Functional characterization of a HD-ZIP IV transcription factor NtHDG2 in regulating flavonols biosynthesis in Nicotiana tabacum

Abstract The HD-ZIP Ⅳ transcription factors have been identified and functional characterized in many plant species. However, no tobacco HD-ZIP IV gene has been isolated, and it is not yet known whether HD-ZIP IV genes are involved in controlling flavonols accumulation in plants. Here, we cloned a HD ZIP gene named NtHDG2 from Nicotiana tabacum, which belongs to the class IV of HD-ZIP family, and the NtHDG2-GFP fusion protein is localized to the nucleus. We further observed that the flavonols contents in the NtHDG2 overexpression leaves increase to 1.9–4.5 folds of that in WT plants, but in the NtHDG2-RNAi plants the flavonols contents reduce to 20.9%–52.7% of that in WT plants. The transcriptions of one regulatory gene NtMYB12, and three structural genes (NtPAL, NtF3′H, NtF3GT), contributing to flavonols biosynthesis, were significantly induced by NtHDG2. However, the transcription level of NtNAC002, a flavonols biosynthesis repressor, was also significantly up-regulated in NtHDG2-overexpression lines, but significantly down-regulated in the RNAi lines, indicating that HDG2 regulates the synthesis of flavonols as a complex regulatory network. Moreover, ectopic expression of NtHDG2 gene promoted the transcription of several AP2/ERF genes, including NtERF1-5, NtERF109, NtDREB1, and NtCIPK11, which participate in regulating root development and resistance to abiotic stresses. Our findings reveal the new function of HD-ZIP IV transcription factors in flavonoids biosynthesis, and indicate that HD-ZIP IV members may play an important role in plant resistance to abiotic stress. The NtHDG2 gene provides a promising target for genetically manipulating to increase the amounts of flavonols in tobacco leaves.