Molecular cloning and functional characterization of NtWRKY11b in promoting the biosynthesis of flavonols in Nicotiana tabacum

ABSTRACT The biosynthesis of flavonols and anthocyanins is precisely regulated by different transcription factors in plants. WRKY11 promotes the biosynthesis of flavonoids in apple, but the molecular mechanism of WRKY11 regulating flavonols biosynthesis, and whether WRKY11 plays the same roles in other plants species remains to be further studied. Here, we cloned four NtWRKY11 genes from tobacco, which all contained the conserved WRKYGQK heptapeptide and a zinc-finger motif. The NtWRKY11b showed higher expression levels than the other NtWRKY11 genes in all the tobacco tissues examined, especially in tobacco leaves. Silencing of NtWRKY11b in tobacco leaves reduced the content of flavonols to 45.2%-69.8 % of that in the WT plants, but overexpression of NtWRKY11b increased the flavonols content by 37.8%-80.7%. Transcriptome analysis revealed 8 flavonoids related differentially expressed genes (DEGs) between NtWRKY11b-OE and WT plants, among which the transcription of NtMYB12, NtFLS, NtGT5, and NtUFGT was significantly induced by posttranslational activation of NtWRKY11b with the presence of protein synthesis inhibitor, indicating a putative direct promotion of NtWRKY11b on the transcription of these flavonoids related genes. Chromatin immunoprecipitation assays further demonstrated that NtWRKY11b could bind to the promoter regions of NtMYB12, NtFLS, NtGT5, and NtUFGT to activate the transcription of these genes. Moreover, ectopic expression of NtWRKY11b also promoted the expression levels of NtCML38, NtCTL1, NtWRKY44, and NtCML37 genes, which have been shown to enhance plant resistance to various stresses. Our findings revealed the molecular mechanism of NtWRKY11b regulating flavonols biosynthesis, and provided a promising target for increasing flavonols content in tobacco.