Proof-of-concept

The utilization of plants for the production of functional metabolites is the focus of scientists' attention. It is also one of the directions of interest in human synthetic biology. The epidermal trichomes of plants are usually considered as the main processing factories for the production of plant metabolites. Therefore, the use of genetic engineering to modify the differentiation fate of plant epidermal cells to increase the density and size of their epidermal trichomes, while immobilizing metabolites in the epidermal trichomes for production, is one of the feasible ways to increase metabolite production.

We found that the CsTBH gene (Tiny branched trichome), a transcription factor encoding a plant-specific HD-ZIPI-type transcription factor, can effectively increase the density of epidermal trichome in cucumber (Zhang et al., 2021). CsTBH was found to bind directly to the promoter of PAL, a key rate-limiting enzyme for flavonoid synthesis. More critically, PAL has been reported to be highly expressed in the epidermal trichome of cucumber (Feng et al., 2023). Therefore, we planned to utilize CsTBH overexpression to produce more epidermal trichome and also overexpression of CsPAL to let it produce more flavonoids. Finally, we chose the CsTBH-CsPAL co-expression model to produce more flavonoids in the epidermal trichome of cucumber based on our theoretical inference.

Supporting Experiment Results

By observing our color development results, we found that the yeast round spots in the positive control group turned blue, the yeast round spots in the experimental group turned blue, while the yeast round spots in the negative control group did not undergo a color change, which proved that our selected CsTBH gene would bind to the cis-acting regulatory element on the promoter of the downstream CsPAL gene, and the two of them would interact with each other.

Fig.1 Yeast single hybrid results show that CsTBH can bind the promoter of CsPAL

Observation of the overexpression plants revealed that their cucumber fruits would grow many small black spines compared to the wild type, which might be the key to the production of flavonoid substances. In order to clarify whether the fruits of the CsTBH overexpression plants that we cultivated by means of gene editing would produce more flavonoids, by referring to the methods of some scientists, we utilized liquid chromatography mass spectrometry to quantify the flavonoids in the fruits of both the wild type and the mutant. quantitatively, and the results are presented below in the form of bar graphs.

Fig.2 Figure 9 Fruit phenotypes of CsTBH-CsPAL overexpression transgenic plants

Fig.3 Mass spectra and bar graph of total flavonoids content in fruits of CsTBH-PAL overexpression plants by LC-MS detection

References：

[1]Zhongxuan Feng, Lei Sun, Mingming Dong, Shanshan Fan, Kexin Shi, Yixin Qu, Liyan Zhu, Jinfeng Shi,Wujun Wang , Yihan Liu , Liyan Song , Yiqun Weng , Xingwang Liu 1, Huazhong Ren. Novel Players in Organogenesis and Flavonoid Biosynthesis in Cucumber Glandular Trichomes. Plant Physiology,2023,192:2723-2736.

[2]Yaqi Zhang, Junjun Shen1, Ezra S. Bartholomew, Mingming Dong, Shuying Chen, Shuai Yin, Xuling Zhai, Zhongxuan Feng, Huazhong Ren, Xingwang Liu. TINY BRANCHED HAIR Functions in Multicellular Trichome Development Through an Ethylene Pathway in Cucumis sativus L. The Plant Journal,2021,106:753-765.