cu tolerance in rice.