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[Display omitted] • Root preferential promoters in rice was used for enhancing function of root preferential genes. • Root preferential promoters in rice was more effective to increase grain yield without side effect frequently observed in those by ubiquitous ones. • Enhanced degradation of RNAs in root is beneficial for crop biomass. • Enhanced ability of ROS scavenge in root is beneficial for crop biomass. Root development is a fundamental process that supports plant survival and crop productivity. One of the essential factors to consider when developing biotechnology crops is the selection of a promoter that can optimize the spatial–temporal expression of introduced genes. However, there are insufficient cases of suitable promoters in crop plants, including rice. This study aimed to verify the usefulness of a new rice root-preferred promoter to optimize the function of a target gene with root-preferred expression in rice. osrns1 mutant had defects in root development based on T-DNA insertional mutant screening and CRISPR technology. To optimize the function of OsRNS1 , we generated OsRNS1 -overexpression plants under two different promoters: a whole-plant expression promoter and a novel root-preferred expression promoter. Root growth, yield-related agronomic traits, RNA-seq, and reactive oxygen species (ROS) accumulation were analyzed for comparison. OsRNS1 was found to be involved in root development through T-DNA insertional mutant analysis and gene editing mutant analysis. To understand the gain of function of OsRNS1 , pUbi1::OsRNS1 was generated for the whole-plant expression , and both root growth defects and overall growth defects were found. To overcome this problem, a root-preferential overexpression line using Os1-CysPrxB promoter (Per) was generated and showed an increase in root length, plant height, and grain yield compared to wild-type (WT). RNA-seq analysis revealed that the response to oxidative stress-related genes was significantly up-regulated in both overexpression lines but was more obvious in pPer::OsRNS1. Furthermore, ROS levels in the roots were drastically decreased in pPer::OsRNS1 but were increased in the osrns1 mutants compared to WT. The results demonstrated that the use of a root-preferred promoter effectively optimizes the function of OsRNS1 and is a useful strategy for improving root-related agronomic traits as well as ROS regulation. [ABSTRACT FROM AUTHOR]