Abstract：Cinnamate 4-hydroxylase (C4H) plays a central role in the phenylpropanoid pathway, catalyzing the conversion of cinnamate to 4-coumarate, which serves as the essential starting substrate for the biosynthesis of secondary metabolites such as lignin and flavonoids. However, the precise functional mechanisms of C4H in okra (Abelmoschus esculentus) remain incompletely characterized. This study isolated and characterized the C4H gene (named AeC4H)(NCBI accession No. SAMN44959506) from the okra cultivar 'Lyuwuxing', conducting comprehensive analyses of its molecular properties, expression profiles, subcellular localization, and biological functions. Bioinformatic analysis revealed that AeC4H possessed a 1 506 bp ORF encoding 501 amino acids, exhibiting the highest sequence identity (97.23%) with its ortholog from Hibiscus cannabinus (Malvaceae). The encoded protein contained conserved P450 family domains, including the characteristic heme-binding motif and substrate recognition sites. AeC4H exhibited preferential expression in young roots, stems, and developing seeds compared with other tissues, as quantified by qRT-PCR. Subcellular localization studies confirmed the endoplasmic reticulum localization of AeC4H, aligning with the known compartmentalization of phenylpropanoid metabolism. Overexpression of AeC4H in Arabidopsis thaliana extremely significantly increased total flavonoids content in transgenic plants (P<0.01). This study provides the systematic characterization of AeC4H in okra, offering novel insights into flavonoid metabolism and valuable genetic resources for molecular breeding programs targeting nutritional quality improvement in this important vegetable crop.
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