Transcription factor NtWRKY33a modulates the biosynthesis of polyphenols by targeting NtMYB4 and NtHCT genes in tobacco

There are abundant polyphenols in tobacco leaves mainly including chlorogenic acid (CGA), rutin, and scopoletin, which not only influence plant growth, development, and environmental adaptation, but also have a great impact on the industrial utilization of tobacco leaves. Few transcription factors regulating the biosynthesis of polyphenols have been identified in tobacco so far. In this study, two NtWRKY33 genes were identified from N. tabacum genome. NtWRKY33a showed higher transcriptional activity than NtWRKY33b , and encoded a nuclear localized protein. Overexpression and knock-out of NtWRKY33a gene revealed that NtWRKY33a inhibited the accumulation of rutin, scopoletin, and total polyphenols, but meanwhile promoted the biosynthesis of CGA. Chromatin immunoprecipitation and Dual-Luc assays indicated that NtWRKY33a could directly bind to the promoters of NtMYB4 and NtHCT , and thus induced the transcription of these two genes. The contents of polyphenols in ntwrky33a , ntmy4 , and ntwrky33a/ntmyb4 mutants further confirmed that the repression of NtWRKY33a on the biosynthesis of rutin, scopoletin, and total polyphenols depends on the activity of NtMYB4. Moreover, the promotion of NtHCT by NtWRKY33a modulates the distribution of metabolism flux into the synthesis of CGA. Ectopic expression of NtWRKY33a inhibit the expression of NtSAUR14 , NtSAUR59 , NtSAUR66 , NtIAA4 , NtIAA17 , and NtIAA19 genes, indicating that NtWRKY33a might be involved in the regulation of plant auxin response. Our study revealed new functions of NtWRKY33a in regulating the synthesis of polyphenols, and provided a promising target for manipulating polyphenols contents in tobacco. • Two NtWRKY33 genes were identified from Nicotiana tabacum , among which NtWRKY33a exhibited stronger transcriptional activity. • NtWRKY33a inhibited the accumulation of rutin, scopoletin, and total polyphenols, but promoted the biosynthesis of CGA in tobacco. • RNA-seq, post-translation activation, ChIP, and Dual-Luc assays indicated that NtWRKY33a directly induced the transcription of of NtMYB4 and NtHCT . • Different mutants were generated to confirm the repression of NtWRKY33a on polyphenols biosynthesis depends on the activity of NtMYB4.