Identification and characterization of flavanone 3 hydroxylase genes reveal a central role for ZmF3H6 in flavonoid regulation and abiotic stress responses in maize.

Flavonoid 3-hydroxylase (F3H) is a key enzyme in the flavonoid biosynthetic pathway; however, its functional roles in maize responses to abiotic stress remain poorly understood. In this study, 14 maize F3H proteins (ZmF3Hs) were identified and characterized through bioinformatics analyses, expression profiling, and functional validation. The ZmF3H genes exhibited structural and physicochemical diversity and were unevenly distributed across seven maize chromosomes. Phylogenetic and synteny analyses classified the ZmF3Hs into four subfamilies and revealed strong evolutionary conservation with homologs from rice, wheat, and sorghum. Conserved motif and promoter analyses suggested functional divergence and multilayered regulatory control. Subcellular localization prediction indicated that ZmF3H6 is localized in the cytoplasm, which was further confirmed by transient expression assays in Nicotiana benthamiana. Expression profiling demonstrated that ZmF3Hs are responsive to multiple abiotic stresses, including cold, heat, and waterlogging. Functional analysis using virus-induced gene silencing revealed that suppression of ZmF3H6 significantly reduced flavonol accumulation and antioxidant enzyme activities while increasing malondialdehyde (MDA) levels under both normal and stress conditions. These findings provide new insights into the biological functions of ZmF3Hs and identify ZmF3H6 as a key regulator of flavonoid biosynthesis and abiotic stress tolerance, offering a promising target for improving stress resilience and flavonoid-mediated defense mechanisms in maize.