Plant-specific transcription factor LrTCP4 enhances secondary metabolite biosynthesis in Lycium ruthenicum hairy roots

Lycium ruthenicum Murr. is an important medicinal plant from traditional Chinese medicine. It contains various biologically active compounds, such as phenolics and alkaloids. These secondary metabolites are used extensively in dietary food and pharmaceutical products. However, these phenolics occur at very low concentrations in the roots, and thus, it is expensive to commercially produce them. The present study was proposed to induce a hairy root culture system for the first time in L. ruthenicum to achieve a high concentration of phenolic polyamines and other non-targeted secondary metabolites. The over-expression sequence of the TCP4 gene was retrieved from the transcriptome data of L. ruthenicum (LrTCP4-OE), and a gene construct (with pCAMBIA 1307) was integrated into the genome of L. ruthenicum by Ri-mediated genetic transformation. A total of 21 metabolites were tentatively identified by using ultrahigh-performance liquid chromatography coupled to photodiode array detector/quadrupole time-of-flight mass spectrometry (UPLC-PDA-qTOF-MS). Transgenic hairy root clones had higher relative abundances of kukoamine A and 17 other secondary metabolites than did control-type hairy roots. After 1month, high-growth transgenic and non-transgenic hairy root lines were subjected to UPLC analysis for absolute quantification (with an authentic standard) of total kukoamine A. Transgenic hairy root lines (LrTCP4-OE) showed higher kukoamine A accumulation (0.14%) than did control hairy roots (0.11%). This enhanced productivity correlated with increased TCP4-OE activity, validating the primary role that TCP4 plays in total kukoamine A synthesis and the efficiency of non-targeted metabolomic techniques in studying plant metabolites.