## 3 Discussion  
In _O. pumila_, WRKY3 transcription factor shared high identity with AtWRKY30, AtWRKY41, and VvWRKY30. Furthermore, OpWRKY3 possesses a highly conserved WRKYGQK motif and a C2H2-type zinc-finger structure, belonging to the group III category. The group III WRKYs, such as _Arabidopsis thaliana WRKY75_ and _WRKY41_, have been shown previously and played essential roles in regulating plant growth and development [30; 31]. In addition, knockout of the _AtWRKY75_ mutant increased the number and length of root hairs [30]. _AtWRKY46_, _AtWRKY54_, and _AtWRKY70_ are involved in both brassinosterol-regulated plant growth and drought response [36; 37]. In the present study, the results of _OpWRKY3_ overexpression and repression revealed that the expression level of _OpWRKY3_ was linear with the biomass of transgenic hairy roots. Repression of _OpWRKY3_ inhibited the growth and development of the root tip and ultimately decreased the biomass of transgenic hairy roots. This is consistent with the _WRKY_ transcription factor gene _CbNN1_ increased rooting ability, promoting adventitious root formation [38]. These results indicated that _OpWRKY3_ is involved in the growth and development of hairy roots. In addition, previous reports shown that group III WRKYs possessed the function of regulating secondary metabolism [39; 40]. In another study has been shown that overexpression of _CrWRKY1-SRDX_ decrease cellular tryptophan decarboxylase (TDC) activity and tryptamine accumulation in transgenic hairy roots [40]. However, their characteristics and functions have not been identified in _O. pumila_. Therefore, the function of WRKYs in _O. pumila_ requires in-depth research, especially _OpWRKY3_. Figure 7: OpWRKY3 binds to the W-box of the _OpCPR_ promoter and activates _OpCPR_ expression. **(A)** The analysis of motifs in _OpCPR_ promoter. **(B)** Yeast one-hybrid assay showed that OpWRKY3 binds to the W-box of _OpCPR_ promoter. Furthermore, overexpression and repression of _OpWRKY3_ affected the expression level of genes involved in biosynthesis of camptothecin, tryptamine, loganin, and secologanin in transgenic hairy roots. The _TDC_ gene expression and loganin concentration were increased significantly while the _SLS_ gene expression and secologanin concentration were decreased significantly in _OpWRKY3_ overexpression hairy roots. This is consistent with a previous report that the accumulation levels of secologanin exhibited a strong negative correlation with the expression level of _TDC_, and that of loganin exhibited a negative correlation with the expression level of _SLS_[17]. These results suggested that _OpWRKY3_ is involved in biosynthesis of tryptamine, loganin and secologanin, which as CPT biosynthetic precursors. Numerous reports showed that WRKY TFs regulate their target genes by binding to the W-box elements containing the TTGAC(C/T) motif [41]. Simultaneously, previous reports indicated that overexpression of _CrWRKY1-SRDX_ repressed the transcription of _TDC_ by binding to the W-box elements of its promoter [40]. _SmWRKY2_ increased accumulation of tanshinones by binding to the W-box of the _SmCPS_[42]. The yeast one-hybrid experiments implied that _OpWRKY3_ regulates the biosynthesis of camptothecin and its precursors by binding to the W-box elements of the _OpCPR_ gene promoter. The total production (hairy roots and medium) of CPT has been increased significantly in OE-8, 13, 14 lines while it was significantly decreased in the S lines. However, the CPT concentrations in transgenic hairy roots were not significantly changed in OE and S lines, compared to control. It may be due to overexpression of _OpWRKY3_ slightly increased the biomass of hairy roots while repression of _OpWRKY3_ down-regulated the biomass of hairy roots. These results indicated that _OpWRKY3_ influenced the production of CPT by regulating the growth and development of hairy roots.