Functional study of NtWRKY6 in response to ABA expression and regulation of polyphenol synthesis

The transcription factor WRKY6 is known to regulate seed germination and seedling growth in plants. However, its role in polyphenol biosynthesis has not yet been reported. To investigate the biological function of tobacco WRKY6, the <italic>NtWRKY6</italic> gene was identified from the Nicotiana tabacum genome. Sequence alignment and phylogenetic analysis revealed that <italic>NtWRKY6</italic> contains a typical WRKY domain and belongs to the Group II subfamily. <italic>NtWRKY6</italic> was highly expressed in tobacco flowers and capsules, and its transcription was significantly induced by several plant hormones, including ABA, BL, and JA. Overexpression of <italic>NtWRKY6</italic> significantly enhanced the resistance of tobacco seedlings to ABA treatment. In three independent <italic>NtWRKY6</italic>-overexpressing (OE) lines, polyphenol content in leaves and roots increased by 18.54%-77.15% and 39.72%-64.07%, respectively, compared to wild-type (WT) plants. In contrast, <italic>ntwrky6</italic> mutants were more sensitive to ABA treatment, and polyphenol content in their leaves and roots decreased by 25.05%-30.67% and 19.85%-29.06%, respectively, relative to WT plants. Furthermore, the expression levels of multiple genes involved in polyphenol synthesis were significantly upregulated in <italic>NtWRKY6</italic>-OE seedlings, while they were significantly downregulated in <italic>ntwrky6</italic> mutants. These results indicate that <italic>NtWRKY6</italic> plays a role in regulating ABA signal transduction and polyphenol biosynthesis, providing a potential target gene for improving tobacco quality.