NtWRKY51 Negatively Regulates the Biosynthesis of Nornicotine by Inhibiting the Expression of CYP82E4

Nornicotine is a harmful alkaloid, and its accumulation in tobacco is associated with leaf maturity and senescence. To reduce nornicotine in tobacco leaves, we explored the regulation of senescence-induced nornicotine synthesis. Based on our prior finding that NtWRKY51 was induced by senescence, we cloned the CDS sequence of NtWRKY51 and conducted analyses including bioinformatics characterization, spatiotemporal expression pattern, subcellular localization, gene function, and identification of downstream interacting gene. The results showed that the full-length CDS of NtWRKY51 was 576 bp, encoding a protein of 191 amino acids, and that it belonged to the WRKYII subfamily. NtWRKY51 was a nuclear localized protein, which was highly expressed in tobacco leaves and stems, with its expression induced by senescence. The nornicotine content in senescent leaves of NtWRKY51-CR lines was significantly increased, reaching more than 1.6-fold that of WT in fully senescent leaves. Conversely, overexpression of NtWRKY51 significantly reduced nornicotine content by up to 40%. These results indicated that NtWRKY51 negatively regulated nornicotine biosynthesis during senescent leaves. Further studies showed that NtWRKY51 binds to the promoter region of the key nornicotine biosynthetic enzyme gene CYP82E4 and inhibited its transcription. The relative expression of CYP82E4 in knockout lines was significantly higher than in WT, whereas its expression in overexpression lines was significantly lower than in WT. Taken together, although NtWRKY51 was induced by senescence, it played a negative regulatory role in the senescence-induced nornicotine synthesis process by inhibiting CYP82E4 expression.