Abstract: The root system is a crucial determinant of maize yield and stress resilience, particularly under drought stress. However, the complex genetic basis governing root system architecture remains largely elusive. To dissect the genetic architecture of the maize root, a transcriptome-wide association study (TWAS) was performed for 16 root traits in a panel of 357 diverse maize inbred lines. TWAS identified 2,978 significantly associated genes, of which 530 showed root-preferential expression patterns, representing high-confidence candidates for root development. Among these candidates, ZmSAUR21, a member of the Small Auxin-Up RNA gene family, was functionally characterized. Both CRISPR–Cas9-mediated knockout and overexpression analyses demonstrated that ZmSAUR21 acts as a key positive regulator of root growth by promoting cell elongation. Furthermore, the transcription factor ZmbZIP89 was identified as a direct upstream activator that binds to the ZmSAUR21 promoter to enhance its transcription, establishing a novel ZmbZIP89–ZmSAUR21 regulatory module. Crucially, ZmSAUR21-overexpressing plants showed substantially enhanced survival rates, improved water use efficiency, and a more vigorous root system under drought conditions. Collectively, this study uncovered a key regulatory pathway controlling maize root development and demonstrates that ZmSAUR21 is a valuable target gene for improving root systems and enhancing drought tolerance in maize breeding programs.

Key words: drought tolerance, maize, root, TWAS, ZmbZIP89–ZmSAUR21 regulatory module